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THE
AMERICAN JOURNAL

PHYSIOLOGY

VOLUME LIIL

BALTIMORE, MD.
1920 -

4

ThE Meee

j

rw
Jodi

f

ade eae

CONTENTS

No. 1. Auaust 1, 1920

Tue TRANSFUSION EXPERIMENT WITH RED BLoop Corpuscises. Aisanobu

me a. umes TOMAS 2. ccck sk viciis oh ss LO IRS RNS. ENTE |
ON THE REGENERATION OF THE Vacus NERVE. F. T. Rogers. ....\\0....4. 15
Errect or VARIOUS SUBSTANCES UPON THE COAGULATION OF CITRATED

Ch. RM MOOP OMA 0c aie css oss bees oe ex A: AN Wee 25
THE INFLUENCE OF PITUITARY EXTRACTS ON THE ABSORPTION OF WATER

FROM THE SMALL INTESTINE. Maurice H. Rees.............. eg Lip ame Sate 43
Tue Speciric INFLUENCE OF THE ACCELERATOR NERVES ON THE DURATION

OF VENTRICULAR SysToLe. Carl J. Wiggers and Louis N. Katz......... 49

Gastric Response To Foops. XIII. THe INFLUENCE OF SUGARS AND

CanpiInsS ON GasTRIC SECRETION. Raymond J. Miller, Olaf Bergeim,

imme, hehfuss and Philip. B. Hawk.2.. 22.666 c ee cee see i ees 65
FuRTHER EVIDENCE ON THE FUNCTIONAL CORRELATION OF THE HYPOPHYSIS
Seen savROID. John A: Larson. 25 6528. RO VO, 89

THE INFLUENCE OF AN ALCOHOLIC ExTRACT OF THE THYROID GLAND UPON
POLYNEURITIC PIGEONS AND THE METAMORPHOSIS OF .TADPOLES. Emily

I A SIE iE ihe eal ie | tes a Sis Salas aighwek in eee Meade WG nae SURE Gres 101
THe ALKALI RESERVE IN EXPERIMENTAL SuRGICAL SuHockx. Bernard
A ath). S's Ms into BS CY. 0 SS Oe ae TR JW aoa, Ona, t 109

Puysico-CHEMICAL STUDIES ON BIOLUMINESCENCE. III. THE PRropUCTION
or Licut By Lucroua VITTICOLLIS Is AN OxipaTion. Sakyo Kanda... 137

No. 2. SEPTEMBER 1, 1920

Bitoop REGENERATION FoLLowine SimpLteE AnemiA. I. Mixep DIET
Reaction. G. H. Whipple, C. W. Hooper, and F. S. Robscheit......... 151
Bitoop REGENERATION FotLtowina Simpte Anemia. II. Fastrna Com-
PARED WITH.SUGAR FEEDING. ANALYSIS OF ‘‘SPARING ACTION OF
CarBoHypRATES.”’ G. H. Whipple, C. W. Hooper and F. S. Robscheit.. 167
Bitoop REGENERATION FoLLowina SimpLte ANeEmIA. III. INFLUENCE OF
BREAD AND MILK, CRACKERMEAL, Rick AND Potato, CASEIN AND
GLIADIN IN VARYING AMOUNTS AND ComBINnaTions. C.W.Hooper, F.S.
AY SE TED, NL OY WANES fs 2S oe ac oso Cah Fs AI 206
Bioop REGENERATION FOLLOWING SimpLE ANEMIA. IV. INFLUENCE OF
Meat, Liver anp Various Exrractives, ALONE OR COMBINED WITH
STaANDARD Diets. G. H. Whipple, F. S. Robscheit and C. W. Hooper.. 236
Bioop REGENERATION FOLLOWING SimpLeE ANEMIA. V. THE INFLUENCE
or Buaup’s Pitts aNnD Hemocuosin. C.W. Hooper, F. 8. Robscheit and
See RL PV APD DIE, 05 as ls hare y ss Se UN OU ALG MRIUNG gic kei ences OR W.» sl Rik Rae 263

1V CONTENTS

PHYSIOLOGIC CHANGES PRODUCED BY VARIATIONS IN LuNG DISTENTION.
III. IMPAIRMENT OF THE CORONARY CIRCULATION OF THE RIGHT VEN-
TRICLE. Ralph Hopkins and Felix P. Chillingworth.............5...... 283

VAGUS AND SPLANCHNIC INFLUENCE ON THE GastTRIC HUNGER MOVEMENTS
or tae Froc. Comparative Stupies III. 7. L. Patterson........... 293

OBSERVATIONS ON THE RELATION BETWEEN EMOTIONAL AND METABOLIC
Spavrrry, .Krederickh S: Hammett... oo... 0 i noc ee ee ee 307

Four Factors Causina CHANGES IN THE TYPE OF RESPONSE OF THE Iso-
LATED| INTESTINAL SEGMENT OF THE ALBINO Rat (Mus Norvecicus

ALBINUS) to Sopium Carponate. S. Hatai and F. S. Hammett....... SIZ. .

Tur ADJUSTMENT OF BLOOD VOLUME AFTER INJECTION oF IsoToNiIc SoLu-
TIONS OF VARIED \ComposiITION. Arthur H. Smith and Lafayette B.
Met gehen. 05 NDS TS Tae Oa aay a piaie aoe ee hale 323

©

No. 3. Ocrossr 1, 1920 ; t

ANTAGONISM OF INHIBITORY ACTION OF ADRENALIN AND DEPRESSION OF
Carpiac VAGUS BY A CONSTITUENT OF CERTAIN TissuE EXTRACTS.

Dis B Colla. ck. PER ARS kn SERS OR er re 343°

REcIPROCAL REACTIONS IN THE CARDIO-VASCULAR SysTEM. Ethel W.
W tchwire rocs ek lee bin Ch wa bw RO Va ee ee i 355
FURTHER OBSERVATIONS ON THE RELATION OF INITIAL LENGTH AND INITIAL
TENSION OF AURICULAR FIBER ON Myo- AND CarpIopyNAmics. Robert

Gesell........ wee AS OPA We Wh ek Re 377
THE ROLE OF THE PANCREAS IN HYPERGLYCEMIA FROM ETHER. Ellison L.
Rees ond. LH. Dots... CUR Re ee eee ep ‘cute aon

PHYSIOLOGICAL STUDIES ON:PLANARIA. IV. A FurTHER Stupy or OxYGEN

CONSUMPTION DURING STARVATION. Libbie H.-Hyman................. 399

VasomMoTorR REFLEXES FROM RECEPTOR STIMULATION IN INTACT ANIMALS.
E.G. Martin, A. C..Franklin and Clarence Hield........... 4 a en 421
STUDIES IN PLACENTAL PERMEABILITY. I. THE DIFFERENTIAL RESISTANCE
TO CERTAIN SOLUTIONS OFFERED BY THE PLACENTA IN THE Cat. R. 8S.

CURBNINGham oor 0s bien e dae vale vole dia ec ale Gis ce Vide sea ey hole ee 439
THE PRODUCTION OF INTRACELLULAR ACIDITY BY NEUTRAL AND ALKALINE
SoLutTions CoNTAINING CARBON Dioxipe. M. H. Jacobs............ . 457

THe Errect or Sautt INGESTION ON CEREBRO-SPINAL FLUID PRESSURE
AND Brain VotumeE. Frederic E. B. Foley and Tracy Jackson Putnam. 464
ANTAGONISM OF DrprRESSOR ACTION OF SMALL Doses oF ADRENALIN BY

Tissur Extracts. J.B. Collipooit ees a ea ee 477
OBSERVATIOHS ON A SEx DIFFERENCE IN THE PRESENCE or NATURAL
HEMOLYSIN IN THE Rat. Yoshio Suzuki......00..5 0000 cc cele dee cveee 483

Stupies oN ABSORPTION FROM SeROUs Cavitigs. III. Toe Errect oF
DEXTROSE UPON THE PERITONEAL MesoTHELium. R, S. Cunningham.. 488
ERE. 6. 5 RR a) aE ae ee a etic, eee mR ape ats 495

ar

_ THE AMERICAN
JOURNAL OF PHYSIOLOGY

VOL. 53 AUGUST 1, 1920 No. 1

THE TRANSFUSION EXPERIMENT WITH RED BLOOD
CORPUSCLES

HISANOBU KAMBE anv ETSUZO KOMIYA
From the Medical Department, the Imperial University, Tokyo, Japan

Received for publication, April 6, 1920

What is the fate of red blood corpuscles if they have been introduced
into an animal body of.same species? This problem has been studied
by numerous investigators (1), (2), (3) and though there are still ob-
jections it is generally believed that the transfused red cells are capable
of functioning in the animal body. Whether these red cells remain and
function in circulation of a recipient animal or whether they are decom-
posed, was formerly studied chiefly by means of the examination of
the changes of urine or the bodily condition of the animal. Recent
opinion indicates that the most satisfactory method of attacking the
problem as to the length of life of the transfused corpuscles is to study
the changes in erythrocyte count following transfusion.

In the earlier work in which this method was used little attention was
given to morphological changes in the corpuscles. This is a point of
considerable importance because from the erythrocyte count alone we
are not justified in drawing conclusions as to the biological phenomena
exhibited by the foreign corpuscle or its host. It is therefore desirable
to collect more data on the subject.

EXPERIMENTAL

In the present experiments full-grown rabbits were used. To obtain

blood from a donor the carotid artery was exposed by usual method

and opened on one side with a sterile scalpel. The blood thus collected

in a sterile, thick-walled flask containing glass beads, was defibrinated

by vigorous shaking, filtered into another sterile flask through double
1

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 1

2 HISANOBU KAMBE AND ETSUZO KOMIYA

gauze, and preserved for injection. Sometimes blood was kept in a
mixture of isotonic citrate and dextrose solution, and when this mix-
ture was used the supernatant fluid was pipetted away. |

To render a recipient animal anemic, blood q. 1. was withdrawn from
carotid artery without anesthesia,-care being taken to avoid the shock
which may be caused by a rapid hemorrhage. The blood obtained from
the donor was then transfused into the ear vein of recipient rabbits
using a sterile syringe. At the time of injection the blood was warmed
to the body temperature. In each series of experiments, the number
of red cells, hemoglobin content and morphology of the blood were
carefully studied. The number of red cells was counted by the Thoma-
Zeiss apparatus, and hemoglobin content was estimated by Sahli’s
hemometer. Blood cells were stained in May-Griinwald-Giemsa’s so-
lution. Each experiment was repeated several times with almost the
same result. To avoid a repetition of data only one instance for eac
case is recorded here. 2

Control experiment. In the first place an experiment was done to
observe in what manner the natural restoration of the anemia caused
by hemorrhage takes place and what changes in the direction of mor-
phology are brought about.

Experiment 1. Rabbit weighing 2500 grams was bled 65 cc. from
the carotid artery. The animal has been always in a good condition.

It may be seen from figure 1 that if the blood of 20-30 cc. per kilo-
gram of body weight is depleted, the number of red cells of the animal
is generally reduced by 40 per cent or more of its original amount and
with a gradual improvement it will return to normal level two weeks
later.

As to morphological changes, polychromatophilia was especially notice-
able. This kind of red cells appeared in twelve or twenty-four hours
after hemorrhage, and gradually increasing in number they reached the
highest point on the third or fourth day, disappearing again in the course
of aweek. These polychromatic red cells are larger than normal red cells,
so the blood has appearance of a remarkable anisocytosis. Erythro-
blasts and basophilic punctates may also be seen more or less in the
early stages of anemia, but they are rather inconstant or few in number.
These results, as is well known, indicate an abnormal activity on the
part of the bone-marrow in the sense of compensation for a loss of
blood cells by hemorrhage. |

Transfusion experiment. From the above recorded fact it is easily
expected that if transfused red blood corpuscles may function in the

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Hb (Sahit)

4 HISANOBU KAMBE AND ETSUZO KOMIYA

animal body and an amount of blood is enough to make up for the pre-
vious deficit and if replacement is done as soon as the animal has been
bled, there will not occur any change in the blood picture, neither
numerically nor morphologically, the bone-marrow being subjected to
no anemic stimulation, while in the reverse case such changes will take
place.

In general we have noted the volume, the erythrocyte count and
the hemoglobin of the drawn blood, and have calculated from these

=
Hp| Red _ pF|26|27/28 129 |30 172) 2 |3 14
cells Ka
2
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4
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Polychromasia [7+] +}4|— || |-|—|—
IR Sehicichis Ite en
Ervibroblasts [hit pot ie
gygoooqqq aasg
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RedCells A 5] of ol YA GSI S
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Fig. 3

the amount of cell suspension of known hemoglobin content that should
be injected to restore the animal’s hemoglobin content to the normal.
Experiment 2. Rabbit weighing 2000 grams was bled (40 ec.) from
the carotid artery, and then 50 ce. of defibrinated fresh blood were in-
jected at once. The animal was very lively.
In the case in which transfusion was done immediately after bleeding,
neither hemoglobin content nor number of red cells indicates any change,

Se Ss

~~

— *

ae

ee ee ai ee ee Se ee Le gs

VIABILITY OF RED BLOOD CELLS 5

as figure 2 shows; morphological figures are entirely the same with the
normal. |

Experiment 3. The used rabbit weighed 3000 grams. Seventy-six
cubie centimeters of blood were taken and after about 20 hours the
same amount of defibrinated fresh blood was injected.

In the case in which blood has been replaced within twelve or twenty-
four hours after bleeding, the hemoglobin content as well as the red cell
count returned to normal level simultaneously with the injection, while
the disappearance of polychromatophilia and anisocytosis took place
after a few days, but in this case the degree of morphological change was
milder and the duration of its appearance shorter than those of the con-
trol experiment (fig. 3). This fact illustrates that the bone-marrow
was subjected to a transitory stimulation, a certain time having elapsed —
before injection. :

As is evident from the results of the above experiments, the trans-
fused red blood corpuscles are capable of functioning.

Among these latter experiments we met with two very interesting
instances, that is, the animal dealt with as just described developed a
sudden anemia on the fourth day after the blood pictures had once
returned to normal. Furthermore polychromatophilia and anisocy-
tosis in a large number, corresponding to the number of red cells,
appeared again in circulation. This anemia returned to normal condi-
tion in the course of fourteen days as in the case of the experiment 1

; (fig. 4).

Experiment 4. Rabbit of body weight of 2750 grams was bled 60 cc.
at one time from the carotid artery. After four hours 65 cc. of de-
fibrinated blood kept in ice for forty-eight hours were introduced.
Animal remained in very excellent condition, very lively.

These results indicate evidently that the tiaretiabdl red cells were
destroyed on the fourth day after transfusion. We shall refer to the
possible reason for this later. :
' Our study will turn now to determine how long these red cells are -
kept alive from the view of their capacity of functioning in the animal
body. lLandois, who has studied this problem extensively, states that

cells kept for two days at 5°C. exert no harmful effect on the animal, but

those kept for three days give rise to albuminuria and hematuria and
cause death on the second day after transfusion. In 1916 Rous and
Turner (4) developed a method for preserving living red blood cells in
vitro and determined the length of their life by means of transfusion
(5). This is the only report which paid attention to the morphology,
so far as we are able to find in the literature.

6 HISANOBU KAMBE AND ETSUZO KOMIYA

They found that erythrocytes preserved in mixture of blood, sodium
citrate and water for fourteen days remained in circulation and func-
tioned so well that the animal showed no disturbance, and the blood
count, hemoglobin and percentage of reticulated cells remained un-
varied, while cells kept for twenty-three days, though apparently intact
and unchanged when transfused, soon left the circulation.

Red SH7] ala hiolufe2hsvals| fat fal lau 25] la [2a
fb cells a
ys
e a =
60!16000.000 J \ Ae
e.] ha k
1S ats Rae Ls
CZ * 7
O OO 000 fb { at ie 4 B ue -|-@+__ ne
: ‘ Ae ae
50 sha \ 7 Be ENCE RY =
\ | a
L jE
_ 184.43 9l00q I [\«
A0|\4000,.000 1 ot.
i rN
Y el A)
V
3013000.000 i
Auisocytosis |=|=|=|—|— [FEE HE] Ht ew ies
Polyeuromasia|—|—| |=] =)-F LE He ery — |" deed ee
lPunctate basophitia |||} | II IT was Bg es
Erythroblasts|—\"|— | 1 Soa oe — pas ei BM EP.
dddgggggdagq ia iS qd Ja fa
Red Cells |A999999949 [9 | 4 Js | 13 [8
addINanag taal is Dd lo SS od lea
per cmm |}qginyyyagqndaq fo o IN o> A le
AA TYNG Ty a | Y | 14 aq |G In
GGYHQOn dF dy | ee © Q@ |
baht) [dada dss la ts lat | la tal Ty
Fig. 4

Our experiments were made with defibrinated blood and blood-
citrate-dextrose mixture to determine the availability for functional
use of red cells kept in vitro.

Experiment with defibrinated blood. When sterile blood was put at
once on ice it showed no trace of hemolysis in the supernatant fluid after —
ten days, while on the twentieth day of preservation a very slight he-
molysis was seen, but the number of red cells did not show a marked

i te te

VIABILITY OF RED BLOOD CELLS

decrease, and the morphological character of the cells seemed entirely
normal. When blood was kept for thirty days, there was some he-
molysis and also an appreciable decrease in number of red cells. In the
fresh and stained preparations the blood appeared normal. In experi-

ments 5, 6, 7 and 8 the blood thus preserved for various periods was
transfused.

112-3 129130
Hb| Red Je de /9\20|Z21 |22/2.3 124 6|2712.3 |29 *
cells ai
EULS
a
[ ie
; \ eo
110| 7.000.000 al | iis
> a bie /
g Y wl K/
\ é. yar"
\ fF yt — At teh
9016.000.000 ie wT. n
4. lé ad ™ Be
BH {! se
‘AN H
ee |
70O|5.000.000 *
HI
\
=
50|4.000.000 q
|
Val
¥
30|3.000.000 t
Auisocytosis§ (> JTyr it tt ttt er fe ft |e} | =
Polycuromasia TET ee
Unc: asophili “a ng ne! OK: aneMe, Gn.et ios Lae wae! evn! ae
s Pe fot WE 1 Ot bh eh ee ae ce OL
aol GO of Sf of of Of of of of of Of OF OF SO
Red Cells ao BF Oo] S} O] a] OF 9] CO] Gf GA) SO] Of ao!lO
€ AAGAAAIDSDA SI o Adgadoalaia
HOY A Sl all Glod NI ood wl ol viea
ee btbetat: GI al So AMIS
NVA TIO Sy WN YG QTd
44. 5] Ol OO] SP OEE SEES BY IN
Hb(Sahli) JAStToMeasiNAViestede
Fig. 5

Experiment 5. Rabbit weighing 2300 grams was bled twice (55 and
35 ec.). After twenty-four hours the equivalent amount (hemoglobin
content 90 per cent) of defibrinated blood kept for three days was
injected.

Experiment 6. Rabbit weighing 2050 grams was bled (40 cc.) and
twenty-four hours later 38 cc. of blood which had been taken eleven

8 HISANOBU KAMBE AND ETSUZO KOMIYA

days previously were injected (number of cells in transfusate

5,456,000 in c.mm.). &
Experiment7. Body weight: 2100 grams. The recipient rabbit was

bled (50 cc.) and at once the same amount of blood preserved for twenty

days was transfused. The animal manifested at no time symptoms of
distress.

Red 27 1231249|30|3/ |412 13/4] |6| |8
Hb cells a e
t
70|6.000.000[a = _i2 ct
. ) Fas Tf aN
IPR RIZC Ie
r NA \
4 f J bs
601S.000.000 [7
: AWA *
50\4.000,000 yi
5 Be
Hh
P’)
4.0|.3,000.000
Anisocyto —_|—|—|—| +] 4-4/4 mn EA tee
Polychromasia aad bar A dec = a ca Bae
Punctate basophil —|— |= IF IE ELF I= cy ee baw 258 Rae
" Ervthroble sfs (oop be ea ee ee,
oOo] ST OF STS; oP Ss sis oO oO
S| S| OJ 3} 3} GS OS
Red Cells |FIIIIAASs fal 1s
ery cmm sa 3s Nqeis 7 2
P aS AS yg gy Is
Oo 4 OO HH Usd [4 [do
’ ~ N 9
Mb(Sahli) FAAZ@aggasdea isi la
Fic. 6

As shown in charts 5, 6 and 7, red blood cells in defibrinated blood
are capable of functioning even when they have been kept in vitro for
three weeks.
~ Experiment 8. Experiments were all done with cells kept for thirty
days in vitro.

A. An animal weighing 3150 grams was bled twice (70 ce. and 30°
ce.), and an equivalent amount of kept cells was injected. The rabbit:
lived for two days but neither the number of red cells nor the hemo- _
globin content returned to normal. In morphological figures there

} he <

ee ee

Ae eee ae oe

SO Oe Oe ee eA ee eS al a ee

VIABILITY OF RED BLOOD CELLS 9

appeared noteworthy alterations corresponding to this change (fig. 8,
A). 3

B. Body weight 1900 grams. The animal was bled 38 cc. and then
50 cc. of blood (cell count in c.mm.: 3,112,000) were introduced. This
rabbit was alive for a-long time after the operation, without showing
any increase in number of red cells and hemoglobin percentage in con-
sequence of transfusion. Thus in natural recovery of above mentioned
anemia, they returned to normal in the course of ten days. At this
time an appreciable change in morphology was noted.

Two other animals died within one-half or one hour after transfusion.

Hb| Red 21 6)'7|.8 49 | oj 23 }IALIS
cells rd
a+ 22
* Ne--e
N a R
20\5.000.000 [7 wee st

er
x
i

60|4.000.000
Anisoevtosis |-I—I-I-I-I-I-F
Polychromasia|—

Runctajé basophiia| |
Erythroblasts

|
|
|
|
|
|
|
|
|
|

]
|
|
|
]
|
|
]

|

Sssggsgssgass
Red Cells |AVTAIIIIII SS
Ga} +} a] af G sl G aa
per emm |Slelqgnd JqgqyaNys
Sa 4 OOTY 4S
SNH a> aS
Meant) | acl da fd oa
Fig. 7.

Both animals contained pitch-dark colored hemoglobinuria in the
bladders.

Transfusion with cells kept in a blood-citrate-dextrose mixture. For the
purpose of preservation three parts of the blood were mixed with five
parts of isotonic citrate and two parts of isotonic dextrose solution.
When they were to be used the supernatant fluid was pipetted off, the
cells suspended in 0.85 per cent sodium chlorid solution until the
original quantity of blood was reached and then used for injection. The
mixture could be kept for one month without showing any trace of he-
molysis. On the thirtieth day there began to appear a very slight
hemolysis.

10 HISANOBU KAMBE AND ETSUZO KOMIYA

Experiment 9. Rabbit weighing 2500 grams was bled (50 cc.) and
replaced after twenty-four hours with 60 cc. blood (number. of blood
corpuscles 4,516,000 in c.mm.) which had been kept for thirty days.

As figure 9 illustrates, the result was entirely similar to those cases
in which fresh blood was transfused.

Experiment 10. A rabbit weighing 2850 grams was used. Two
bleedings (60 cc. and 35 cc.) were effected; after having left the animal

Red 23/24) | [8-1/4 [20/2)|22l23/24/25]26] |28) Bo} |
Hb} cells 1 ol
v
H
i
60|6.000.000 4 ry
A
| | ay ete
Kap so) gear
000.000 ' we.
5015 , et
\ \ lal Ye
\ 2 \ are s
40|4.000,000 i LY W
es ri
3 013.000.000
Anisocytosis |—|> [Ft [tt wel ol ad Ld BLL Wa —|—
Polychromasia |= HEY |} a te —|—
SPT RES! aes bas Raed Fags —— ee ee se eS ee — — —FP—
yvthroblasts j=|=}—} + {+ meee Kania as: D rs ae
ia Q/Sio/ 0 9} S| al a] a] of 9] B
o| 9 S oO] 8
Red Cells [sigsisi | |3idasassis fa iis
9159 | [eas ga4sSs [4 |os
per cmm. [9] 9} «9 VINgsasug le wl +
3/9] 4 yrytiys +a is 14 4
1 rv)
Hib(Sahli) |eisigis an SISAH Ss HA Ls lie
Fig. 8

in anemic condition for forty-eight hours, there appeared a large num-
ber of young cell forms in the circulation. After transfusion with an
equivalent amount of blood cells preserved in ice for forty days, these
cells disappeared within a few days from the circulation, indicating
that the transfused blood corpuscles function entirely normally (fig. 10).

In this experiment it happened that the red cell count showed a grad-
ual decrease from the fifth day after transfusion, reaching its maximum
drop on the ninth day, after which a gradual recovery ensued. Mor-

VIABILITY OF RED BLOOD CELLS a:

phological figures ran parallel to the changes in number of red blood
corpuscles. Marked polychromatophilia and anisocytosis, etc., which
once had been restored to normal, appeared again on the tenth day, but
at the time of recovery from anemia had disappeared altogether.

The experiments so far have shown that red cells may preserve their
vitality in vitro for a long period, namely, in defibrinated blood for
twenty days, in the mixture of blood-citrate-dextrose even for thirty
days or more, and may function normally in the animal body when
transfused. Our results therefore differ somewhat from the findings of

Hb Red Fyvolst 12 IZ |g iS|/6| [18
cells it
R
4 \
Fa
70|5.000.00 0 left? nf Be =
J ra a rN
\ \ DUN
"7 Te irk
W\ \ ay
60|/4.000:000 \ ei
1 .
4
\!
7
5013,000-000 é
Anisocytosis |~|7|7IF IF IF ]= |7} [=
Iychromasial | TIF | |
Functate. basophilial (7 | ee
Evvthyoblasts 17 it ich al
| AEEERRRERE
Red Cells S33} 3] a aS 9
per cmm I9anNasyqy js
ANS SIAN 12
44 of og tO WG [6
Hb (Sahli) JSS aN ads lz
Fig. 9

Rous and Turner who concluded that cells kept for twenty-three days
in their preservative mixture, though apparently intact and unchanged,
soon left the circulation.

Preceding the conclusion of this work we will touch briefly on the
cause of the peculiar cases of acute anemia above mentioned (exper. 4),
’ which have developed at a certain period after transfusion. At first we
assumed that this anemia may occur due to the destruction of red cells,
being caused by the injurious effect on the function by cold, because

12 HISANOBU KAMBE AND ETSUZO KOMIYA

such transfusion experiments with blood preserved in ice showed in
succession the same results, having initiated a sudden drop of hemoglo-
bin on the fourth day after transfusion. But it became evident after- .
wards, as a number of experiments proved, that this destruction of red
cells is not simply due to the preservation or the refrigeration by ice.
Rous and Robertson (6) made a report similar to ours, having injected
10 ec. of blood every other day. They stated that in-several animals

1S)9 Dole eces|e4ie526e 1 eajeqBo37| [2|o 14>
Hp) Red 4
cells , 18 19
| £. Kec. *
_eV\ 4 1s js
60|.5,000.000 [7 yn aes te
é “ \ ; \ ¥
| Th ry
\ | k Way * ¥.
000.000 ~
50|4.0 , ae
bt &
j
A0|3.000,000
Wi
M
30|2.,000.000 %
Anisocytosis —l— hae ae a Re eee ee ree
Poly chromasia —l—| I+t+tlH—-|-l-} I-Ie ie ole ote
Pinctate bas ophilia| ~|— —|—}—j -|-|—}| —|-l—]—} —|- 4 d=
Ervthroblasts (otal eee eee eS
$ o| O sa os 99 39 8's 8 Ol o °
Red Cells | 3a J33 8/3) 95948 sj} [8
% ANyasgss aay Shak 8
peremm [aq [AI GNaaydae ala jx
Hf ol ty 4) St SSS 99 0) od oe ES
bina S
Mb(sahh) [aa idadenasasa isl [2
Fig. 10.

in which the agglutinin was strongest, the plethora was suddenly suc-
ceeded by severe anemia, despite continued transfusion.

Not having made any serological researches, we hesitate to assert
whether or not in our cases such strong agglutinin was also existing.
We could, however, demonstrate afterwards occasionally in a number of
transfusion experiments such hemoagglutinin, though not so strong,
similar to that pointed out by the above authors with regard to the
character of temperature control of the agglutination, the persistence

Ps
,

VIABILITY OF RED BLOOD CELLS 13

of the agglutinating principle and many other points. Among those
eases we had one instance (exper. 10) which developed anemia on the

fifth day after transfusion, but more slowly than the acute cases above

stated, reaching its maximum in the course of four days.

The plasma of this rabbit agglutinated not only the cells of its own,
but also those of the same species and showed this activity even when
diluted 150 times; it was also disposed to have stronger activity, in
spite of a gradual recovery of anemia.

Rous and Robertson reported:

We have chilled, without result, two plethoric rabbits possessing a weak agglu-
tinin in the hope of initiating a drop in the hemoglobin. The chilling was accom-
plished by means of ice-cold water, in which the well shaved ear of the rabbit was
submerged for 4 or 1 hour. Throughout this period the circulation in the cold
ear was a enbicnctts good. The rectal temperature fell to 37°C., considerably
below the normal for the rabbit, but not enough to produce the in vitro agglu-
tination of blood corpuscles.

It seems probable that the agglutinin above described including also’
the case of Rous has no direct causal relation to such anemia and the
question arises whether an uncommon hemolysin may play perhaps an
important rdéle.

Furthermore the fact that such occurrence has been noticed only in
the experiments performed in cold winter time and not in other seasons,
leads us to believe that it may have some connection win paroxysmal
hemoglobinuria.

SUMMARY

1. Transfused red cells even preserved in ice for a long time, not to
mention fresh blood, are capable of functioning if transfused into the
animal body of same species.

2. Erythrocytes preserved as defibrinated blood maintain their nor-
mal vitality for twenty days, in the mixtures of isotonic sodium citrate

_and isotonic dextrose, even for thirty days and more; thus they cael be

used to replace the blood lost.

3. A sudden anemia occasionally devils a few days after trans-
fusion. There is a possibility that this is due to the isolysin, even
though it occurred only in isolated cases. We are, however, unable to
draw a sure conclusion from the present work, but will pursue further
our studies which may throw some light on this point.

14 HISANOBU KAMBE AND ETSUZO KOMIYA

The authors desire to express their thanks to Prof. Dr. T. Irisawa
for his suggestions and encouragement throughout the course of this
work; and also to Prof. 8. Mita for his kind advice.

BIBLIOGRAPHY

(1) Panum: Virchows’ Arch., 1863, xxvii, 240.

(2) Ponrick: Virchow’s Arch., 1873, lxii, 273.

(3) Lanpotrs: Transfusion des Blutes, Leipzig, 1875. _

(4) Rous anp Turner: Journ. Exper. Med., 1916, xxiii, 219.
(5) Rous anp Turner: Ibid., 1916, xxiii, 239.

(6) Rous anp Rosertson: Ibid., 1918, xxviii, 509.

ON THE REGENERATION OF THE VAGUS NERVE

F. T. ROGERS
From the Hull Physiological Laboratory, University of Chicago

Received for publication April 30, 1920

Three years ago, in connection with work on nerve crossing between
the phrenic and vagus, attention was again directed to the doubt
existing as to the possibility of functional return after section and
suture of the vagus. This, according to Langley (1) and Tuckett (2),
and more recently by Schaffer (3) is doubtful or, if it occurs, it does so
only after the lapse of years. Asa control on other work it was decided
to repeat the old experiment of section and suture of one vagus leaving
the other intact and after as long a time for recovery as practicable in
the laboratory, test the nerve for functional activity. In order to
avoid the uncertainties involved in testing functional regeneration by
electric stimulation, resort was made to the old suggestion of subse-
quently cutting the intact vagus leaving the regenerating nerve to
exert such action as it might. This conclusion was forced by the
negative results following stimulation of the nerve when only a few
months were allowed for regeneration. The results of this series of
experiments confirmed the similar experiments made by others using
the same method (1), (4). |

The first series of experiments consisted of four cats and one dog in
each of which one vagus and cervical sympathetic were sectioned and
sutured just below the level of the thyroid gland. After time intervals
of from three weeks to six months the nerve was tested electrically with
the animal under ether anesthesia and with arterial pressure recorded
from the carotid artery (table 1). In every case it was found that
while stimulation of the normal nerve produced the usual cardiac
inhibition and fall in blood pressure, the stimulation of the previously
sectioned and sutured nerve caused no cardiac inhibition and no gastric
motor effects when stimulated on either side of the point of suture.
Stimulation of this trunk above and below the scar did cause the usual
respiratory inhibition and gave reflex effects on the blood pressure.
With reference to the actual recovery of efferent fibers, Tuckett states

15

16 F. T. ROGERS

that after three years this does occur. The only other similar positive
statement that I have found is that of a single observation of Stewart
(5), of which he states that ‘‘some regeneration appeared to have taken
place (after three hundred days) since stimulation of the nerve caused
slowing and weakening of the heart.”

TABLE 1

In each animal of this series one vagus was sectioned and sutured, except in the case
of cat 17 in which the nerve was crushed by hemostatic forceps and not sectioned.
After the time intervals indicated in the table, the animals were etherized, carotid
blood pressure tracings made and both the normal and the previously sectioned —
vagi were stimulated with tetanizing current. Stimulation was applied above and
below the scar of union, but in no case did the effects differ. In all the animals
of this series it was found at autopsy that the two ends of the nerve were united by
a small neuroma

TIME EFFECTS OF STIMULATING THE SUTURED VAGUS
ALLOWED
ANIMAL FOR
aT? Heart Respiration Blood pressure Stomach
months —
Cat 28 2 No inhibition | Inhibition '
Cat 12 4. No inhibition | Inhibition No contrac-
tions

-Cat 18 5 No inhibition | Inhibition | Weak stimulation
pressor _ effect,
strong stimula-
tion depressor
3 effect
Cat 17 6 No inhibition | Inhibition | Depressor effect
Dog 40 4 |.No inhibition | Inhibition | No contrac-
tions

Of the second set of animals only two dogs survived the long time
set for the experiment (tables 2 and 3.) In two dogs, nos. 95 and 96,
twenty and sixteen months respectively elapsed between the section
of the one nerve and the section of the remaining vagus. In one case
the animal lived thirty-four days and in the other sixteen days after
cutting the second nerve. The cause of death in both cases seemed to
be starvation due to paralysis of the esophagus and continued vomiting
which followed attempts to eat. Respiration continued at a normal
rate and amplitude during this interval of life, save that at times both
dogs showed a hiccough-like disorder associated with cough that
seemed to be due to irritation of the respiratory tract by vomited mate-
rial. This continuation of normal breathing might be considered

= ee 2

:

REGENERATION OF VAGUS NERVE 17

confirmatory of Schaffer’s recent findings that section of the vagi in
the cat causes little change in the respiratory rhythm provided asphyxia
be prevented by keeping the larynx open. The fact noted in the
tabulated findings that in one of my animals the section of the regen-
erated nerve after the previous section of the other vagus was followed
by a slowing and deepening of the breathing, indicates regeneration
had occurred, of either or both, the afferent pulmonary fibers or motor
fibers to the laryngeal muscles. According to Vanlair (6), functional
regeneration of the motor fibers of the recurrent laryngeal nerve can
be demonstrated after one year. The facts just stated above seem to
confirm this finding of Vanlair but unfortunately the writer was unable
in this dog to make any observation as to the part played by the larynx
in this change of respiration.

With reference to the heart an interesting condition was found
Electrical stimulation of the regenerating vagus, with the animal
under ether anesthesia, caused no cardiac inhibition. Sectioning of
the normal nerve so as to leave only the regenerating nerve in relation
to the heart, was followed by a marked increase in the rate of the heart
beat. These facts of negative results to electric stimulation and an
immediate increase of the heart rate after cutting the remaining normal

nerve indicate that the regenerating nerve. was not functional for it is

common knowledge that section of only one vagus leads to only a slight
cardiac acceleration. This conclusion was subsequently confirmed by
cutting the regenerating nerve which caused no change in the heart

- rate (table 2).

In spite of these indications of absence of function in the regenerat-
ing fibers, the rate of the heart beat daily became less and in two weeks
the rate was that of a normal animal. In other words, with one vagus
degenerated and the other not functional, the cardiac rhythm returned
to a normal rate. This fact was also noted by Stewart.

When this stage of recovery had been reached, the injection of atro-
pine gave a tremendous increase in the heart rate. This effect was
evidently due to some other factor than that of paralysis of vagus
fibers. The writer hesitates to speculate on the mechanism of this
atropine effect. It recalls the observation of Carlson (7) that atropine
stimulates the heart ganglion of Limulus and suggests that the usual
effect of atropine in the normal animal is twofold, paralyzing the
extrinsic inhibitors and stimulating the automatic nerve mechanism.

In dog 95 a fortunate incident gave direct ocular proof of the fact
that the vagus inhibitory fibers to the heart can regenerate. As stated

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 1

18

F. T. ROGERS

TABLE 2
Dog 96 :
July 4, 1918. Section and suture of the right vago-sympathetic. The dog is
three and a half months of age
October 15, 1919. Gastric fistula is made
DATE .HOUR PROCEEDING RESPIRATION sie vig REMARKS
Nov. 1 | 2:00 p.m. | Dog quiet 12 per min. 84
Nov. 1] 5:00p.m.j| Left vagus
sectioned
Nov. 1 | 10:00 p.m. 12 per min. | 230 | Vomiting during
San. the night
Nov. 2 | 11:00 a.m. 20 per min, 162
Nov. 2] 9:00 p.m. 9-12 per min.
irregular
Nov. 3 14 per min. | 130] Eating without
irregular vomiting
Nov. 4 13 per min. | 142 | Eating and vomit-
irregular ing
Nov. 5 8 per min. | 142| Vomiting and
labored cough
Nov. 6 9 easier 146 | Alert, wags tail.
Vomits and
coughs
Nov. 7 10 per min. 136 | Eats nothing
Nov. 11 | 3:00 p.m. | Stomachtrac- | 10 per min. 116 | Getting very thin
ing made
Nov. 11 | 4:30 p.m. | 0.3 ec. of 0.1
per cent
atropine
Nov. 11} 5:00 p.m. 149
Nov. 12} 1:00 p.m. 10 per min. 114 | Very thin, eats
nothing
Nov. 12 | 5:00 p.m. | Cut the right ce
vagus
Nov. 12 | 11:00 p.m. 5 per min. 112
Nov. 14] 2:00 p.m. is 6 per min. 92
Nov. 14 | 3:00 p.m. | 0.3 cc. atro-
pine 0.1 per
cent
Nov. 14 | 3:15 p.m. 6 per min. 160
Nov. 14 | 6:00 p.m. 6 per min. 122

Nov. 16, 2:00 p.m. Dog dead. Lungs, hyperemic patches with two pus
pockets, }.inch diam. Scraps of food in stomach.
united.

Observations at time of cutting the second vagus, November 1, 1920.

The two ends of the vagi

SES | Se Ce

|
= ee a a ee Se ee CS Se oe Ce ae eS
ns ane > 4

REGENERATION OF VAGUS NERVE 19

Blood pressure recorded from the left carotid artery.

Balloon in stomach connected with water manometer, to record gastric con-
tractions and respiration.

Dog under ether anesthesia: stimulation with tetanizing current.

1. Effects of stimulating left vagus before the nerve was cut.

Heart, inhibition and fall of carotid pressure (fig. 1, A). Respiration,
inhibition. — ;

Stomach, strong contraction followed by weaker ones (fig. 1, A).

2. Effects of stimulating ’the right nerve which had been cut and sutured.

Stimulation central to the scar of union.

Heart, no inhibition. Rise in blood pressure.

Stomach, no contraction.

Respiration, inhibited.

Stimulation peripheral to scar of union.

Heart, no change. Rise in blood pressure (fig. 1, B).

Stomach, no contraction (fig. 1, B).

Respiration, inhibited.

3. Repeat the stimulation of the normal nerve.

Normal effects on heart and stomach, as in paragraph 1 above.

4. Cut the left vagus and closed the wound in the neck.

previously, the dogs seemed to die of starvation. Two or three days
before death the animals became inactive and went into a comatose
condition. In the case of this animal, I chanced to find him when
death was imminent. The dog was cold to touch, breathing was barely
preceptible at three or four times per minute. Since the animal was
dying, it was killed by opening the thorax without anesthesia. The
heart was beating slowly at the rate of 40 per minute. The regenerat-
ing vagus trunk was stimulated by tetanizing current, twice above and
twice below the scar marking the point of suture. Each time of stimu-
lation the heart ceased beating for five to ten seconds and resumed
beating at a slower rate than that preceding the stimulation. Unless
there be accessory cardiac fibers outside the main vago-symipathetic
trunk, which had escaped section, this observation indicates that
regeneration of the vago-inhibitory fibers may occur if sufficient time
be allowed. And it lends force to the criticism that electric stimulation
of regenerating nerves in anesthetized animals is not a wholly reliable
test.

In dog 96 a gastric fistula was made a month before cutting the
second vagus. When this animal was etherized at the time of cutting
the second nerve, comparative graphic tracings were made of the motil-
ity changes in the stomach after stimulating the normal and the regen-
erating nerve. Under light anesthesia, stimulation of the normal

20 F. T. ROGERS

vagus caused a strong contraction followed by smaller peristaltic
waves. Stimulation of the regenerating vagus a few minutes later
caused no detectable contraction of the stomach (fig. 1). Two weeks

TABLE 3
Dog 95. Adult brown bull dog

March 7, 1918. Sectioned the right vago-sympathetic and accu the ends
together in the carotid sheath. The sheath was then closed bas two
stitches, but none were taken through the nerve ends

November 5, 1919. The left vago-sympathetic was sectioned

DATE HOUR. PROCEEDING Bay St PULSE REMARKS
Nov. 5]. 1:00p.m. | Before operating | 15 74 | Dog active
Nov. 5| 2:00p.m. | Cut left vago-
sympathetic eich eh
Nov. 6 14 147 | Eating. Chases guinea
pig ,
Nov. 7 12 142 | Drinks water; vomits
solid food
Nov. 15 12 124 | Eating and vomiting —
Noy. 17 Dog emaciated 15 | 146| Drinksmilk ~~
Nov. 18 | 12 | 112] Frequent hiccough —
Nov. 19 | 2:00 p.m. 12 | 108 | Dogshivering, hiccough
Nov. 19 | 2:45 p.m..| Given 0.3. cc. of
eS atropine sulph.
Noy. 19 |. 3:15 p.m. iz 186
Nov. 19 | 7:30 p.m. . 12 120
Nov. 20 | 16 129 | Dog eats and then vom-
its. Is very thin
Nov. 21 Dog is fed soft Vomiting reduced
food only
Dec. 1 Continued progressive
emaciation
Dec. 8 Will not eat Lies quietly; does not
é move about —
Dec. 9 Thorax opened, Dog comatose
vagi stimulated
Dec. 9 Autopsy

Lungs have scattered hyperemic areas but no consolidation. One small pus
pocket found. Stomach empty, normal appearance. Heart normal. The two
ends of left vagus united by a smaller strand of tissue.

after cutting the second vagus, tracings were made of the hunger con-
tractions in this dog (fig. 2). These were similar to those occurring
before cutting the second nerve but not so vigorous. These contrac-

REGENERATION OF VAGUS NERVE 21

tions were promptly abolished by atropine. This is suggestive of
direct paralysis of the vagus fibers in the regenerating nerve but it does
not prove that regeneration of the gastro-motor fibers had occurred,
for this inhibition might have been due to any of the following possible
factors: a, a direct action on the intrinsic plexuses as suggested by
Magnus for the intestine; 6, inhibition through the splanchnics as
result of central stimulation by the atropine; c, or some possible rela-

!

il

sy aTarmashoteh
Blood Paraawt — » ‘¢
“Batson w Tarecatde

Fig. 1. A. Stimulation of normal vagus, dog 96. Carotid blood pressure and
balloon in stomach to record respiration and stomach contractions. The stomach
tracing is above the blood pressure record on the left side of figure A and drops
below on the right side. Cardiac inhibition, respiratory inhibition and contrac-
tion of the stomach followed stimulation of the nerve.

B. Stimulation of the right vagus sixteen months after section and approxi-
mation of the ends of the nerve. No cardiac inhibition, no stomach contrac-
tion, inhibition of respiration and a rise in blood pressure followed stimulation
of the nerve below the scar of union. Ether used as anesthetic.

tion to the secretion of epinephrin. All other evidence in this dog
indicated that these gastric fibers were not functional and hence: the
observation indicates that atropine will inhibit gastric contractions
independently of whether or not the vagus fibers are active. At any
rate, electric stimulation of the regenerating nerve in the anesthetized
animal gave no gastric motor effects and atropine ‘abolished gastric
motility in the unanesthetized condition.

22 F. T. ROGERS

In these dogs in which a year and a half was allowed for the regenera-
tion of one vagus after section and suture, the subsequent division of
the remaining nerve was followed by no appreciable change in the
rate of breathing or in the amplitude of the respiratory movements, so
far as could be judged by ocular observation. Stimulation above and
below the scar of suture with a tetanizing current caused the usual

ny At ‘ A fn f\ ‘
Metall aio i

Wy" Teen

Fig. 2. Tracings numbered I, II, and III in order from above, downwards.
I. Gastric hunger contractions, dog 96, October 28. Right vagus sectioned
and sutured sixteen months previously. Left vagus intact.

II. November 11, gastric hunger contractions after cutting left vagus leaving
only the regenerating nerve intact.

ITI. November 11. Continuation of tracing JJ. Thirty minutes after a sub-
cutaneous injection of 0.3 cc. of 0.1 per cent atropine sulphate.

nhibition of breathing. Although causing no cardiac inhibition when
electrically stimulated in the etherized dog there was an immediate
pressor effect on the blood pressure (fig. 1). Regeneration of afferent
fibers in the vagus had therefore occurred.

After death the regenerated nerve of dog 95 was excised for a dis-
tance of half an inch above and below the point of suture. This was

i =

REGENERATION OF VAGUS NERVE 23

stained by Ranson’s pyridine silver method for medullated and. non-
medullated fibers. Save that the arrangement of nerve fibers in
fascicles below the scar was not evident, there was no distinct difference
in the number of nerve fibers in the regenerated part as compared
with that above the point of section.

In this report no reference is made to the changes in the sympathetic
nerves of the neck which were cut simultaneously with the vagi. |

SUMMARY

One vagus nerve was sectioned: and the ends approximated so.as to
allow regeneration to occur in a series.of dogs and cats. The regenerat-
ing fibers were stimulated electrically at time intervals varying from
one to sixteen months after cutting. These tests made with the ani-

mals under ether anesthesia gave no evidence of the regeneration of

either cardiac inhibitory or gastric motor fibers.

In one dog twenty months after one vagus was sectioned, this nerve
was stimulated with the dog in a comatose condition but no ether
anesthesia. Distinct cardiac inhibition followed.

In two dogs, section of the remaining normal vagus, sixteen and
twenty months after previously sectioning and suturing the other, led
to death in sixteen and thirty-four days respectively. Apparently
death was due to starvation resulting from difficulty in swallowing and
frequent vomiting. During the period of life following section of the
second vagus, the following facts were noted:

1. An immediate marked increase in pulse rate followed section of
the second vagus. This slowly declined and after eleven to fourteen
days the rate was that of a normal animal. At this stage atropine
caused a great increase in the rate of the heart beat. These effects
occurred in a dog in which the regenerating nerve was not functional
for subsequent division of the nerve caused no change in the heart
rate.

2. With only the regenerating nerve intact, but with no evidence of
it being functional, atropine reduced the gastric motility.

3. The rate of breathing with only the regenerating nerve intact
was the same as it was with one vagus intact. Cutting the regenerat-
ing nerve led to the classic picture of slow labored breathing. Stimula-
tion of the regenerating nerve above and below the scar caused the
normal respiratory inhibition and pressor effects on the blood pressure.
Regeneration of the vagus fibers necessary to maintain the normal

24

respiratory rhythm had therefore. ‘
motor to the oe or ay, ae from

MS

1913,

3

EFFECT OF VARIOUS SUBSTANCES UPON THE COAGULA-
TION OF CITRATED PLASMA!

BEN KARPMAN

From the Department of Pharmacology, University of Minnesota

Received for publication May 3, 1920

Although an enormous literature has arisen upon the coagulation of
the blood, and the réle of inorganic salts, lipoids and tissue extracts has
been extensively investigated, little has been done along the line of the
organic substances. I have therefore undertaken, in this paper, a
systematic investigation of the effects of as many classes of organic
compounds as possible upon the coagulation of citrated blood plasma.
It was hoped to determine by this method whether any substances or
radicals could be found which might be regarded as specifically favoring
or specifically inhibiting the act of coagulation, and which might thus
throw light upon the intimate chemical mechanism of coagulation.

METHOD

Fresh beef blood taken from the abattoir at the time of slaughter
was put into a jar containing enough sodium citrate solution to make
the final mixture contain 0.5 per cent of the salt. .This was centrifuged,
put in the ice box and used the same day.2 A 1 per cent solution of

1 Thesis submitted for the degree of M.D., University of Minnesota.

2 Effect of age on the activity of citrated plasma. I. Citrated plasma was
tested for coagulation time,—when fresh, and also when 1, 2, and3daysold. Co-
agulation time was found to be 5 minutes, 35 seconds; 8 minutes; and 7 minutes,
15 seconds, neepeutively. The precipitate formed during these intervals was not
removed.

II. Citrated plasma was tested for coagulation time when fresh, 11 days, 14
days, 16 days and 21 days old. Precipitate formed removed in the intervals men-
tioned. Coagulation time was 5 minutes, 35 seconds, and 4 hours for the first
two; the rest formed within a day a small amount of solid material which was
suspended in the fluid.

III. Citrated plasma was divided into three portions and tested for coagula-
tion time in the intervals indicated. Precipitate nemOVed each time. Normal
clotting time, 13 minutes.

a. Tested when 2, 6, 7,8 and 9 days old. Coagulation time 15 minutes, 55 sec-

25

26 BEN KARPMAN

dry crystals of CaCl, in distilled water was made, and this was added
to the plasma at the same time as the substance whose effect on coagu-
lation was being studied. | |

The tubes used were mostly small Wassermann tubes. Where larger
tubes were used they were, whenever possible, selected of the same
size to insure uniformity of contact with foreign material. Observa-
tions were all made at room temperature, usually 72°F. It is to be
noted, however, that during the winter and spring months the average
coagulation time of controls was 8.1 minutes (the highest being 13.4
minutes and the lowest being 3.6 minutes), while during the summer
months the average coagulation time of the plasma was 5.6 minutes

onds; 19 minutes, 15 seconds; 21 minutes, 36 seconds; 3 hours and 6 hours respec-
tively.

b. Tested when 6 and 8 daysold. The first gavea Pivatwlion time of 40 min-
utes, 15 seconds; the second was still fluid after 12 hours.

c. Tested when 8 days old. Still fluid after 24 hours.

IV. Citrated plasma was tested for coagulation time when fresh, 4 days, 5
days, 9 days and 10 days old. At the intervals indicated, the citrated plasma
was divided into two portions; a larger one containing the clear supernatant fluid
and a smaller one containing all of the precipitate formed during the interval.
The former was treated again in the same manner on successive days as more
precipitate formed. Normal coagulation time 4 minutes, 10 seconds. The larger
(supernatant) fraction gave a coagulation time of 3 minutes, 24 seconds; 8 min-
utes, 28 seconds, and 8 minutes, 42 seconds, on the 4th, 9th and 10th days respec-
tively. The first small (precipitate-containing) portion gave a coagulation time
of 2 minutes, 37 seconds on the 4th day, and spontaneous clotting on the 5th day;
the second small portion, 3 minutes, 47 seconds on the 9th day; the third small
portion, 5 minutes, 52 seconds on the 10th day.

V. An extract was prepared from fresh citrated plasma by a method modified
from Wright (1). The plasma to which this extract was added showed some
reduction in coagulation time. The results were vitiated by the fact that the
extract was used in a solution of sodium carbonate; as the latter substance
interferes with coagulation, it masked the effect of the extract.

The above observations suggest that the coagulation time of citrated plasma
increases considerably with age. This increase is more marked when the precip-
itate formed in the intervals is removed, but when the precipitate is present in
larger amounts, the coagulation time is markedly shorter. This fact suggests
that the precipitate contains something which aids coagulation; but whether
this is due to the presence in the precipitate of a definite thromboplastie sub-
stance, or is merely due to hydrolytic dissociation of calcium citrate with liber-
ation of calcium ions, or to some other entirely different factor, has not been de-
termined. A substance extracted from citrated plasma also showed coagulating
power; but it is not clear yet whether this substance is identical with other keph-
alin like substances that can similarly be extracted from various organs and
tissues,

_—, =

CHEMICAL FACTORS IN COAGULATION OF CITRATED PLASMA 27

(the highest being 8 minutes and lowest being 2.3 minutes). Whether
such marked variation is due to heat or to some nutritional factor that
in some way affects coagulation time in winter as compared with sum-
mer, has not been determined. To insure uniformity and greater ac-
euracy, the plasma, CaCl, solution and the substance used (when liquid)
were all measured from microburettes graduated to 0.1 cc. The plasma
was measured out first and then the Ca solution was added. Imme-
diately afterward the chemical to be tested was added, and the tube
moderately shaken and mixed. The end point was judged by Howell’s
method inverting the tube, and time noted in minutes and seconds.
Occasionally we experienced a little difficulty with this. criterion, since
some of the substances would give only partial or incomplete coagulation. |

A new control tube was used for each set of experiments and for
each sample of plasma.

Throughout the experiments care was hake to test the effects of
single substances rather than of mixtures, but this was not always
possible, a fact which must be borne in mind in the interpretation of
results. The effect of water on coagulation time of plasma has been
determined in a series of experiments, and it was found that there is a
definite, although not very marked, diminution in coagulation time.
It is clear, however, that these results cannot be applied entirely to
solutions of substances, since these in solutions behave somewhat dif-
ferently than when pure, even aside from the mere factor of water.
Where mixed salts were used, such as choline HCl or tyramine HCl,
the results cannot be applied entirely to choline or tyramine without
reservation, since the acid factor might mask their effect.

In the descriptions which follow, the word ‘‘clot” is used to desig- _
nate the formation of a single coherent jelly-like mass which adheres
to the walls of the tube; the word “precipitate” is used to designate
the formation of discrete flocculi which do not cohere to one another
and do not form a jelly. This use of these terms must be borne in
mind in the interpretation of the results recorded in this paper.

RESULTS OF EXPERIMENTS
Group I. Altphatic series

1. Formic acid. All tubes formed thick viscous fluids. No distinct
precipitate could be seen. The time of solidification (coagulation) was
in direct proportion. to concentration. The solid resembled more a
precipitate than a clot and appeared homogeneous and structureless.

28 BEN KARPMAN

2. Acetic acid. All tubes gave a very flocculent precipitate, the
amount of the latter being in direct proportion to concentration. With
the exception of the 0.8 per cent which remained permanently fluid,
the supernatant fluid of the rest of the tubes solidified after a few days;
it was of a soft mushy consistency and no fluid could be expressed from
it.

3. Propionic acid. All tubes became cloudy with the formation of
a precipitate. The 0.85 per cent tube was still fluid after 2 hours;
the rest of the tubes solidified in 2 minutes, 10 seconds; 15 seconds and
5 seconds, (control 5 minutes, 45 seconds), the substance being very
soft and mushy.

4. Butyric acid. The 0.85 per cent became cloudy and emulsion-like
with a fine flocculent precipitate, and remained permanently fluid.
The 2 per cent gave a very soft solid after 2 days; the rest of the tubes
formed almost instantaneously thick solid precipitates.

5. Valeric acid. The 0.4 per cent mixed well but remained perma-
nently fluid with formation of a precipitate and cloudy supernatant
fluid. The rest of the tubes did not mix well, forming thick emulsion-
like fluids above and a large precipitate on the bottom. Of these, the
0.85 per cent was still fluid after 3 days; the 2 per cent was practically
solid in 15 minutes; and the 4 per cent and 7.5 per cent formed almost
instantaneously thick solid precipitates. :

6. Oleic acid. On mixing an emulsion formed in the tubes. The
‘contents of all tubes were still fluid after 24 hours with an evident tend-
ency to form solid emulsions.

7. Formaldehyde. The clots were very soft, practically semi-solid*
and very transparent, the transparency being more marked in higher
concentrations. No precipitate was visible and no fluid could be ex-
pressed. The solid was easily broken up into small particles.

8. Alcohol. All tubes became cloudy and milky in proportion to
concentration, and a white precipitate separated out afterwards. The
7.5 per cent and 14 per cent tubes solidified in time equal to that of
control. The other tubes remained pornanen fluid. In firmness
the clots resembled the normal.

9. Glycerine. In concentration of 0.85 per cent to 3 per cent showed
some retardation, this being more marked in the lower percentages;
in concentration between 4 per cent and 7.5 per cent no marked devia-
tion from control was evident, while in concentration above 7.5 per

3 See footnote 4.

She a, Tage! ERE Sik plone ert Se kg aaa Mae hearted

CHEMICAL FACTORS IN, COAGULATION OF CITRATED PLASMA 29

cent it again showed retardation, the effect increasing with increased
concentration. The clots were very firm and elastic, and no serum
could be expressed.+*

10. Ether. The 7.5 per cent and the 14 per cent mixtures showed
considerable retardation, the clots resembling normal in firmness and
consistency. The 20 per cent and 30 per cent coagulated but partly
in 30 minutes, the rest remaining fluid. As the substance was added
it did not mix with the plasma and was seen gradually to rise to the
top. In all cases, coagulation proceeded from the bottom.

11. Acetone. The 4 per cent, 7.5 per cent and 14 per cent tubes formed
clear mixtures, which coagulated in 1 hour, 1.8 hour and 2.5 hours
respectively. The 20 per cent, 25 per cent and 29 per cent formed
turbid, emulsion-like mixtures with precipitation. The first two were
found to be solid after 2 days, while the last one remained fluid. The
clots were soft and mushy and no fluid could be expressed.

12. Urea. There was no precipitate visible. Clot softer than normal.
Considerable retardation.

13. Hexamethylenamine. In all tubes, with the exception of 0.1 per
cent, there was a settlement of the substance in proportion to concen-
tration. There was no apparent difference whether it was used in
solution or in pure form, for although the deviations in the latter were
much more marked, coagulation time was practically normal. Clots

were somewhat harder.

14. Hexamethylenamine and phosphoric acid, 50 per cent of each. All
tubes were fluid at first with no evident signs of either coagulation or
precipitation. In 24 hours became thick and semi-solid. Were all
found to be solid in 15 hours, soft in consistency, with no fluid expres-
sible, the solid resembling more a precipitate than a clot.

15. Choline hydrochloride. All tubes gave delayed coagulation time
in proportion to concentration. The 5 per cent was still fluid after a
half-hour.

16. Glycocol. In small concentrations up to 3 per cent the tubes
beeame cloudy while in higher concentrations the tubes formed a dis-
tinct precipitate. After 18 hours the 0.3 per cent was solid, the 0.6
per cent semi-solid, the rest solid white precipitates.

4In the course of the experiments it was frequently noted that there was a_
considerable difference in the effect of various substances on the character of the
clot. Thus, glycerine gave a very firm and elastic clot; dinitrobenzol a soft clot,
while resorcin gave a firm clot in concentration up to 0.5 per cent and a very soft
one in higher percentages, and no fluid could be expressed.

30 BEN KARPMAN

17. Chloroform. Did not mix with the plasma and was seen floating
in it as oily drops, gradually settling to the bottom. Coagulation pro-
ceeded from the top. The clots were soft, clear and jelly-like.

1. The first members of the saturated fatty acid series show consider-
able similarity in their effect on coagulation of plasma. They all inter-
fered with coagulation by precipitation, the amount of the latter being
in direct proportion to concentration; and the higher the member the
more pronounced was the reaction.

2. Oleic acid interfered with coagulation by emulsification.

3. The representative members of the other group, namely, formalde-
hyde, alcohol, ether and acetone, have all produced the effect of re-
tardation, interference, or both. The retardation produced by for-
maldehyde can hardly be explained by precipitation since it did not
show any visible change; however, the character and consistency of
the solid suggest that it was more like a precipitate than a clot.

4. Glycerine did not show any appreciable effect on coagulation,
although its power of retarding coagulation i in very low and very ni
concentration is suggestive.

Of the rest, glycocol markedly interfered with coagulation even in
small concentrations. The effect of choline HCl and urea is consider-
able less marked while hexamethylenamine was practically without any
effect. Chloroform has shown a definite retardation and possible
inhibition.

Group II. Aromatic Series

1. Benzol. On mixing, all tubes assumed a milky emulsion-like ap-
pearance. The tubes solidified later, some showing a separation into
three layers, benzol, emulsion and plasma, from the top down.

2. Phenol. In concentrations of 0.2 per cent to 0.85 per cent,
normal or only partial coagulation was obtained, whether the phenol
was used in pure form or in solution, although the process was more
complete when the same strength was used in solution than in erystals.
The clot formed was normal in appearance and spread from the top
down. The 2 per cent to 8.5 per cent showed emulsification and pre-
cipitation, solidifying later with separation of fluid.

3. Resorcin. The 0.1 per cent to 0.4 per cent gave firm clots with
delayed coagulation time; the 0.85 per cent gave a soft clot. The 2
per cent to 25 per cent assumed an emulsion-like appearance with sub-
sequent separation of a precipitate, not unlike phenol, but the tubes
remaining permanently fluid.

CHEMICAL FACTORS IN COAGULATION OF CITRATED PLASMA 31

4. Benzaldehyde. On mixing all tubes became turbid and emulsion-
like with formation of a precipitate, the thickness of the emulsion and
the amount of the precipitate being in direct proportion to: concen-
tration. All tubes remained permanently fluid except the 0.85 per
cent, which solidified very slowly.

5. Benzoic acid. The 0.1 per cent to 0.4 per cent and 2 per cent showed
delayed or partial coagulation, in direct proportion to concentration,
the clots being less elastic. The 0.85 per cent gave a precipitate and
remained fluid.

6. Benzyl alcohol. All tubes showed emulsification and precipitation,
in direct proportion to concentration. :

7. Nitrobenzol. On mixing, all tubes became turbid, separating later
a precipitate which settled to the bottom as a white solid mass, the
amount of the precipitate being in direct proportion to concentration.
The supernatant fluid was quite clear and coagulated, the coagulation
time not varying markedly from normal. |

8. Dinitrobenzol. In spite of shaking, a considerable part of the
crystals settled to the bottom. Coagulation time was normal. No
precipitate was visible. i

9. Cinnamic acid. The 0.85 per cent gave normal coagulation time.
The 2 per cent to 4 per cent formed a hard surface scum, the material
below remaining fluid for a considerable time. Were all found to be

clotted in 60 hours, the clots being paler in appearance and not as firm

in consistency as normal.
10. Aniline. The 0.85 per cent gave delayed coagulation, the 2 per

cent partial coagulation. The 2.4 per cent to 29 per cent formed
emulsion-like mixtures.

11. Phenylhydrazine. On the addition of the substance there was an
almost instantaneous precipitation and solidification, the solid being
soft and mushy with no fluid expressible. The solid was very much
unlike a clot and was easily broken up into small masses.

12. Pyridine. Tubes became cloudy, pale and soon solidified, the
solid being easily broken up into small bits and masses, and resembling
more a precipitate. No fluid could be expressed.

13. Quinoline. All tubes showed a precipitate on the addition of
quinoline, the amount being in direct proportion to the amount of sub-
stance used.

14. Tyramine hydrochloride. Clots were apparently normal both as
to color and consistency. 3

15. Antipyrine. Alltubesformed aclear solution with no settlement
of antipyrine or precipitate visible. The 4 per cent was still fluid

a BEN KARPMAN

after a week, the 7.5 per cent was solid in 14 hours, the solid being very
soft and flocculent. The rest solidified promptly on the addition of
the substance, the solids being of soft gelatinous consistency and with
no fluid expressible. The retardation was inversely proportional to
concentration.

16. Caffeine. There was a settlement of caffeine on the bottom in
proportion to concentration. Clots very clear, soft and no fluid could
be expressed.

The benzol series showed a variable effect:

1. Benzol, aniline and benzaldehyde interfered with coagulation by
emulsification. With phenol the effect is changed to that of precip-
itation, due to a change in solubility by the introduction of the OH
group. This effect is considerably weakened by the introduction of
(OH)s, since resorcin even in very high concentrations did not give
sufficient precipitation to cause solidification, an effect which phenol
produced in much lower concentration. This difference is also shown
by the fact that in very low concentrations resorcin showed retardation
against incomplete coagulation of phenol for the same concentration.
It should also be noted that phenol when used in solution is more effec-
tive than when used in pure form.

2. Comparing benzoic and cinnamic acids it is seen that both retard
coagulation, but the action of the latter is far more marked than that
of the former. Here too, probably, one of the causes of the difference
may lie in their different solubilities, cinnamic acid being the less soluble.

3. However, that solubility alone cannot account for all the effect of
a substance on coagulation is seen in the case of benzyl alcohol which,
although much more soluble than either of the above acids, has shown
interference even in small concentrations.

4, Nitrobenzol and dinitrobenzol have shown rather indifferent
effect. .

5. Phenylhydrazine gave an almost instantaneous solidification in
whatever concentration used. The effect of pyridine was somewhat
less marked, requiring higher concentrations and more time for solidi-
fication; while the quinoline precipitate remained fluid in high concen-
trations. In the case of these three substances, the difference in action
can hardly be explained by differences in solubilities since pyridine is
very soluble, quinoline somewhat less, while phenylhydrazine is: least
of all. .

Antipyrine and caffeine have both definitely retarded coagulation,
although neither produced any visible change in plasma. Tyramine
was practically without any effect.

"q

ne er eT er eee re a A ee
= NI Tee dn a . _

ry

~~
oe. — 4 ee 7 =
Seen ae a ee pia eS) Cre Aas Ne Ria i a

=

CHEMICAL FACTORS IN COAGULATION OF CITRATED PLASMA 33

Group III, Alkaloids

_ 1. Quinine alkaloid, pure. On mixing, all of the quinine came to the
top. Clots were all normal in consistency, nor was there any difference
in coagulation time as compared with control.

2. Quinine bisulphate. On the addition of the salt, there was an
almost instantaneous precipitation and solidification. The 2 per cent
precipitate soon settled to the bottom, leaving a fluid above; the 4 per

cent formed a solid precipitate throughout.

8. Strychnine alkaloid, pure. On mixing, the tubes became cloudy;
later there was a settlement at the bottom in proportion to concentra-
tion. In time and consistency the clots were practically normal.

4. Atropin alkaloid, pure. The 0.2 per cent formed a solid scum
which on inverting would prevent flowing out, though the rest of the
tube remained fluid for a considerable time. Noted to be completely
solid after a week. Clot normal, somewhat softer. The 0.4 per cent
showed considerable retardation.

5. Nicotine. The 2.4 per cent was still fluid after 17 hours and but
partly solidified in a week. The rest of the tubes showed marked re-
tardation in coagulation, which was inversely proportional to concen-
tration. The clots were very soft, mushy, with no body, and but little
fluid could be expressed. No precipitate was visible.

Of the alkaloids used quinine and strychnine showed themselves to
be without any effect on coagulation of plasma;-atropin showed some
retardation while nicotine gave most definite and marked retardation.
Here the difference in action lies perhaps both in the different solubilities
and alkalinities since quinine and strychnine show least of these proper-
ties; atropine somewhat more and nicotine most of all.

Quinine bisulphate must, of course, be interpreted in terms of its
acid content which interfere with coagulation by precipitation.

Group IV. Inorganic substances

1. Ammonia. On the addition of ammonia, all tubes showed a hazy
cloudiness, which within an hour settled to the bottom as a very light
precipitate,> the amount of the latter was in direct proportion to con-
centration. The supernatant liquid remained permanently fluid.

* This precipitate was tested and was shown to have the following properties?
It was insoluble in water and alkalies and in 80 per cent alcohol; soluble in acids
and may be reprecipitated by alkalies; not coagulated by heat in acid solutions.

Hence the substance is very likely a metaprotein compound of ammonia. ,

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 1

34 BEN KARPMAN

2. Sodium carbonate. All tubes gave a whitish precipitate. After
4 hours all were still fluid and somewhat gelatinous in appearance. The
0.1 per cent was found to. have become solid in 18 hours; the 0.25 per
cent and 0.35 per cent in 5 days, the rest remaining fluid.

3. Hydrochloric acid. On the addition of the acid all tubes bisdaae
immediately cloudy with the formation of a precipitate, the amount —
of the latter being in proportion to concentration. The 2 per cent and
2.3 per cent were practically all precipitate. After 18 hours the 0.2
per cent and 0.4 per cent were still fluid; the rest of the tubes apparently
solid, but the solidity easily disturbed by moderate shaking.

4. Sulphuric acid. On the addition of the acid all tubes became
cloudy, some separating later a white precipitate. The 0.2 per cent
and 0.3 per cent were found solid after 3 days and after 45 minutes
respectively; the rest, 0.4 per cent to 1.25 per cent formed thick viscous
fluids which later solidified, the solid being very soft and resembling
more a precipitate.

5. Phosphoric acid. All tubes were still fluid after 15 hours; no pre-
cipitate visible. They became solid after 10 days, the clot being soft,
with no fluid expressible.

In this group, ammonia and HCl have shown marked interference
even in small concentrations; Na,CO; and H.SO, showed both retarda-
tion and interference, while H;PO, showed the least effect, producing
retardation even in high concentrations.

If we now sum up the effect of various chemicals on coagulation of
citrated plasma, we may offer provisionally the following grouping.

I. No effect. 1. Some substances such as alkaloids, dinitrobenzol,
tyramine hydrochloride, etc., have no effect on coagulation in what-
ever concentrations used. They do not produce any visible change in
the plasma, although some may produce such change (nitrobenzol).

2. Some substances may have no effect in certain concentrations,
while producing a definite effect in other concentrations (retardation,
etc.) as glycerine, alcohol, ete.

II. Retardation. The effect of retardation may show itself either in
prolongation of coagulation time or incompleteness of the process.

1. Prolongation of coagulation time varies considerably with each
substance and concentration. In most instances, the degree of re-
tardation was directly proportional to concentration used. In some
_ eases, however, notably with antipyrine and nicotine, this was inversely
proportional to concentration; the proportional decrease of retarda-
tion with increased concentration may be so progressive that the coagu-

ee ah a TT EE TT, ae Ee ae ne a ee

CHEMICAL FACTORS IN COAGULATION OF CITRATED PLASMA 35

lation time will finally fall below that of the control and thus assume
the form of hastening. In yet another case (formic acid) retardation
started in lower concentrations in direct proportion to concentration,

ending with apparent hastening in higher concentration.

Although some substances do not show any other effect but that of
retardation (formaldehyde, etc.), other substances will retard in some
concentration while producing a different effect in other concentration
(glycerine, resorcin, NasCOs, etc.), most frequently incompleteness or
interference with coagulation.

2. Incomplete coagulation. This is not as frequent as the preced-
ing but there are several chemicals which sometimes effect only partial
coagulation. As a rule it is accompanied by other effects such as
retardation (ether, chloroform, urea, etc.) or precipitation (phenol),
and a visible change. From the fact that some cases of incomplete

- coagulation finally coagulate after a lapse of considerable time, we may

regard incomplete coagulation as the next step of a markedly retarded

- coagulation.

III. Interference. Interference with coagulation may manifest
itself in several ways.

1. Precipitation. This is the most common occurrence, the amount
of precipitation varying widely with different substances and concen-
trations used. Thus the members of the saturated fatty acid series,
as well as phenylhydrazine, pyridine, HCl, NasCO;, NH3, benzoic acid, -
etc., will interfere with coagulation even in very small concentrations;

_ other substances (resorcin, etc.) may require somewhat higher concen-

trations, while still others (acetone, urea, etc.) require relatively high

__ concentrations to produce the same effect.

2. Emulsification. This is of somewhat less frequent occurrence
than the preceding and is noted in such substances as oleic acid, benzol,
aniline; others (benzaldehyde, acetone, etc.) show a mixed effect.
Both precipitation and emulsification are frequently accompanied by
retardation or incomplete coagulation.

3. Inhibition. Such substances as nicotine, antipyrine, caffeine,
etc., occasionally show apparent inhibitory effects, i.e., no coagulation
takes place and no change in the plasma is visible. It usually accom-
panies retardation and as a factor it probably stands between retarda-
tion and interference. To this group probably belong also chloroform
and ether. |

IV. Acceleration. We have not encountered a single case of genuine
hastening of coagulation. Some substances do hasten the process

KARPMAN

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38 | BEN KARPMAN

more than the controls (phenylhydrazine, pyridine, etc.) but the char-

acter of the solid formed resembles more a precipitate than a clot,
although no distinct precipitate is visible. It is possible that, as in
case of interference, there is more than one mechani by which coagu-
lation is brought about.

It should be said here that some substances belong ecgene to this
group although not causing any visible change (formic acid, formalde-
hyde, etc.). There are good reasons to believe that although no change
is visible, their apparent retardation is really an interference.

It seems thus quite clear from the above considerations that a close —

relation exists between the various factors discussed. Thus, sub-
stances having no effect on coagulation of plasma in certain concen-
trations will, in other concentrations, retard the process; while sub-
stances retarding in some concentrations will, in higher concentrations,
interfere or inhibit. These phases are apparently intimately related,
one often passing insensibly into the other, forming a progressive
chain of events (no effect—retardation—incomplete coagulation—
inhibition—interference) and all probably operating on the basis of
some common property.

What then are the conditions that will determine the_ particular
effect. of a substance upon coagulation of plasma?

The substances having no appreciable effect on coagulation may
act in a particular manner because of one or more of the following
reasons.

1. They are not soluble in plasma (alkaloids, dinitrobenzol).

2. They may be soluble in water, but. the medium is not favorable
for their action (formin requires an acid medium).

3. Their effect is neutralized by an opposite property (tyramine
HCl-acid-alkaline-neutral.)

4. The changes produced do not sufficiently alter the plasma so as
to interfere with coagulation.

As we pass to the next phase, that of retardation, it would seem that
the retarding substance induces certain definite changes in the plasma
and these may be due to any one or more of the following conditions:

1. The substances are not soluble in plasma.

2. They do not react chemically with plasma nor form easily a phys-
ical mixture (chloroform, ether).

3. They act by dehydration, absorbing water from plasma (glycerine).

4. Their acid or alkaline reaction (caffeine, antipyrine, nicotine,
urea, NazCOs). |

eh

CHEMICAL FACTORS IN COAGULATION OF CITRATED PLASMA 39

5. Their effects neutralized by an opposite property, cholin HCl.

6. May be strong reducing agent (formaldehyde). :

The same mechanism is probably at work in case of interference,
only the changes are so pronounced as to interfere entirely with the
process of coagulation:

1. Many of the substances are quite soluble and, uniting with some
substances of the plasma, cause precipitation iphanol resorcin).

2. Their reaction is markedly acid or alkaline (the fatty acids,
HCl, benzoic acid, pyridine, ammonia, NazCOs, etc.). Here evidently
solubility in water as such does not play an important réle, for the
effect is the same whether the substances are very soluble in water—
as pyridine, HCl, etc.—or increasingly less soluble—as the fatty acids.
3. They may be ‘insoluble in water but form an emulsion, thus
depriving the plasma of water (oleic acid, benzol, etc.).

4. They may have a marked solvent or precipitant action on some
plasma constituent (alcohol, acetone).

5. Being neither soluble in plasma nor active shdinienliy. their mere
presence inhibits coagulation by preventing the aggregation of fibrin
threads and crystals into gel formation (chloroform, ether).

That the precipitate is most likely a new chemical compound is
readily seen from a qualitative analysis of one of the precipitates.
(ammonia q.v.); a further analysis of each individual precipitate formed
would probably show that the precipitate formed in each case is dif-
ferent. Protein behavesin an acid.solution like a cation, and anions
render it insoluble; in an alkaline medium, it behaves like an anion,
migrating to the anode, and cations render it insoluble. Although in
general the rate of precipitation is proportional, ceteris paribus, to the
molecular conductivity of the added salt, it would seem that while it
is true for the inorganic acids, it is not exactly true for the fatty
acids, for their ionization decreases as we go up the scale while the
precipitation increases at the same time (2).

The process of emulsification is a much simpler one and the disturb-
ance is more of a physical than of a chemical nature.

As it has been observed in numerous instances that the reaction
between the substance used and the plasma is a quantitative one,
the degree of reaction will obviously depend partly on the properties
of the substance, and on the condition of the plasma. It is quite
evident that the more soluble a substance is, the less likely it is, ceteris
paribus, to interfere with coagulation. Thus, the precipitating action
of the fatty acids rises as we ascend the scale, while their solubility

40 BEN KARPMAN

decreases at the same time. Phenol in solution interferes with coagu-
lation much less than when used in crystal form, while resorcin inter-
feres less than phenol. Quinine and strychnine are insoluble and have
no effect. Finally, it may be said that in general the substances belong-
ing to the second group are considerably more soluble than those of
the third. This, perhaps, will explain why a substance will interfere
in higher concentrations while only retarding in lower concentrations,
since smaller quantities are more easily dissolved. On the other hand,
that water per se is not the determining factor is quite evident from the
consideration that some substances interfere markedly with coagula-
tion, although they are very soluble (phenylhydrazine, urea, pyridine,
etc.).

It has long been observed that the reaction of the blood has a con-
siderable effect on its coagulability. An increased acidity leads to an
increased aggregation and finally precipitation of colloidal particles of
fibrin, and similarly increased alkalinity may in smaller concentration
change the form of the clot from a crystalline form to a structureless
mass, and in higher concentrations cause a total failure of clotting (3).

The present work abundantly verifies these observations. Sub-
stances having a distinctly acid or alkaline reaction have, in all cases,
failed to cause normal clotting, even when used in smaller concen-
trations. If the alkaloids are cited as exceptions, it should be remem-
bered that those that had no effect on clotting are totally insoluble
(quinine and strychnine) while the more soluble ones had a distinet
effect (atropine and nicotine). It is possible that the manner in which
acids or alkalies interfere with coagulation is in a way comparable to
coagulation of protein by heat, since coagulation of blood is due to the
formation of an insoluble fibrin compound. According to Chick and
Martin (4), in heat coagulation of protein there is first a denaturation
or reaction between the protein and hot water, and second, agglutina-
tion or separation of the altered protein in a particulate form, the
reaction velocity increasing with an increase in acidity or alkalinity.
From the purely physical point of view the addition of acids or alkaline
ought to disturb the plasma equilibrium since protein salts have a
greater attraction for water than electrically neutral protein and,
according to Fisher (5), the presence of acid or alkali greatly increases
the power of protein to imbibe water. The semi-solid character of
some of the clots and the inability to express water from them is prob-
ably due to absorption of water by fibrinogen and hence may be re-
garded as an incomplete clot.

CHEMICAL FACTORS IN COAGULATION OF CITRATED PLASMA 41

It is also a common observation in the laboratory that old poorly-
preserved kephalin loses its thromboplastic properties and may even
retard coagulation; and according to McLean (6) the loss of thrombo-
plastic power goes hand in hand with the development of acid reaction.
According to W. H. Heard (7) certain concentrations of alkaline earth
cause marked retardations of coagulation. It is conceivable that
these variations may be accounted for by variations in their respective
hydroxyl ions.

Whether certain chemical groupings have a more intimate relation
to coagulation of plasma than others, cannot be said definitely. The
introduction of either H or OH ions, as stated above, definitely inter-
feres with coagulation; the introduction of the phenolic OH seemingly
has a favorable effect on coagulation; while the presence of nitrogen
group by effecting a change in the reaction of the substance, interferes
with the process.

SUMMARY

The effect of various chemicals on coagulation of citrated plasma has
been studied and it has been shown that a chemical may have one of
the following effects on plasma.

. No effect.

. Retardation of the process by:

. Prolongation of the coagulation time.
Incomplete or partial coagulation.

. Interference by:

. Inhibition.

. Precipitation.

. Emulsification.

. Acceleration.

Reasons have been advanced to show that an intimate relation
exists between the factors mentioned and by gradations one may pass
into another, suggesting that they all probably work by reason of
some common mechanism.

The various properties which may be responsible for the particular
effect a substance will have on coagulation have been considered and
it was suggested that such effect may depend on the solubility of the
substance in plasma, its alkaline or acid reaction, on its dehydrating
power, reducing power, etc.

It has also been observed that in the interaction between the chem-
ical and the plasma, new chemical compounds are formed, and the
relation is probably a quantitative one.

BRaoeae wore nr

It is a pleasure. to express my ‘aa
given and valuable suggestions received from Dr.
I also wish to thank Dr. E. D. Brown for helpful «

the aepabieaeae
BIBLIOGRAPHY -

(1) Wricut: Brit, Med. Boren, 1801, ii, 641. i
(2) Rosertson: Journ. Biol. Chem., 1911, ix, 303.
(3) Howe: This Journal, 1916, xl, B2T4 20 525) aed
(4) Cuick anp Martin: Journ. Physiol., 1912, xly, 612. C
(5) Fisner: Edema, New York and London, 1915, se :
(6) McLean: This Journal, 1919, xliii, 586.

(7) Hearp: Journ. Physiol., 1917, li, 295.

*

beth ae kee |

SATE ae ae eR

THE INFLUENCE OF PITUITARY EXTRACTS ON THE
ABSORPTION OF WATER FROM THE SMALL
INTESTINE

MAURICE H. REES
From the Physiological Laboratory of University of South Dakota

Received for publication May 12, 1920

Recent investigations in both the experimental and in the clinical
fields indicate quite conclusively that pituitary extracts produce at
least a temporary (seven to eight hours) antidiuretic effect when
administered subcutaneously. The question arises as to how this
antidiuretic action is brought about. Is it a direct or an indirect action
on kidney excretion?

Motzfeldt (1) concludes from his experiments on rabbits that pitui-
tary extracts produce an antidiuretic action by stimulating the sympa-
thetic nervous system and bringing about a vasoconstriction within the
kidney. | | :

Dale (2) working with perfused kidneys of the dog and the cat, found
that pituitary extracts caused a vasoconstriction of the renal vessels.
Houghton and Merrill (8) arrived at a similar conclusion. On the
other hand, King and Stoland (4) found a vasodilatation of the renal
vessels and an increased flow of urine.

The literature regarding the effect of pituitary extracts on the intes-

tine is rather contradictory. Fodera and Pittau (5) in 1909 noted that

intravenous injections caused defecation. Increased peristaltic waves
following intravenous injections were noted by Bell (6) and by Ott and
Scott (7). Shamoff (8), working on isolated loops of the rabbit’s
intestine, found that posterior lobe extracts gave a relaxation of the
intestine. 2
The writer suggested in previous work (9) that the antidiuretic action
of pituitary extracts may be due to an interference with the absorption
of water from the intestine. It was noted that rabbits quickly devel-
oped a diarrhea following subcutaneous injection of pituitary extracts.
Cats showed a marked tendency to vomit following similar injections.
These observations suggested the advisability of investigating the
43

44 MAURICE H. REES

effect of pituitary extracts on the absorption rate of the intestine and
also on the emptying time of the stomach.

In the present work we have attempted to find out whether subcu-
taneous injections of pituitary extracts cause any variation in the rate
of water absorption from the small bowel.

METHODS AND RESULTS

Dogs and cats were used in our experiments. One commercial
pituitary extract was used, namely, pituitrin (Parke, Davis & Company).

The injections of pituitrin were subcutaneous in every case, and were
given four to five minutes before the beginning of the test experiments,
that is, at the close of the control period.

In the experiments recorded in tables 1, 2 and 4 the animals were
kept under an anesthetic (ether) during the entire experiment.

The small bowel was exposed with as little trauma as possible, and
the lumen was washed with warm tap water. A measured amount of
warm tap water was then introduced into the cannulated loop of the
bowel. At the end of a half-hour period the water remaining in the
bowel was removed and measured, and amount of absorption noted.
Four or five minutes before the close of this control period the test
animals received a subcutaneous injection of pituitrin. The same
amount of warm tap water was introduced into the loop of bowel at
the beginning of the second and at the beginning of the third half-hour
periods and the amount of absorption noted in each case.

In table 1 (first period) it will be noted that normal rate of absorp-
tion varies widely in different animals. This is probably due in part
to the varied lengths of bowel used.

Following the injections of pituitrin there was delayed absorption in
all but two of the fourteen dogs experimented upon. In one of these
two dogs (no. 10) the amount of pituitrin used was probably too small
to be effective. In the other case (no. 8) the intestinal mucosa was
found to be greatly inflamed and this may account for the failure of
the pituitrin to delay absorption.

With cats the results were not so uniform since only four out of the
six experimented upon showed a delayed absorption after pituitrin
injections. The intestines of cats are much more susceptible to trauma
than are those of dogs. This may have been a factor in the variation.

The question arises as to whether the decreased absorption noted in
the seeond and third half-hour periods may not be due to the continua-

.

PITUITARY EXTRACT AND INTESTINAL ABSORPTION

TABLE 1

45

Summary of experiments on the effect of pituitary extracts on the rate of absorption

of water from the small intestine.

The second column of the table shows the amount

of water injected into the washed bowel at the beginning of each 30-minute period.
Dogs were used except in nos. 15 to 20 in which cats were used. The pituitrin
was injected subcutaneously at the close of each control period

ip eiat da manta SECOND PERIOD THIRD PERIOD
AMOUNT | AMOUNT
Shine. igor Ww hoo oll pumice Water absorbed Water absorbed Water absorbed
INJECTED | INJECTED
First loop ig ag First loop ww eoag First loop o
ce, cc. ae. ce. ce. ce. ce. ce.
1 250 1.0 200.0 210 160.0 175 155 170
2 250 1.0 120.0 155 115.0 135 100 102
3 300 0.5 255.0 255 235.0 240 ;
4 — 200 0.5 100.0 90 70.0 68
5 100 0.5 60.0 58 50.0 55
6 200 1.0 125.0 100.0 86
7 300 1.0 192.0 170.0 120
8 150 0.5 100.0 105.0
9 200 0.5 40.0 30.0
10 100 0.25 62.0 75.0
11 100 0.5 85.0 18.0
12 200 1.0 130.0 105.0 100
13. 300 1.0 160.0 135.0 120
14 300 1.0 170.0 130.0 ~ 100
15 25 0.5 21.5 13.5 i 12
16 25 0.5 21.0 14.0 11
17 50 1.0 31.0 31.5 27
18 50 1.0 33.5 41 26
19 50 1.0 12.0 0 7
20 ° 50 1.0 15.0 6.0 5

tion of the anesthetic and to the operative procedure. To determine
this point we took the normal absorption rate on several animals with-
out pituitrin injections. An inspection of table 2 will show that in the
absence of any pituitrin injections the absorption rate may be even
greater in the second and third half-hour periods than it is in the first
half-hour or control period. In no case was there a marked decrease
in the second period, and in only one case (no. 21) was there a marked
decrease in the third period.

In order that we might still further rule out the possible effect of the
anesthetic we repeated the absorption experiments on four decere-

46 , MAURICE H. REDS

TABLE 2

Control experiments: To show the normal rate of the absorption of water from the
small intestine during the first, second and third half-hour periods of the experi-

ment. Dogs were used in experiments 21, 22 and 23. Cats were used in the

remaining experiments

ecrasisiaw ci. abet ae ie eek AMOUNT OF WATER ABSORBED PER HALF HOUR
EXPERIMENT INJECTED
First period Second period Third period
COs): C05, -)%: en: Osis
21 200 85.0 88 65.0
22 50 42.0 43 32.0
23 50 42.0 40 28.0
24 25 13.0 15 13.5
25 25 8.0 7 14.0
, 26 25 12.5 | OS 10
oT 26 9.0 | 8 eS
TABLE 3

Showing the rate of water absorption from the small intestine before and after the
injection of pitutitrin in decerebrated dogs. The dogs were decerebrated three hours
previous to the beginning of the experiments on absorption.

Bae eo ad, AMOUNT OF WATER ABSORBED P50 RAL
NUMBER OF
WATER PITUITRIN
SXEERie ee INTRODUCED INJECTED First half-hour| Second half- | Third half-
period,control | hour period hour period
ce. ce. cc. ce. ce.

28 300 0.50 150 135 100

29 300 0.50 135 100 85

30 450 1.00 225 60

31 200 0.50 125 20 414

TABLE 4

Effect of pituitary extract on the flow of blood from the mesenteric veins; 0.5 cc. of
pituitrin was injected subcutaneously in each experiment. Dogs were used

FIVE MINUTES AFTER INJECTION
OF PITUITRIN

BEFORE INJECTION OF PITUITRIN,

NUMBER OF EXPERIMENT CONTROL

gtt. per minute gtt. per minute

32 30 26 :
33 160 80 :
34 70 61
35 140 130

i — a

PITUITARY EXTRACT AND INTESTINAL ABSORPTION 47

brated dogs. The findings in these experiments are recorded in table
3. In operating on these animals hemorrhage was kept down to a
minimum and sufficient time (three hours) was allowed for the animal
to recover from the anesthetic and, in so far as possible, from the shock
of the operation. It will be noted that in every case there was a de-
crease in the absorption rate during the second and third periods, that
is, following the injection of pituitrin. This shows that the anesthetic
could not have been responsible for the decreased absorption following
the pituitrin injections.

It was thought that vasoconstriction of the vessels of the intestinal
wall might be a factor in reducing the absorption after pituitrin injec-
tions. This possibility was investigated by placing a cannula in one
of the mesenteric veins, noting the rate of blood flow by the drop
method. By referring to table 4 it will be noted that there is a reduc-
tion in the number of drops per minute after pituitrin injection. The
reduction was not pronounced except in one case (no. 33); in fact,
they do not go much beyond the limit of error due to the difficulty of
preventing the blood from clotting in the cannula.

DISCUSSION AND CONCLUSIONS

Our investigation leads us to the conclusion that subcutaneous injec-
tions of pituitrin bring about a delay in the aeons of water from
the small intestine.

This delay does not seem to be sufficient, in most cases, to entirely
account for the delay in the excretion of water from the kidneys which

_has been found to result from pituitrin injections.

It is possible that the subcutaneous injection of pituitrin may cause
some vasoconstriction of the intestinal vessels. This can not be
pronounced or very extensive since it has been repeatedly shown and —
was verified by ourselves in this and previous work, that subcutaneous
injections of pituitary extracts do not cause a variation in the general
blood pressure.

Motzfeldt (1) suggested that the antidiuretic action of pituitary
extracts is due to splanchnic stimulation, causing a vasoconstriction
within the kidneys. It is possible that this mild splanchnic stimulation
also extends to the vessels of the intestine.

6) BELL: Brit. Med. ig 1909, Pa
(7) Orr anp Scorr: Amer. Med., 1911, vi, 154
(8) SHAMOFF: ‘This Journal, 1916, xxxix, 268)"
(9) Rees: This Journal, 1918, xlv, 471.

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i

THE SPECIFIC INFLUENCE OF THE ACCELERATOR
NERVES ON THE DURATION OF VENTRICULAR
SYSTOLE

CARL J. WIGGERS anv LOUIS N. KATZ

From the Physiology Laboratory of Western Reserve University School of Medicune,

Cleveland

Received for publication May 12, 1920

INTRODUCTION—PREVIOUS WORK

Ever since the discovery of the accelerator nerves in 1867 by v.
Bezold and Bever (1) and the Cyon brothers (2), a sporadic interest
has been manifested in the question as to whether, in addition to
altering the heart rate, these nerves also specifically affect the strength
and duration of ventricular contraction. It does not seem to have
been realized, however, that a careful study of their effects on the
duration of ventricular systole is capable of disclosing whether the
normal ventricular beat is controlled entirely in a mechanical way or
whether it is also specifically controllable through nervous influences.

The idea that the mammalian ventricle is controlled in a simple
mechanical fashion received its greatest support from the oncometer
experiments of Henderson and his co-workers (8). They found that,
under normal conditions of venous pressure, the volume curves of
the ventricles at all heart rates are practically superimposable on
portions of a standard curve obtained during a long vagus beat. This
led to the formulation of the law of ‘‘uniformity of behavior’ accord-
to which the systolic volume discharged is entirely a function of the
heart rate. Later Henderson and Barringer (4) presented work which

indicated that this law also holds when the heart rate is increased by

excitation of the accelerator nerves.

Although emphasis has not been specifically laid on the fact by
Henderson and his co-workers, it is evidently a corollary of the “uni-
formity of behavior’’ law that the duration of systole is fixedly related
to the cycle length under all conditions which produce a change in the
heart rate. A review of other experimental work indicates, however,

49

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 1

50 CARL J. WIGGERS AND LOUIS N. KATZ

that when the accelerator nerves are stimulated, systole and diastole
vary quite independently. Thus Baxt (5) in 1878 reported that
stimulation of an accelerator nerve chiefly reduces the phase of systole.
His technical procedures were, however, crude and entirely unreliable.
Contrary effects on the duration of systole were reported from the
stimulation of the vagus nerve by Klug (6) in 1881, and by Mac-
Williams (7) in 1888. The inability to decide questions of this nature,
by their methods, is now obvious. The first experiments, therefore,
that today would be regarded as accurate and at all decisive were
made by Hiirthle (8) in 1891. This investigator recorded the arterial
pulse tracings with his membrane manometer and used the interval —
from the primary rise to the dicrotic notch as an index of the duration
of systole. He found that the period of systole, so determined, is
slightly abbreviated when cardiac acceleration is induced by stimula-
tion of the accelerator nerves or when the vagi nerves are sectioned.
Accelerator nerve stimulation, after sectioning of the vagi nerves, pro-
duces a marked decrease in the duration of systole; stimulation of
the vagi, on the other hand, affects systole very slightly, but exerts its
chief influence on the duration of diastole. In 1897 Frank (9) not only
substantiated this work but reported, in addition, that by simultaneous
stimulation of the vagi and accelerator nerves with suitably adjusted
currents, it is possible to decrease the duration of systole even when
the length of the diastolic phase is unaltered. In the comprehensive
investigations of the accelerator and vagi nerve action, carried out by
Reid Hunt (10) in 1899, can be found, among other data, the following
observations: Section of the accelerator nerves causes a prolongation
of both systole and diastole, the former being lengthened rather more
than the latter. Stimulation of the accelerator nerves causes a short-
ening of both systole and diastole. Stimulation of a vagus nerve
chiefly prolongs diastole, affecting systole relatively little. Under
certain conditions, simultaneous. stimulation of vagus and accelerator
nerves produces a shortening of systole while diastole remains unaffected.
The conclusion that such results are not in accord with a mechanical
regulation of the heart beat does not necessarily follow. Since the.
rate of systolic ejection diminishes toward the end of systole the dura-
tion of systole must by the law of ‘‘uniformity of behavior’ become
increasingly abridged as the cycles shorten more and more. ‘Thus, in
the volume curve reproduced in figure 1, a reduction in cycle length
from 0.8 to 0.7 second entails a reduction in the ejection phase of
systole from 0.215 to 0.21 second; while an equivalent reduction in ~

ACCELERATOR NERVES AND VENTRICULAR SYSTOLE 51

the cycle length from 0.4 to 0.3 mathematically decreases the ejection
phase from 0.175 to 0.15 second. Inasmuch as vagus section and
vagus stimulation ordinarily do not alter the heart rate beyond ranges
where slight variations might be expected, whereas accelerator stimu-
lation quickens the beat so much that a more pronounced shortening
of systole might be anticipated, it follows that the mere demonstration
that accelerator stimulation shortens the systole is proof neither of any
specific influence of these nerves over ventricular contraction, nor does it
prove that the heart deviates from a mechanical scheme. Only if it can
be shown that the periods of systole during accelerator nerve stimulation
vary materially from those which may be accounted for on the basis of
volume curves, can any inference be drawn as to a selective action of the
accelerator nerves on the ventricle.

METHODS OF INVESTIGATION

In order to determine whether the lengths of systole and diastole
during accelerator stimulation conform to or deviate from a mechanical
regulation of the normal heart beat, we first established a plot of the
theoretical systoles that should obtain at different cycle lengths and
then compared, in the form of a plot, the actual systoles at different
cycle lengths with these theoretical values.

Experimental procedures. In order to accomplish this it was neces-
sary to determine accurately the duration of systole and diastole while
the circulatory conditions were as nearly normal as possible. It was
especially important, for example, to avoid opening the chest and the
institution of artificial respiration—events which in themselves alter
venous pressure relations considerably.

We therefore determined the systole and cycle lengths by optically
recording the heart sounds by means of the direct sound recording
capsules of Wiggers and Dean (11). The main vibrations of the first
sound correspond to the first rise of intraventricular pressure while the
first vibration of the second sound is synchronous with the incisura of
the aortic pressure curve, events which mark the onset of systole and
diastole respectively (12).

Dogs anesthetized with morphine and chloretone were used as
experimental animals. The vagi and accelerator nerves together with
the stellate ganglion were first prepared for section and stimulation,
the latter being dissected without opening the thorax. The thorax
was then shaved and a sound receiver adjusted over the apex region

52 CARL J. WIGGERS AND LOUIS N. KATZ

by an elastic band encircling the thorax. This receiver was connected
with the sound recording capsules by a tube having an adjustable
lateral opening. The vibrations of a 50 v. d. tuning fork were simul-
taneously recorded. In order to gauge the appropriate time for taking —
sound tracings on bromide paper, a carotid-pressure curve was con-
tinuously traced on a long paper kymograph. |

From the optical records, the lengths of consecutive cycles and cor-
responding systoles were subsequently determined. This was done in
about 3000 cycles recorded during many experiments on ten different
dogs.

A

413

\

I

i

' | 4
l i is eer ket eT ae a Oe eas ee Me a
° J 2 3 4 a5 PRE ies 8 cc) Lo 4 12 13 it 1 Le

Fig. 1. (4 original size.) Diagram constructed for experiment C 207, deter-
mining the relation of systole and cycle length at various heart rates—also the
method of adapting volume curves to vagal beats with different periods of systole.
Shows arcs used when ejection phases AB, AB’ and AB? are equal to 0.25, 0.20 and
0.15 second respectively. B-1’, B-2’, B-3', etc., indicate duration of cycle. 1-1’
2-2’, 3-3’, ete., the period of systole. Abscissa = 0.1 second. Detailed description
in text.

Methods of constructing a curve expressing the theoretical duration of
systoles at different cycle lengths. In order to obtain a curve of the ~
theoretical systoles at all heart cycles according to Henderson’s mechan-
ical conception of cardiac control, it was first necessary to plot a theo-
retical volume curve for each animal. This construction, however,
is beset with a number of difficulties which, we believe, are not insur-
mountable. We started out according to a very simple plan: On large
sized codrdinate paper, a volume curve similar to that plotted as a
standard by Henderson (13) was laid off. A reduced reproduction
with codrdinates omitted, is shown in figure 1 (A, B, C). At varying
points, e.g., at 2, 3, 4, 5, etc., arcs of the standard ejection curve A B

Ree, ea SE ea REL Pee Mey Miele RNS ae ial ei ak

ACCELERATOR NERVES AND VENTRICULAR SYSTOLE 53

were drawn. The abscissal distances B-—1’, B-—2’, etc., then denote
the duration of the cycle and the abscissal distances 1-1’, 2—2’, etc.,
measure the corresponding systole lengths. These intervals can
obviously be readily and accurately determined on large coérdinate
paper for a consecutive range of cycles. In figure 1, one such cycle
B-3’ and its corresponding systole 3-3’ is indicated by dotted lines.
So far the process is not dissimilar to that employed by Henderson
except that the relation of systole to each cycle length was determined

, pa , eystole 2 Fre
in cycle lengths differing by 0.1 second. This seals ratio will here-

after be referred to briefly as the s/c ratio.

The data so obtained were plotted by dots on coédrdinate paper, as
shown in figure 4, the ordinates representing the duration of systole,
the abscissae, the cycle lengths. By connecting these data by lines a
curve s/c is obtained from which the theoretical s/c ratio at any heart
rate can be derived.

It soon became obvious, however, that such a theoretical curve of
s/¢ ratios could not be applied to different animals, inasmuch as cer-
tain variable factors were not taken account of. In the first place we
found that the duration of long vagal systoles varied from 0.22 to 0.32
second in different animals with corresponding variations at more
rapid rates. It therefore became necessary to construct for each ani-
mal a separate hypothetical volume curve based on its vagal systole
and from it to derive a curve of s/c ratios applicable to that animal.

This we did after the following manner: The duration of systole was —
first determined during a long vagus beat occurring after slowing had
been established for some time. Inasmuch as only the interval of
systolic ejection and not the total period of systole is concerned in the
construction of the volume curve, an interval of 0.05 second was de-
ducted from the vagal systole for the isometric period.!

The resulting interval of systolic ejection was then laid off on the
abscissae of large-sized coérdinate paper and an arc having the same
contour as that given in Henderson’s standard curve was drawn to fill
this time. Thus, in figure 1, the ares AB, AB! and AB? have the same

1 This we believe to be allowable for, according to the investigations of Hiirthle
(8), de Heer (14), Garten (15) and Frank (16), this is an average period which is
not affected in length by such changes in the circulation as occurred during the
course of our experimentation. Evenif this period does vary slightly in differ-
ent dogs, no significant error can be introdued since the same figure was again
added before the plot of s/c ratios was made.

54 CARL J. WIGGERS AND LOUIS N. KATZ

conformation but correspond to ejection periods of 0.25, 0.20 and 0.15
second, respectively. In this particular instance, the are AB, with a-
duration of 0.25 second was subsequently used as a pattern for the
smaller segments 1—1’, 2~2’, 3-3’, etc. It is obvious that if the systolie
ejection time were either 0.15 or 0.20 second, the ares AB’ or AB?
must be used as a pattern for the smaller segments.

The further criticism may be anticipated that belief in a “uniform-
ity of behavior’ law does not necessitate the assumption that hearts
of different animals with the same systole lengths necessarily have the
same contour of ejection curve. According to Henderson’s (4) results,
however, volume curves taken under normal conditions show that the

A 32 13

.

}
| j | | Li L l I l 1 l i i i
° a 2 3 4 7 a1 6 7 rt a x) 1.00 11 1.2 13 1.4 is 1.6

Fig. 2. (+ original size.) Diagram constructed for experiment C 210, showing
volume curve with ejection phase of 0.215 second and three possible diastolic

filling curves C, C’ and C%. The effect of diastolic filling rate on the length of - |

systole is indicated by the systoles 4-4’. Abscissa = 0.1 second. Detailed de-
scription in text.

curve of systolic discharge is a straight line at all points except at the
extreme lower end. A variation which could occur only at this place
would therefore affect the duration of only the very shortest cycles,
e.g., 1-1’ in figure 1. Since cycles of such short duration were never
obtainable in any of our experiments, however, it is clear that this
criticism is of no practical importance.

A second practical difficulty arose, however, in the correct projection
of the diastolic filling curve. Even though all possible precautions
were taken to maintain an effective venous pressure sufficient, accord-
ing to Henderson and Barringer (4), to insure maximal filling, it would
not be in disagreement with the idea of superimposable beats to sup-
pose that the filling curves of different animals are dissimilar. Indeed

ACCELERATOR NERVES AND VENTRICULAR SYSTOLE 55

it is quite conceivable that the heart within the intact thorax has a
filling curve quite different from any capable of registration by on-
cometric methods. Such differences in filling, however, will affect
considerably the relation of systole to cycle length at different heart
rates. This is illustrated in figure 2 where one may suppose the heart
_ te be filled according to any of three curves C, C’ or C2. Reference to
the dotted lines bracketed as 4in figure 2 or to the three corresponding
plotted curves of s/c ratios in figure 3, indicates that the theoretical
curves depend fundamentally on the unknown character of the filling
curve. | .

Confronted with the necessity of having some gauge as to the type
- of filling occurring in each animal’s heart, we selected that line of s/c
ratios which most nearly coincided with the long vagal beats and the
normal beats of the anithal.

2s : =

\lo

Fig. 3. Three curves showing relation of systole to cycle length at different
heart rates when the rate of diastolic filling differs. Constructed from data of
table 1 and volume curves of figure 2.. C, C’ and C? are corresponding curves.
Abscissae represent cycle length; ordinates, systole lengths, in seconds. Circles,
normal s/c ratios; crosses, s/c ratios of vagal beats. Detailed description in text.

The entire method of deriving our theoretical curve of s/c ratios,
thus outlined in principle, may be further clarified by following the
consecutive steps in a typical experiment: |

In experiment C 210, stimulation of the right vagus caused a slowing
of the ventricles from which a cycle having a period of 1.55 second
and a systolic period of 0.265 second was selected. Deducting 0.05
second for the probable isometric interval, leaves an ejection phase of
0.215 second. ‘This distance is laid off on codrdinate paper and an
are AB drawn. A reduced figure with coédrdinates omitted for reasons
pertaining to reproduction is shown in figure 2. In this case, three
possible filling curves, C, C’ and C? are drawn. By inscribing ares of
the ejection curve A B, at intervals of 0.1 second from points /, 2, 3, 4

56 CARL J. WIGGERS AND LOUIS N. KATZ

and so forth, and measuring the horizontal distances between 1-/,
2-2’, 3-3’, for each type of filling curve (illustrated by dotted lines
bracketed as 4) three possible durations of the ejection phase for each
cycle length are obtained. Now adding again 0.05 second previously
deducted gives the theoretical systoles for each cycle under three con-
ditions of ventricular filling. This is shown in the following table:

TABLE 1
DURATION OF THEORETICAL EJECTION DURATION OF TOTAL SYSTOLE
DURATION OF PHASE IN FIGURE 2 ACCORDING TO FIGURE 3

CYCLE

Curve C Curve C’ Curve C2 Curve C Curve C’ Curve C2
0.2 9.0 9.0 8.5 14.0 14.0 13.5
0.3 13.5 12.5 10.75 18.5 17.5 15.75
0.4 15.75 14.5 12.0 20.75 19.5 17.0
0.5 17 .25 16.5 13.75 22.25 21.5 18.75
0.6 18.0 17.5 15.0 23.0 22.5 20.0
0.7 19.0 18.5 16.0 24.0 23.5 21.0
0.8 19.5 19.0 17.0 24.5 24.0 22.0
0.9 20.0 19.5 18.0 25.0 24.5 23.0
1.0 20.5 20.0 19.0 25.5 25.0 24.0
131 21.0 20.5 20.0 26.0 25.5 25.0
by: 21.5 21.0 20.7 26.5 26.0 25.7
1.3 21.5 21.5 21.25 26.5 26.5 .| 26.25
1.4 21.5 21.5 21.5 26.5 26.5 26.5
1.5 21.5 21.5 21.5 26.5 26.5 26.5

Plotting these theoretical systoles in relation to the cycle, as in
figure 3, the three curves C, C’ and C? are obtained. If now we plot
as small circles the actual s/c ratios found during the natural heart
cycles of the animal, it will be seen that they follow the line C’ most
exactly. This line is therefore adopted as the theoretical line of s/e
ratios for other comparisons. In a similar way, a line of s/e ratios
was derived for all experiments and in the rest of the plots the line
selected is alone reproduced.

It is of interest to add that, according to these analyses, we found
that the hearts of our animals followed ‘a type of filling not dissimilar -
to that described by Henderson as typical for the normal heart. In
this instance we believe, for example, that the volume curve of the
ventricle corresponds to the line C’ in figure 2. _

Te eee
3 2

ACCELERATOR NERVES AND VENTRICULAR SYSTOLE 57

EXPERIMENTAL RESULTS

_ Comparison of the actual s/c ratios during accelerator nerve stimulation
with the theoretical values at corresponding heart rates. After the theo-
retical curve of s/c ratios had been thus determined for each animal,
we plotted the actual s/c ratios obtained under different experimental
conditions in relation to it, especial attention, of course, being directed
to the influence of the accelerator nerves. As the results require de-
tailed presentation, an analysis of three experiments, typical of all
cases, is appended.

_Experiment C 207 (fig. 4). The theoretical s/c ratio curve selected
as applying to this animal’s heart is shown as a solid line (s/c) connect-
ting dots at 0.1 second intervals. The small circles close to the line

‘cz I
tT -
ap |
my mt
—
=. — |
mS oS
5) aan o
71 or
eo hear ah
Lice ot
ie Nghe
ac Wh bs v n
4. » cs
‘ rs ’
ry Nw A e
bi a? A
v2
7 :
Va y
76.
ge’ :
2 3 4h} 5 6 Z 8 3 1} i perl 41\3 1

Fig. 4. Plot from experiment C 207, showing relation of actual s/c ratios to
the theoretical curve. Abscissae, cycle lengths; ordinates, systole lengths, in
seconds. Detailed description in text.

show normal s/c relations. The right stellate ganglion was stimulated
first, but we shall defer the discussion of this effect. Then both vagi
nerves were sectioned. The s/c ratios of 13 measured cycles are indi-
cated by crosses of group J. It is evident that when vagus control is
suddenly removed, and the accelerator control alone remains, the s/c
ratio is below that of the theoretical line. Within a few minutes,
however, the duration of systole increases again, as shown by the
crosses of group IZ, which represent measurements of 13 cycles ten
minutes after vagotomy. The left vagus nerve was then stimulated
but, with the exception of a few beats, the cycles were so long that they
were omitted from this plot for reasons of reproduction. It may be
noted, however, that the systoles following immediately after stimula-
tion began were slightly below the theoretical line, while those occur-

58 : CARL J. WIGGERS AND LOUIS N. KATZ

ring later coincided with it. Immediately following this stimulation,
12 measured cycles showed s/c ratios represented by the crosses of
group III. The obvious after-effect seems to be a slight increase in
the heart rate during which the systole lengths are somewhat longer
than the theoretical curve calls for. Such results indicate that perhaps
vagus stimulation is not entirely without an influence on ventricular
systole,—whether direct or indirect we are not able to say. A detailed
discussion of this questiqn is, however, not within the province of the
present communication. |
We may now return to the effects of accelerator nerve stimulation.
The dots of group IV represent the s/c ratios of 17 measured cycles
recorded during accelerator stimulation while the vagus nerves were

™ | Pe
. Lt dled JACEE ICC re re
— Y @ i!
os e : Th ,
rg be cle EL. : cd
a io aanas4
7 . xe + == SJ
a te : iy |e T oJ S
\ Y ie S
iB Al deat | tet | i h
A ue Pm lon 4
im Bait bere: hae — =—+ wa
T
‘
'
15) '
we ie uf =
ipm iy Lor*
H Pi. Sr er
tie 4
4
10 te
4 5 g' 7 8 f 9 Lie di dja 13 di

Fig. 5. Plot of data from experiment C 209, showing relation of actual s/e
ratios to theoretical curve. Abscissae, cycle lengths; ordinates, systole lengths
in seconds. Description in text.

still intact. The dots of group V show the s/c ratios of 14 measure-
ments shortly after the cessation of stimulation. The dots of group
VI, representing 32 measured cycles indicate the greater effect of
accelerator stimulation when the vagi nerves had been divided.

The data plotted in relation to the theoretical s/c ratios make it
obvious without further comment that the stimulation of the accel-
erator nerves reduces systole a great deal more than can be accounted
for by a mechanical abbreviation of the systolie portion of the volume
curve. . :
Experiment C 209 (fig. 5). The line of theoretical s/c ratios in this
experiment was derived from a vagus. beat having a cycle length of
0.98 seconds and a systole length of 0.285. ‘The small circles arranging
themselves around the theoretical line are normal cycles representative

— le ee ee

ACCELERATOR NERVES AND VENTRICULAR SYSTOLE 59

of 40 measured beats. The large dots represent ratios occurring during
vagus excitation. The small dots of group J indicate the duration of
systole during mild stimulation of the stellate ganglion; those of group
II, the effect of exciting the ganglion with a stronger current. In
both instances the vagus nerves were intact. It is evident that a
reduction of s/c ratios below the theoretical value occurs.

In order to determine the effect of accelerator stimulation on the s/c
ratio when cycles of longer duration occurred, we tested the effect of
simultaneous stimulation of the accelerator and vagus nerves. This
could, of course, be accomplished by simultaneous electrical stimula-
tion of these nerves. We found it, however, more expedient to induce
the vagus stimulation through chemical means. Pituitary extract
when injected, produces such a stimulation in some animals as was
fortunately the case in this experiment.

The dotted circles of group J/JJ, arranging themselves somewhat
below the theoretical line, show the duration of systole during such
pituitary slowing. That this slowing is at least predominantly due to
vagus stimulation, is evidenced by the fact that subsequent section of
the vagi restores the normal rate and duration of systole. This is
shown by the crosses of group JV which represent the systoles of 18
measured cycles after the vagi nerves were cut and while pituitrin was
still acting. ,

While the heart rate remained slow due to the pituitary extract, the
stellate ganglion was again stimulated. Although this caused some
increase in rate, the heart rate did not equal that natural to the ani-
mal. The s/c ratio decreased not only far below the theoretical ratios
for such cycles, but also far below the s/c ratios of other much shorter
eycles. This is evident on comparing the dots of group V, representing
9 measurements of beats occurring during accelerator nerve stimula-
tion, either with the dots of group J, with the crosses of group IV or

with the circles representing normal s/c ratios. Finally, after the vagi

had been divided and s/c ratios represented by the crosses of group IV
had been attained, the stellate ganglion was again stimulated. The
results of 27 measurements are shown by the dots of group VJ.
These results indicate clearly that the s/c ratio is much reduced
below the theoretical expectations by the influence of the accelerator
nerves, not only at rapid, but at slower heart rates as well.
Experiment C 210 (fig. 6). In this experiment the curve of the theo-
retical s/c ratio was derived as analyzed in detail in an earlier portion
of the paper. The small circles represent the actual s/e ratios of 60

60 CARL J. WIGGERS AND LOUIS N. KATZ

normal beats. The small circles in group JIJ are representative of
cycles following the injection of ;}> grain of atropine sulphate.

The dotted circles show the s/c ratios of 13 cycles obtained during
the action of pituitary extract early in the experiment. Section of the —
vagi abolished this slowing and established a normal rate. Seven
measurements of right vagus and 18 observations of left vagus stimula-
tion are shown by crosses. Under all of these conditions of varying
vagus activity the s/c ratio does not deviate in any pronounced fashion
from the theoretical line.

On accelerator nerve stimulation, this conformity is no longer ob-
served. The dots of group J represent measurements of 53 cycles
during and immediately after accelerator nerve stimulation. The
dots of group IJ show the results of 33 measurements of cycles obtained

| x

i

oll,

oe 9

\:

+ all

a

15S top
Mf od

,
West ts

3 $ 5 6 z 3 9 A ili wal [| i i

Fig. 6. Plot of data from from experiment C 210, showing relation of actual
s/c ratios to theoretical curve. Abscissae, cycle lengths; ordinates, systole
lengths, in seconds. Description in text. '

while the accelerator nerves were stimulated and the heart slowed by
pituitary extract. With the exception of three dots in this group
which, as a matter of fact, correspond to cycles at the very onset of
stimulation, the s/e ratios are far below the theoretical line.

Finally, when the vagi nerves had been sectioned, 3 ec. of a 1: 50,000
solution of-epinephrin, which presumably also affects the sympathetic
endings in the heart, were injected. The crosses included in group J
illustrate the s/c ratio of 44 rapid beats thus produced. It is obvious
that epinephrin like accelerator stimulation acts to abbreviate systole
much more than can be explained by a mechanical shortening of systole
. at these rates.

The mechanism of accelerator action. Discussion of results. The
experiments above cited indicate clearly that, while under normal

ni... Ce a ee ee

- ACCELERATOR NERVES AND VENTRICULAR SYSTOLE 61

conditions changes in the duration of systole may conform reasonably
to the changes anticipated if the ventricle beats according to a uniform
plan, the abbreviation of systole induced through accelerator activity
both at rapid and slower heart rates is in excess of that which may be
accounted for by a mechanical shortening. In some way the accelera-
tor nerves exert an influence on ventricular systole which can be termed
specific. Before it may be assumed, however, that such an influence
is exerted directly on the ventricular muscle and acts to abbreviate the
contraction process, diligent inquiry must be made for the involvement
of some possible mechanical mechanism. :

The possibility suggests itself at once that the rapid heart action
induced by accelerator stimulation causes an increase in the minute
volume discharged which might conceivably operate to reduce the
venous pressure and auricular filling. If this were the case, the rate of
ventricular filling, especially in early diastole, would decrease and this
might mechanically abbreviate systole. In other words, it is conceiv-
able that under rapid heart action we would have an entirely different
filling curve and that therefore the theoretically derived curve would
no longer apply. Unfortunately, we did not follow the venous pres-
sures throughout the experiment nor do such observations during
accelerator stimulation seem to have been reported. In the volume
curves recorded by Henderson (4) during accelerator nerve stimula-
tion, there is no such indication of reduced filling; indeed the rate of
filling appears to us slightly increased. Such an explanation, however,
could not ac¢ount for the greatly abbreviated systoles occurring when
the accelerator nerves affect beats maintained at or below the normal
rate by simultaneous vagus action. |

The further suggestion that the reduction of systole by accelerator
stimulation is due to a shortening of the isometric period rather than
the ejection phase, might be entertained were the reduction itself not
frequently far greater than the entire isometric interval. By no plau-
sible conception at present presenting itself can the effect of the accel-
erator nerves be explained otherwise than through a specific effect on
the duration of muscular contraction itself.

This explanation is further substantiated by the lack of a fixed rela-
tion between the duration of systole and diastole when we follow them
from beat to beat during increased accelerator activity. Thus, accel-
erator nerve stimulation usually causes an immediate shortening of
diastole while the period of systole shortens gradually and progressively
as stimulation continues. In some experiments, systole continues to

62 CARL J. WIGGERS AND LOUIS N. KATZ

shorten even when diastole is again increasing. -We have plotted
experiments in which, during continued stimulation, diastole actually
lengthened again so that its duration was greater than normal, yet
systole continued at its shortest length. When stimulation ceases the
systolic period usually remains shortened for as much as 20 seconds; a
shortened diastole, on the contrary, at once begins to increase in length.
The general trend of such experiments is shown in an abbreviated plot

in figure 7. At A, a few consecutive normal cycles are indicated. At |

B, the 20th and subsequent beats during accelerator stimulation are
shown. A moderate decrease in systole and a marked decrease in
diastole are evident at this time. As stimulation continues (B-C),

0.5)
\
IN
I
1 i x
re \ A : a
\ F
03 [\*t
y
‘ \l.
ban Pa
a:l he zs a wr
: =
fele o x = e|*ye
oa TCE
l Db Li

Fig. 7. Plot from experiment C 209 IT, showing effect of accelerator nerve stim- -

ulation on duration of systole and diastole in consecutive heart beats, each
represented by a dot. Upper plot, duration of diastole; lower curve, systole;
ordinates represent time in seconds. Description in text.

systole decreases more than diastole. Between C and D, 25 beats are
omitted. While accelerator stimulation continues at D, the length of
systole remains practically unaltered while the period. of diastole has
recovered considerably. At EH, stimulation ceased. Between # and
F, 6 beats were omitted. By this time, F, diastole has regained its
normal length, but systole continues shortened. G is a later normal
control.

Another illustration is shown in the case of epinephrin stimulation
in figure 8. The plot starts with a few normal control data. At A,
the systole and diastole after the 20th beat following epinephrin injec-
tion are plotted. Both vagi nerves had been severed and a previous

dose of ;4,’grain of atropine sulphate had been administered. In this -
100.

EE —— ae

2
| A
:
:
:
;
j
,

ACCELERATOR NERVES AND VENTRICULAR SYSTOLE 63

instance, diastole first lengthens while systole progressively decreases
(A B). Between B and C, 45 beats are omitted and by that time
systole has again begun to lengthen while diastole decreases further.

‘This again demonstrates the lack of a fixed relation between systole

and diastole when the accelerator endings are stimulated.

In view of these facts and since cardiac acceleration under normal
conditions is undoubtedly often due to accelerator nerve activity, the
hypothesis that the ventricle normally beats according to a ‘‘uni-
formity of behavior’ law at these rapid rates should be submitted to
further experimentation.

s
e ° -¢
20 5 E ry +" * ee elete »
1 beet lt | pee ebe? “1
'

ae | [

Fig. 8. Plot from experiment C 210 VII, showing effect of epinephrin on dura-
tion of systole and diastole in consecutive heart beats, each represented by a dot.
Upper plot, duration of diastole; lower curve, systole; ordinates represent time
in seconds. Description in text.

SUMMARY AND CONCLUSIONS

Although it had been shown by previous investigators that stimula-
tion of the accelerator nerves causes a marked reduction in the dura-
tion of systole, it had not been demonstrated that this reduction was

greater than could be accounted for on Henderson’s law of “uniform-

ity of behavior,’ and consequently no clear demonstration existed of
any specific effect on the ventricular musculature.

-By determining the duration of systole as well as the cycle length in
a slow vagal beat, we found it possible to construct a probable volume
curve for each animal and from it to derive a plot of the theoretical
relation that should exist between cycle and systole lengths at any
heart rate if the heart, during changing nervous action, beats accord-
ing to a uniform law.

The actual systole and cycle lengths were determined from the
recorded heart sounds during a wide range of heart rates obtained

through vagus sectioning, vagus stimulation and accelerator excitation

64 CARL J. WIGGERS AND LOUIS N. KATZ

and these values were then plotted on coédrdinate paper in relation to

the theoretically constructed curve of Se ratios.
Upon doing this, it was found that while the actual a ratios
cy

obtained during normal conditions and during vagus stimulation coin-
cided reasonably well with the theoretically derived values, the length of
systole during accelerator stimulation and during the action of epinephrin
were markedly less than those indicated by the theoretical curve. Inasmuch q
as this occurred whether the heart rate was actually increased or main- _
tained slow by the simultaneous action of pituitary extract, it is diffi-
cult to refer this to any mechanical effect on the venous pressure and
ventricular filling. :
Consequently, the conclusions are reached a, that the accelerator
nerves have a specific effect on the ventricular musculature which
operates to reduce the contraction period; and b, that, in view of these
observations, the hypothesis that under normal conditions the ventricle
operates according to a uniform mechanical law, should be subjected
to further investigation.

BIBLIOGRAPHY

(1) v. Bezoup anp Brver: Untersuchungen aus d. physiol. laborat. im Wiirzburg,
1867, ii, 235.

(2) CYon AND CYon: Centralbl. f. d. med. Wissensch., 1866, 801; Archiv f.
Anat. u. Physiol., 1867, 389.

(3) HENDERSON, ET AL: This Journal, 1906, xvi, 325; 1913, xxxi, 288, 352.

(4) Naa ianaew AND BARRINGER: This Journal, 1913, xxxi, 297.

(5) Baxt: Arch. f. Physiol., 1878, 122.

(6) Kuve: Arch. f. Physiol., 1881, 260.

(7) MacWiturams: Journ. Physiol., 1888, ix, 359.

(8) Hirraze: Arch. f. d. gesammt. Physiol., 1891, xliv, 89.

(9) Frank: Sitzungsb. d. Gesellsch. f. Morph. u. Physiol., Miinchen, 1897.

(10) Hunt: This Journal, 1899, ii, 395.

(11) Wiacers anD Dran: Amer. Journ. Med. Sci., 1917, cliii, 666.

(12) WiacERs AND Dgan: This Journal, 1917, xlii, 478.

(13) HenpERson: This Journal, 1909, xxiii, 354, fig. 3.

(14) p. Heer: Arch. f. d. gesammt. Physiol., 1912, exlviii, 1

(15) Garten: Zeitschr. f. Biol., 1915, Ixvi, 52.

(16) Frank: Zeitschr. f. Biol., 1905, xlvi, 495.

o_o =

a

GASTRIC RESPONSE TO FOODS!

XIU]. Tue INFLUENCE OF SUGARS AND CANDIES ON GASTRIC
SECRETION

. RAYMOND J. MILLER, OLAF BERGEIM, MARTIN E. REHFUSS
AnD PHILIP B. HAWK

From the Laboratory of Physiological Chemistry of Jefferson Medical College,
Philadelphia

Received for publication May 21, 1920

The widespread use in the diet of large quantities of refined sugars
and candies is a comparatively modern development. Because the
races of men have lived for ages without general access to sugars in
concentrated form, a question has naturally arisen as to whether the
use of such foods may not in some instances give rise to harmful effects.
It was perhaps natural also that the rapid development of the glucose
industry should bring forth opponents and proponents of its wide use
in cooking and in confections.

It is not necessary at this date to ieeave statements with reference
to the alleged harmful character of cane sugar or glucose per se, inas-
much as it is well known that all digestible carbohydrates are absorbed
from the intestine in the form of simple sugars and in the main as
glucose formed from the starch of foods.

Certain objections to the use of sweets must, however, be considered.
It cannot be denied that the eating of candies before meals decreases
the appetite for other foods in general and that thus, particularly in
the case of children, the intake of foods containing essential proteins,
vitamines and inorganic salts may be reduced below the optimum
requirements for growth. Purified sugars can obviously furnish but
the single dietary essential, carbohydrate. This depression of appe-

_ tite may be associated with the rapid absorption which sugars undergo

in the intestine as well as with the depression of gastric secretion which
is indicated by data presented in this paper. Such work as has been

1 The expenses of this investigation were defrayed from funds furnished by

Mrs. M. H. Henderson, The Curtis Publishing Company and Doctor L. M. Halsey.

65

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL, 53, No. 1

66 MILLER, BERGEIM, REHFUSS AND HAWK

carried out on the assimilation of carbohydrates as measured by the
rise of blood and urinary sugar following their ingestion indicates that
pure sugars are absorbed sooner from the intestine than the glucose
formed from starch ingested (1). The assimilatory power of the nor-
mal human body for glucose would not appear, however, to be readily
overtaxed.

It is known that in diabetes the weakened assimilatory function of
the system for carbohydrate is still further impaired by the ingestion
of large amounts of such food, and it might be supposed that habitual,
long-continued use of many sweets might lead to the aggravation of a
diabetic tendency which might not otherwise manifest itself. Such a
suggestion has been made, but there is little concrete evidence to sup-
port it.

Sugars and candies, on the other hand, are particularly suited: to
furnish to the body, in convenient form, an additional quota of readily
assimilable energy. Thus it has been pointed out that a single caramel
may furnish 45 calories or sufficient energy for a mile walk (2), and -
that other confections yield similar amounts of energy. The view
that these preparations are of negligible food value must, therefore,
be discarded. Surely there is a physiological basis for the candy
craving of children which cannot be disregarded, unless energy in
abundance is furnished by other foods, nor does there seem to be any
good reason for replacing more of the food carbohydrate by fats (except
those high in vitamine) which are less readily assimilated and for which
children generally have less desire.

In the cases of gastro-intestinal derangements more common in
adult life, the influence of diet on the secretory, motor and fermenta-
tive processes of the digestive tract may overbalance considerations
of energy value.

In the present paper we have endeavored to determine the influence
of certain sugars, candies and other confections on the secretory and
motor responses of the stomachs of normal adults.

The experiments were carried out on normal medical students and
members of the staff of the department. They reported about nine
o’clock in the morning, and any residuums which were present were
removed from the stomach. The sugar solutions or candies were then
given and samples of stomach contents removed at 15-minute intervals
until the stomach was empty. Free and total acidities, pepsin, trypsin —

and amino acid nitrogen were determined by methods previously
described (3).

GASTRIC RESPONSE TO SUGARS AND CANDIES 67

The response of the stomach was studied following the ingestion
of cream candies, hard candies, chewing candies, fresh and stale can-
dies, chocolate and candy combinations. Inasmuch as most sweets
enter the stomach essentially as sugar solutions, a preliminary study
was made of the influence of concentrated and dilute solutions of cane
sugar and glucose.

The response of the stomach to dilute and concentrated solutions of
sucrose, glucose and maple sugar. Nine experiments were made on
dilute and concentrated sugar solutions. The results of these experi-
ments are charted in figures 1 to 9. One subject was given 250 cc.
portions of 4 per cent glucose and cane sugar solutions; another sub-
ject, 150 cc. portions of 6 per cent glucose and cane sugar solutions,
the total amount of sugar given in each case being about 10 grams.
. The same evacuation time (1 hour and 45 minutes) was obtained in
each of the four experiments, and the acid responses were very similar.

No distinction could, therefore, be made between the responses of
the stomach to dilute solutions of glucose and cane sugar nor, as the
curves show, could there have been any distinct depression of gastric
secretion by the dilute sugar solutions in the quantities given.

Maple sugar in dilute solution was given to the same subjects and
was found to leave the stomach in from 45 minutes to an hour and 15
minutes without distinct depression of gastric secretion. The fact
that this solution left sooner than the cane sugar or glucose may have
had some relation to the more pleasant taste of the maple sugar. The
cases are not quite comparable, however, as the glucose and cane sugar
solutions were given without removing residuums.

Concentrated sugar solutions were given the men who had previously
received dilute solutions. One subject was given 100 grams each of
_ glucose and cane sugar in 59 per cent solutions. The other subject
was given 100 grams of glucose in 40 per cent solution. Such solu-
tions remained in the stomach from one-half to an hour longer than
similar volumes of the dilute sugar solutions. In the case of the glu-
cose solutions, the secretion of gastric acid was markedly depressed for
an hour and a half or until much of the glucose had left the stomach.
The secretion of pepsin was inhibited also. Cane sugar in concen-
trated solution appeared to have somewhat more stimulatory power,
but its evacuation was likewise delayed. It is possible that the sweeter
taste of cane sugar or its less rapid absorption from the inte tine may
influence the response of the stomach to its concentrated solution, but
more evidence on this point would be required. It is clear that con-

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GASTRIC RESPONSE TO SUGARS AND CANDIES 73

centrated sugar solutions markedly depress gastric secretion and delay
evacuation. :
Soft candies. Under this heading were included chocolate creams,
fudge, bonbons and wafers. The contents alone of chocolate creanis,
and plain milk chocolate were also studied.
The interiors of chocolate creams (consisting mainly of glucose)

were given in 100-gram portions to two subjects (see figs. 11 and 12).

It will be noticed that gastric secretion was markedly inhibited and
evacuation much delayed by the ingestion of this amount of cream
candy which was given without water and formed a concentrated sugar
solution in the stomach. The same depressing action was noted where
soft creamy bonbons were given (see fig. 13). These contained some-
what more cane sugar and were more highly flavored than the creams
previously tested. This may possibly have accounted for the slightly |
more rapid evacuation.

Soft creamy wafers of strawberry flavor were given to one subject,
100 grams of the candy being ingested. As might be expected, the
gastric secretion was depressed by the large amount of sugar present.
Evacuation, however, was completed in moderate time (12 hour),
showing perhaps some influence of the fruit flavor on gastric motility.

‘Wafers of the same type but with strong peppermint flavor remained
three-quarters of an hour longer in the stomach of this subject than did
the strawberry wafers and gave rise to somewhat more acid secretion.
The delayed evacuation may have been due to irritation of the duo-
denal mucosa by the oil of peppermint used as a flavoring agent.

Chocolate fudge remained in the stomach of one subject half an
hour longer than a strawberry-flavored cream candy (see fig. 16).,

_ There was also a distinctly higher acid production in the case of fudge.

Both of these effects must be related to the presence in the fudge of
butter fat and chocolate.

That chocolate stimulates gastric secretion is indicated by our
experiment in which milk chocolate was given (see fig. 17), an acidity
twenty points higher being attained than in the case of creams. Milk
constituents are probably responsible in part for this effect. Milk
chocolate left the stomach in 2 hours or half an hour sooner than cream
candy. It must, therefore, be considered as throwing less of a burden
on the stomach than the latter.

Chocolate creams were compared in two cases with the contents
of similar creams. Our best subject (see fig. 18) showed practically
the samé evacuation time for both but a somewhat higher acid develop-

74

MILLER, BERGEIM, REHFUSS AND HAWK

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GASTRIC RESPONSE TO SUGARS AND CANDIES

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MILLER, BERGEIM, REHFUSS AND HAWK
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GASTRIC RESPONSE TO SUGARS AND CANDIES
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77

78 MILLER, BERGHIM, REHFUSS AND HAWK

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GASTRIC RESPONSE TO SUGARS AND CANDIES 79

ment on chocolate creams in agreement with our findings on chocolate
alone. Our second subject also gave higher acidities on chocolate
creams but evacuation was delayed due to considerable intestinal regur-
gitation characteristic of this subject. He was also given, for com-
parison, chocolate creams which were 8 months old and very stale and
not appetizing. These showed delayed evacuation and high acidity
as compared with cream candies, and it appears probable were less
easily handled by the stomach than similar candy in the fresh condi-
tion. Interpretation is somewhat difficult on account of the excessive
regurgitation of this subject.

Hard candies. Two men were permitted to suck continuously for
15 minutes on hard candies (lemon-flavored stick candy). The candies
were weighed before and after, and it was found that one man suc-
ceeded in obtaining 15 grams of candy, the other only 7 grams. No
water was taken by either during the course of the experiment so that
the sugar must have entered the stomach as a solution of moderate
concentration. In each case a slight gastric secretion of moderate
acidity was developed, and the stomach was empty inan hour. Neither
was there any distinct continued secretion afterward. It is clear that
the burden placed on the stomach by sucking the hard candy was
very much less than that produced by the liberal or moderate eating of
cream candies. |

Chewing candies. Caramels, salt water taffy and gum drops were the
chewing candies studied (see figs. 22 to 25). Caramels gave rise to a
much greater acid production than cream candies, although evacuation
times were about the same. This acidity may have been due to the
greater chewing psychic secretion as well as to direct stimulation by
ingredients of the caramels other than sugar. At any rate the marked
depressing action of pure sugar candies was not noted. The marked
differences between free and total acidities were due largely to the
action of the gastric acid on the phosphates of swallowed saliva.

In the case of gum drops experimental difficulties were met with as
the gelatinous mass formed in the stomach clogged the aspiration

tube. It is clear, however, that the gum drops left the stomach in

moderate time and produced little acid stimulation and were thus
handled without difficulty so far as the stomach was concerned.

Salt water taffy left the stomach of one subject sooner than caramels
or creams but developed a much lower acidity than caramels. It may
be that the chewing psychic secretion caused by eating caramels was
greater due to their flavor more nearly approximating that of pala-
table food normally giving rise to a gastric stimulation.

80

MILLER, BERGEIM, REHFUSS AND HAWK

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GASTRIC RESPONSE TO SUGARS AND CANDIES

81

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82 MILLER, BERGEIM, REHFUSS AND HAWK

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GASTRIC RESPONSE TO SUGARS AND CANDIES

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84 MILLER, BERGEIM, REHFUSS AND HAWK

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GASTRIC RESPONSE TO SUGARS AND CANDIES 85

Marshmallows and licorice. 'Toasted and untoasted marshmallows
were given to one of our subjects. The gastric response was markedly
different in the two cases. The toasted marshmallows left the stomach

an hour sooner and gave rise to an acid development forty points
higher than plain marshmallows. The psychic stimulation due to the
more appetizing flavor after toasting may be partly responsible for
such differences, as well as the alteration in texture and in the condi-
tion of the egg white which they contain. It is known that raw egg
white has very little stimulatory power as compared with the cooked
product. |

A man was allowed to suck a stick of licorice for 15 minutes, obtain-
ing in this time about 5 grams of the substance. It gave rise to a
fairly abundant secretion of moderate acidity and remained in the
stomach for 2% hours. | |

_ Pop-corn preparations. Sugared pop-corn was given to two subjects
in amounts of 50 and 38 grams respectively. Such pop-corn was
found to leave the stomach in about 1 hour and to develop a moderate
acidity. Buttered pop-corn gave a very similar response in another
subject (see fig. 31), leaving for the most part in an hour and a half. —
It must be borne in mind, however, that certain of the larger and
harder particles of corn which could not be aspirated remained in the
stomach somewhat longer. Practically all of the corn left in moderate
time. | 3

Bread and honey. Sugars being very frequently given in the form
of syrups added to bread and other foods, we endeavored to determine
what effect such additions might have upon the gastric response.

A man was given 40 grams of whole wheat bread without additions,
and after this had left the stomach he was given the same amount of
bread with 20 grams of honey. The addition of honey depressed
gastric secretion slightly but did not delay evacuation, although the
food value of the preparation had been greatly increased. A moderate
amount of honey added to bread cannot, therefore, be considered
harmful.

86 MILLER, BERGEIM, REHFUSS AND HAWK
TABLE 1
The response of the human stomach to candies

TOTAL
dust PREPARATION ron mel Some, | Se
HOUR

1. Rud | Dilute glucose’ solution........ Pe Achhioe 1:45 | 107.5 90.0
2. Ree | Dilute glucose solution.................. 1:45 57.5 48.0
3. Rud | Concentrated glucose solution.......... 2:15 | 106.0 28.0
4. Ree | Concentrated glucose solution.......... 2:30 86.5 10.0
5. Rud | Dilute cane sugar solution...............] 1:45 82.0 68.0
6. Ree | Dilute cane sugar solution.............. 1:45 71.0 58.0
7. Ree | Concentrated cane sugar solution........ 2:45 99.5 95.0°
8. Rud | Dilute maple sugar solution............. 1:15 88.5 86.0
9. Ree | Dilute maple sugar solution............. 0:45 32.5 32.5
10, Sea | Ch@eolate 6reaims 5 oO oy cau ccs ce oo eee 2:15 56.5 56.0
11. Sar. | Ol@polate creams... . CAG aa 3:15 79.5 54.0
12;\Bar PO reanis 26.65... i ee, BRI ae 2:45 36.0 24.0
13. Beas ioGreaha cd .. cai. ci nc gee okesivee REG 2:30 |. 57.5,] 44.0
14.. Whi, |. Cha@eolate fudge... .......7-2-sdasbs eatin ke es Le 73.0 61.0
th: GOB. 1 BAe CHOCOINEE oo. cos soc s ssa eneeee se 2:00 69.5 69.0
16, Sar | Bonbons..... 2.0.0 as te ee eee 2:30 33.0 24.0
17. Whi | Cream wafers, peppermint..............| 2:30 42.5 40.0
18. Whi | Cream wafers, strawberry............... 1:45 37.5 29.0
£9.) Wha 4 Lemon eticks::o:. ss). fejpe. fc sees’ ee eelem ep 1:00 52.0 6.0
20 Sid, 4 QMO ARES 8 fie aalt ac sete sage 1:00 | 62.0 18.0
2h PORE: | COOPRIOGIB coi soe hoe Ce eee 2:45 80.0 | 71.0
See WG | CALSMOW 32.065 chtentia on eer 2:45 92.0 86.0
98: Sar | Taffy; salt water... 2527. YU RV a On Bee 26.0
28 TOL) Diesrice $3) 0064 a. os BE ei 2:45 64.0 54.0
25. Fees) | Apynd eee oud 2. ps s-cs page ped ob nile wate 1:30 38.5 24.0
26. Fle | Marshmallows, plain..................- 3:15 61.5 43.0
27. Fle | Marshmallows, toasted.................] 2:15 | 103.0 88.0
23. pea. | Bresd- 8nd ROnCY oo. ceils: wean eee wane 1:30 75.0 64.0
29. Whi | Pop-corn, sugared’)... 20... fee 1:30 65.5 58.0
30.‘Hol | Pop-corn, sugared:.. i). cia. dats. deul Wis 1:30 78.5 64.0
31.,:Fle |: Pop-corn, butteted «ce ci siegs dased oandhore 1:30 56.5 56.0
32. Sar | Stale chocolate creams.............5.-. 3:15 72.5 64.0

SUMMARY AND CONCLUSIONS

Large amounts (100 grams) of cane sugar or glucose in concentrated
solution markedly depressed gastric secretion and delayed evacuation
of the stomach. ;

Small amounts (10 grams) of cane sugar or glucose did not appreci-
ably inhibit either gastric secretion or evacuation.

GASTRIC RESPONSE TO SUGARS AND CANDIES 87

Candies depress secretion and delay evacuation in proportion to

their sugar content and the amounts of them ingested. This tendency
is influenced, however, by flavoring substances, and particularly by
added food ingredients such as milk, eggs or chocolate, which stimulate
gastric secretion.
. Candies should be eaten not before but after meals. Hard candies
which must be sucked are preferable to cream candies for children
- because of the smaller quantity of less concentrated sugar solution
derived from them. .

Cane sugar and maple sugar elicited much the same response from
the human stomach as glucose, although the possibility that the greater
sweetness and less rapid absorption of the first mentioned sugars gives
them a slight advantage is not excluded.

Soft candies such as bonbons, soft creamy wafers and the interiors of
chocolate creams when given in 100-gram portions exerted the same
depressing action on gastric secretion and evacuation as concentrated
sugar solutions.

Peppermint oil used as a flavoring agent delayed evacuation while a
strawberry fruit flavor appeared to accelerate it.

Chocolate appeared to stimulate gastric secretion as indicated by
experiments on milk chocolate, chocolate fudge and chocolate creams,
which gave higher acid figures than plain sugar candies. Stale choco-
lates remained in the stomach relatively long.

The sucking of hard candies introduced but a small amount of
sugar into the stomach which was readily evacuated and exerted little
depressing action on gastric secretion.

Chewing caramels gave rise to a more voluminous gastric secretion
than cream candies, but evacuation times were about the same. Salt
water taffy gave rise to less secretion, while gum drops left the stomach
rapidly with little acid production.

Plain marshmallows remained in the stomach rather ie: but after
being toasted these confections left the stomach TAY and gave
rise to high intragastric acidities.

Licorice gave rise to a fairly abundant secretion and remained in the
stomach for nearly 3 hours.

Sugared or buttered pop-corn developed a moderate acidity and
left the stomach rather quickly.

The addition of honey to bread did not delay evacuation, although
acid production was somewhat depressed.

(1) Jacopson: Biochem. Weekes 1913, lvi, 1p Pa
(2), BENEDICT AND BEneDIcr: Boston Med. Surg Jo im.

elxxxi, 415. oy Sheree tee lini;
(3) Surrn, FISHBACK, Buronm, LicHTENTHA La, Re

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FURTHER EVIDENCE ON THE FUNCTIONAL CORRELATION
OF THE HYPOPHYSIS AND THE THYROID

JOHN A. LARSON

From the Rudolph Spreckels Phystological Laboratory of the University of
California

Received for publication May 26, 1920

In a former paper (1) I presented evidence to show that the admin-
istration of the anterior lobe of the pituitary has a very beneficial
action upon maintenance and growth of thyroidectomized rats. In
that paper I pointed out the importance of using animals of ‘‘approxi-
mately. the same size, age and strain, and wherever possible from
the same litter.” At that time however, I was unable to meet these
conditions in a wholly satisfactory way. I have now repeated the
experiments with a larger number of animals and with strict regard to
the comparison of individuals from a single litter.

There were in all seventy-two litters used. These were selected of
as nearly the same date of birth as the conditions of the work would
permit. The litters were kept separated and directly upon the day of
weaning were ear-marked and subdivided into the respective series.
In this arrangement extreme care. was taken in regard to sex, weight
and even color. All runts, individuals below the average weight and
size of the litter, were eliminated. In anticipation of possible acci-
dental deaths from ether, etc., the members in the operated series
greatly outnumbered those in the normal series. There were four
main series of animals:

A. Thyroidectomized animals fed upon the normal diet in addition
to kidney.

B. Thyroidectomized animals fed upon the normal diet in addition
to anterior lobe.

C. Normal, that is, unoperated, animals fed upon the normal diet in
addition to anterior lobe.

D. Normal, that is, unoperated, animals fed upon the normal diet
in addition to kidney.

89

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 1

90 JOHN A. LARSON

To these series a fifth, E, was added, consisting of only a very few
thyroidectomized animals hich were fed upon the normal diet in
addition to thyroid. As was to be expected from the results of thyroid
feeding in cases of thyroid deficiency in other animals and also from
the work of Cramer and McCall (2) on the rat, the animals in group E
differed scarcely at all from the normals. The individual results will
not be given in the tables, but the final results will be recapitulated
with the other four series. |

In the subdivision of the litter one rat of the same sex and color
was placed in each of the four main series. This was possible in most
cases, since the number in the litters ‘varied from four to twelve. But
if there happened to be, say, two males and two females in one litter,
those of the same sex were placed in the operated series and the others
in the unoperated. After picking out one animal for each series all of
the remaining litter mates were distributed strictly according to sex
in the operated groups, there being sometimes four from one litter in
each operated series. Any odd member was placed in the fifth group.
The result was that at the end of the division every member in each of
the four series had a litter mate of the same sex in every other series.
I have thus been able to give tables in which it is possible to see the
results in each single litter although as a matter of fact the whole
number of animals used was so large that a statistical treatment would
have served the same purpose.

So far as possible, litter mates were operated upon on the same day.

The experiment lasted from August 1, 1919, the date of the first
operation, until March 1, 1920, the time of the last weighing. The
animals were weighed separately and at the same time, in relation to
feeding, once a week until through December and then every two
weeks until January 19, and the final weighings were made at the i
of the experiment on March 1.

The rats were kept five in a cage. The cages contained only males
or females in order to avoid distortion of growth curve through preg+
nancy. The food which I have spoken of above as “normal diet”
consisted of the following ingredients:

Corn mealice occ bo fos be NS ae clas vn i wk 6.0 parts

BRIOG hia ce mak Wiaddr are «bert o PN AM ROR ey ee 2.0 parts
Barley os Be oS ws a 5 ch eae bee alate ce ke 2.0 parts
INICAE THOME ii 58k do 56 Ae ne LAR Batt Ri Sana 4.5 parts
EBC Se aie oe wees Sire tek ees pst art geet ah re Rigas aaa 5.5 parts
NAGI iy, o's aie cine leincpin's «ecole c's atin e @ Skrale 8 lve ae okie nin Ung ne at 1.0 per cent
CATs ES vkce Koo ond bon one's bee ah o BEM eed we celle aaa 1.5 per cent |

Greens ad libitum

FUNCTIONAL CORRELATION OF HYPOPHYSIS AND THYROID 91

In addition to the normal diet each animal received as already stated
either anterior lobe of the pituitary or an equivalent amount of kidney.
The members of group E received 0.2 gram of fresh beef thyroid daily;
whenever possible the dosage of anterior lobe was one entire lobe
daily to each animal in the two series, and whenever there were too
few glands for that dosage they were divided equally between the
different members. The anterior lobes were administered in such a
way that each animal received at least one-half a gland at each feeding.

The histories. of all the members of the series are recorded in two
main tables. Throughout the following discussion no reference is
made to animals which were killed by ether or in a few cases, lost.
Such individuals were discarded.

In the first table the histories of all of the animals still living at the
termination of the experiment are exhibited apart from those rats
which died. These latter are treated in tables 3 and 4.

Table 1 shows the sex, number, date of birth, date of operation,
initial and terminal weights as well as the gain. Up to no. 65, the
number, sex and date of birth are the same for all of the individuals
represented in a horizontal row, since all of the animals therein are
members of the same litter. In litters 65, 66 and 67, the members
are of the same sex in the operated group but of a different sex from
that in the normal groups, which is the same, however, in the two
unoperated series. The members of groups 73 to 81 are from different
litters owing to deaths of the other litter mates. :

Table 2 presents the averages of the data shown in detail in table 1.
The homogeneity at the outset of the experiment is indicated by the
average initial weights which are 32.9, 32.5, 31.8 and 31.8 respectively.
In contrast with these the average terminal weights (and the average
gains) show marked differences. Growth was slower in the thyroid-
-ectomized animals fed on the control diet than in the thyroidectomized
animals which received pituitary, the gain of the former being only
123.7 grams as compared with 171.3 grams. This result is in con-
formity with that contained in the previous paper. It must be remem-
bered, moreover, that these figures deal only with the animals which
survived at the close of the experiment. If in calculating the effect
upon the rate of growth the animals which did not survive to the end had
been included, the difference would have been still more marked.

It is also of interest to note that in the normal series the gain in
weight was greater in the pituitary animals than in those receiving
the control diet. Under the circumstances there can scarcely be a’

LARSON

A.

JOHN

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FUNCTIONAL CORRELATION OF HYPOPHYSIS AND THYROID
/

95

TABLE 2
Summary of results shown in table 1
grams grams grams
OT ae 80 32.9 156.6 123.7
ee eee 81 32.5 203.8 171.3
eis a. 4.0. 67 31.8 212.7 180.9
MOLIOG Ms... 66.5... 67 31.8 198.5 161.7
Oe eg han 2 12 33.7 . 193.3 159.6
TABLE 3a
Average gain for each sex
Ree tamer | tise asrencion | Semen | om) moe +
C a. ee a ‘do = C. a =| o ds ‘ q
foi f| 2} e9| #2) 2 | fs] #2| 2 | eel eel #
ee ipal £ | EE) ea) epee | Ba | Bee Pees
< < 4 |< < a |< < <q |< < 4
grams | grams | grams | grams | grams | grams | grams | grams | grams | grams | grams | grams
Males..:.... 34.4)173.8)1389.4) 34 |206.8/172.8) 33.3/252.7/219.4| 33.1/225.9/192.8
Females....} 31.3)158.6)127.3) 32 |184.1|152.1) 32.4/182.3)149.9| 31.3)165.4/144.1
TABLE 33

Total percentage gains; comparisons are made between members of the same sex
under different conditions

B. THYROID | DIFFERENCE|C. NORMAL DIFFERENCE
A. THYROID D. NORMAL |:

: + ANTERIOR| BETWEEN |-+ ANTERIOR BETWEEN

‘+ KIDNEY LOBE A AND B LOBE + KIDNEY C AND D

per cent per cent per cent per cent per cent per cent
Mammeso ss sy 405.2 508 .2 103.0 658.8 582.4 76.4
Femalés...... 306.2 406.7 - 100.0 462.6 425.7. 33.9

TABLE 3c

Percentage gains of males over females; comparisons are made between the males
and females receiving the same treatment. The figures here recorded
represent the gains of the males over the females

A. THYROID -++ KIDNEY

B. THYROID + ANTE-

Cc. NORMAL + ANTERIOR

D. NORMAL -+ KIDNEY

RIOR LOBE LOBE
per cent per cent per cent per cent
9.5 13.6 46.3 36.3

96 JOHN A. LARSON

TABLE 4

Animals dying during experiment, showing days of life after operation

NUMBER OF
LITTER

NUMBER OF

LITYER DAYS OF LIFE

SEX DAYS OF LIFE SEX

Series A

1 rot 204 36 of 61

2 of 33 38 9 27

3 9 201 42 rot 117

4 of 152 43 Q 118

5 Q 189 44 Q 47

6 2 37 45 of 383i

Z. Q 44 48 9 169

8 9 46 49 of 78

9 Q 24 51 9 55

10 Q 168 52 rot 201
11 Q 192 53 Q 32
12 Q 133 54 ot 198
13 Q 24 55 of 198 i
14 of 24 56 of 1
15 of 196 57 rot 31 :
16 of 38 58 rot 55 )
17 of 44 59 fot 171
18 Q 23 60 fot 2
19 Q 76 61 o. 34
20 Q 131 62 rot 1
21 Q 23 63 9 3
22 rot J 166 65 9 191
23 fo) 195 66 Q 4
24 Q 168 — 67 Q 22
25 of 127 70 Q 8
26 Q 24 71 Q 13
27 Q 31 72 Q 217
28 Q 182 4A of 93
31 Q 61 5A Q 162 -
34 9 71 56 A fot 93

Series B

=

Q, 4 AQ 4 40

= NR wo &
rs

40 Qy 40 410 AQ,
&

FUNCTIONAL CORRELATION OF HYPOPHYSIS AND THYROID 97

doubt that the difference is due to a stimulating influence of the anterior
lobe.

It is conceivable that the results might be different for the two sexes.
I have tabulated the gains for the males and females separately in
table 3. Here again the anterior lobe exerts a beneficial action upon
the operated animals. In addition a comparison of the two sexes of
the normal animal seems to reveal an increased effect of the anterior
lobe upon the growth of males.

As stated above, tables 1 and 2 include only animals which were
alive at the end of the experiment and are therefore useful in showing
the influence of the gland substance on the rate of growth. Another
important feature of the effect of the gland substance is seen in its
influence upon the duration of life. Table 4 gives the length of life
after the operation up to death and table 5 shows the total number of
animals in each series as well as the number bee died and the per-
centage of deaths.

TABLE 5

Tabular summary of deaths

IN SERIES IN SERIES | IN SERIES IN SERIES IN SERIES
A B Cc D E
mee er... ss. sess ee 140 91 67 67 12
Number deaths................ 60 10 00 00 00
Number alive................. 80 81 67 67 12
me OGRE GOaths,....5... sac sews 42.8 10.9 0 0 0

DISCUSSION OF RESULTS

The experimental results show definitely that the administration
of the anterior lobe of the hypophysis increases the growth and pro-
longs the life of thyroidectomized rats. Also the pituitary substance
accelerates the growth of normal animals. These results at once sug-
gest two possible interpretations: either a, that this beneficial effect is
due to the direct substitution of the pituitary substance for that of the
thyroid; or b, to a favorable action of the hypophysis upén the organ-
ism as a whole. The latter alternative is supported by the increased
growth of the normal animals to which pituitary had been admin-
istered. On the other hand the difference in the two groups of operated
animals is so much greater than in the two groups of normal animals
that it hardly seems possible that this variance can be accounted for by

98 JOHN A. LARSON

a general improvement of the state of the animals but rather that it is
due in part, at least, to an actual substitution of the hypophyseal
substance for the lacking thyroid secretion. This possibility is ren-
dered more probable by the effect of the pituitary on the prolongation
of life of the operated animals. For a further discussion of these
possibilities and the literature, reference is made to the previous
paper (1). '

Since the publication of the previous paper three investigations
have appeared which have an interesting bearing. All three deal
with the effect of the administration of pituitary to thyroidectomized
frog larvae. Smith and Allen (4) working independently report nega-
tive results. Hoskins and Hoskins (3) on the other hand, not only
obtained beneficial effects but found that pituitary causes a hastened
metamorphosis. They interpret these results as indicating either a
substitution or an effect of the pituitary upon the whole organism.
Thus the results in the previous work, the first positive indication of
the possibility of a substitution, have been confirmed by the author
and by Hoskins on different animals.

The question now arises as to the cause of death of the operated
animals. Post mortems were made of all the animals and in not a
single case was there any macroscopic evidence of infection in the
operated region. Invariably the animal was extremely emaciated,
undersized and with scanty hair. The cause of death may be fairly
attributed to metabolic disturbances and interference with the normal
bodily functions due to lack of thyroid secretion.

Careful pathological examinations of three animals were made by
three different pathologists. In one, no pathological condition save
that of extreme emaciation could be detected, while in another evi-
dences of a pneumonia were present. In the last one examined but
little of significance was found, the pathologist agreeing with me that
even if the animal had died of some terminal disease such as pneumonia
the “evidence is sufficient to make a diagnosis of terminal pneumonia
developing in an animal whose resistance had been lowered by abnor-
mal disturbances of metabolism.’”’ Many of the animals which later
died had remained for months in an emaciated, dwarfed condition.
This condition did not develop till some time after the operation. As
stated above, all so-called runts were excluded at the beginning. The
retarded development then must have been due to thyroid deficiency.

Kiven if the deaths had been due to infection of any kind it would
still be necessary to show why these infections attacked only the oper-

FUNCTIONAL CORRELATION OF HYPOPHYSIS AND. THYROID 99

ated animal. Such a result would still be attributable to disturbed
metabolism or lowered resistance. Again the scattered distribution of.
deaths would contra-indicate the incidence of an infection.

That the deaths are primarily due to a thyroid rather than a para-
thyroid deficiency might be inferred from the longevity of many of the
animals after operation. As in the previous research in the cases of
very young operated animals which died two to four days after opera-
tion some evidences of tetany were observed, only three cases inthe
present experiment. However the general condition of the animals
up to death indicates a thyroid deficiency. Many of the operated
animals exhibited what might be termed myxedematous symptoms.
In addition to a dull lethargic condition there was a marked difference
in the state of the coats of the operated animals and the normals.
This condition in the operated individuals varied from dirty yellow
coats to lack of hair in many spots. Three very unusual conditions
were noted which the writer has not seen mentioned elsewhere. The
most striking was in some individuals a complete loss of hair along the
midline of the back. This gave the white rat a weird appearance, due
to the bright red skin along the back in contrast to the white coat
elsewhere. The bare place ran along the back and top of the head,
being widest at the head. This condition was detected in about
twelve of the operated animals. It appeared about the second week
after operation and disappeared within four weeks, new hair growing
and the general condition of the animal improving. This subsequent
reappearance of hair is in accordance with the findings of Cramer and
McCall (2) who state that after thyroidectomy in rats there is at first
a fall in metabolism which is later followed by a return to normal:or
nearly normal condition. In eight other operated animals there was a
petuliar dirty, pinkish coloration on the top of the head forming a V-
shaped pattern. This appeared about four months after the operation
and upon animals in poor condition, and persisted until death. In
other animals, chiefly in those operated animals which received kidney,
the hair disappeared completely in large irregular patches about the
body. As time went on this condition grew worse. Seven of the
operated rats became blind in one eye and two blind in both eyes. Of
these one was in the pituitary group and the others in those receiving
normal diet. Blindness has frequently been described in connection
with pituitary disorders.

100 JOHN A. LARSON

SUMMARY

The results of the present series of experiments show::

1. That the effect of administration of anterior lobe of the pituitary
to thyroidectomized rats tends to prolong life and to accelerate crow
This confirms the results stated in the previous paper.

2. That pituitary feeding also noticeably increases the rate of growth
in normal rats.

3. Anterior lobe seems to exert more influence upon the growth of

the normal males than that of the normal females.

The writer wishes to express his gratitude and appreciation of Prof.
S. S. Maxwell’s interest and advice during the entire research.

BIBLIOGRAPHY

(1) Larson: This Journal, 1919, xlix, 55.

(2) CrAMER AND McCauu: Quart. Journ. Exper. Physiol., 1918, xii, 81, 97.
(3) Hoskins anpD Hoskins: Endocrinology, 1920, iv, no. 1.

(4) AuLEN: Anat. Rec., 1919, xv, 353.

Ss

— a ee a ee

THE INFLUENCE OF AN “ALCOHOLIC EXTRACT OF THE
THYROID GLAND UPON POLYNEURITIC PIGEONS
AND THE METAMORPHOSIS OF TADPOLES!

EMILY C, SEAMAN_

From the Department of Experimental Medicine, Cornell University Medical
College

Received for publication May 26, 1920

-. It has been found by Eddy (1) and other workers (2) that water-

soluble vitamines can be extracted from various animal tissues. A
study was made in this laboratory to ascertain whether the thyroid
gland contains these substances and what physiological action, if any,
they possess. :

I, EFFECT OF EXTRACT ON POLYNEURITIC PIGEONS

Fresh glands were ground in a meat chopper and extracted with 95
per cent alcohol made 0.8 per cent acid with HCl. To 1000 grams of
the chopped glands were added 2 liters of the acid alcohol. The jars
containing the material were shaken in a shaking machine for two or
three hours, and allowed to stand forty-eight to seventy-two hours.
The extract was filtered, the bulk of the alcohol distilled off and the
residue evaporated to dryness before an electric fan. The dry residue ~
was dissolved in hot water, filtered, evaporated to dryness a second
time and re-dissolved in 500 cc. of hot water and filtered. This filtrate
was a dark brown solution, yielding a positive reaction with Biuret and
Millon reagent, and a strong deep blue color with the special phospho-
tungstic acid reagent, the color reaching its maximum density after
standing from two to three hours, One cubic centimeter of the extract
contained 0.05 mg. of iodine and 0.435 per cent of nitrogen. Later
attempts to isolate the vitamine fraction failed to separate it from the
one containing iodine. The vitamine quality of the extract was studied
by the curative effects on polyneuritic pigeons and, as a control on the

1 From the Johnston Livingston Fund for Experimental Therapeutics.
101

102 ; EMILY C. SEAMAN

effect of the iodine content, the “‘residue’’ as prepared in the labora- —
tory was used in corresponding doses. In every case the residue
failed to produce any, curative effect on the polyneuritic pigeons,
demonstrating that it was not the iodine content which produced
results. The extract showed rapid and marked curative properties in
every case in which the whole extract was used. Separate extracts
from different lots of glands were made. It was found when the same
proportions of glands and acid alcohol were used that the final solution
was not always of the same strength. This strength was standard-
ized according to the iodine content and the final solution either evap-
orated or diluted until 1 cc. contained 0.05 mgm. of iodine. There was
a slight variation in the nitrogen content.

The extract was divided into four portions:

a. The extract used as a whole. ;

b. Precipitation by phosphotungstic acid to isolate vitamine fraction
after the method of Funk. | 3

c. Extract treated with Lloyd’s reagent and dry powder used.

d. Portion reserved for experiments on metamorphosis of tadpoles.

Results

The whole extract when used on polyneuritic pigeons gave marked
and rapid curative results. The extract which had been treated with
phosphotungstic acid, barium sulphate, mercuric chloride and_ HS,
as outlined by Funk and Eddy (1), gave no results in any case. The
administration of the powder, obtained after shaking the extract with

2 The thyroid ‘‘residue,’’ as described in a previous communication from this
laboratory, is the non-coagulable portion of a slightly alkaline extract of the
glands after the nucleo-proteins have been removed. The hashed fresh glands are
extracted for twenty-four hours at room temperature in tap water made faintly
alkaline with NaOH. After straining through cheese-cloth, the filtrate is
treated with 10 per cent acetic acid to precipitate the nucleoproteins. The acid
filtrate is heated to boiling to remove the acid heat-coagulables; the filtrate
from this made alkaline and again brought to boiling and filtered. This filtrate
is neutralized and concentrated until 1 cc. of the filtrate contains 0.05 mg. of
iodine. This thyroid ‘‘residue’’ produces, when injected into dogs, quite defi-
nite physiological reactions. In the stomach and pancreas it appears to stimu-
late the functions believed to be performed by the terminals of the gastric and
pancreatic fibers of the vagus nerve. A neutral alcohol extract and the acidu-
lated alcohol produce closely similar physiological effects, but clinically the
acid alcohol extract does not seem as useful as either the neutral alcohol extract
or the ‘‘residue.”’

NUTRITIONAL VALUE OF ALCOHOL EXTRACT OF THYROID 103

Lloyd’s reagent, gave positive results but the improvement was slower.

The protocol for the administration of the whole extract given to

polyneuritic pigeons is as follows:

Six pigeons were fed upon polished rice for weeks until extreme symp-
toms of polyneuritis occurred. Two pigeons were fed on normal pigeon
food and used as controls. Two of the polyneuritic pigeons were given
the “residue” by mouth in amounts corresponding to the iodine con-
tent of 4 cc. of the extract. In both cases no curative effect was pro-
duced in twenty-four hours after three doses of the residue. One
pigeon who received only the residue died; the second, who received
the residue and later the extract, survived.

With the four pigeons which were given the extract marked improve-
ment was seen within six hours; demonstrating quite conclusively that
the extract possessed curative properties for polyneuritic pigeons
which the residue at this time did not possess.

Pigeon I. (Vitamine extract). Bird prostrate in cage and apparently dying.
Weight dropped from 325 grams to 296 grams.
February 23, 10:00 a.m. 4 cc. extract by mouth
2:00 p.m. Feathers smoother, bird moving head, and eyes
brighter; 2 cc. of extract by mouth
5:00 p.m. Bird crouched on legs, still unable to stand; 2 cc.
extract given by mouth .
February 24, 9:00 a.m. Walked strongly about cage; 4 ec. extract given by
mouth
2:00 p.m. Bird apparently normal; flies across cage. When
, placed on floor of room, able to walk but totters a
little after a dozen steps; 4 cc. extract given by
| mouth
February 25, 9:00 a.m. Bird walked strongly about floor of room. Dosage
discontinued
Pigeon IT, (Vitamine extract)
February 25. Weight dropped from 300 grams to 232 grams. Marked symptoms
of paralysis.
11.00 a.m. 3.5 ce. of extract
4:50 p.m. Bird raising itself on legs if touched; 2 cc. extract given
by mouth
February 26, 10:00 a.m. Marked improvement; bird perched on food pan,
feathers smooth and bird apparently normal. No
further dosage
March 15. Bird in good condition, flies and walks strongly. Put back on
normal food
Pigeon III. (Vitamine extract)

February 25. Weight dropped from 257 grams to 221 grams. General paralysis.

11:00 a.m. 2 cc. extract given by mouth
4:30 p.m. 4 ce. extract given by mouth

104 EMILY C. SEAMAN

February 26, 10:00 a.m. Marked improvement; 2 cc. extract given by mouth
3:00 p.m. Bird normal; 2 cc, extract given by mouth
February 27. Bird in such good condition, dosage discontinued
Pigeon IV. (Vitamine extract)
February 27. Weight dropped from 295 grams to 213 grams. Total paralysis.
9:00 a.m. 4 cc. extract given by mouth 1
2:00 p.m. Bird crouched on legs, unable to stand; 2 ce. extract
given by mouth
February 28, 9:00 a.m. Bird walking about cage; 2 cc. extract given by mouth
March 1. Bird walks strongly with head erect
9:00 a.m. 2 ce. extract given by mouth
2:00 p.m. Bird normal; dosage discontinued
_. Pigeon V. (Residue)
February 26. Weight dropped from 312 grams to 241 grams. Extreme symptoms
of polyneuritis.
10:00 a.m. 4 ce. residue given
February 27, 9:00 a.m. Bird dead
Pigeon VI. (Residue and extract) eats
March 1. Weight dropped from 297 grams to 231 grams. Extreme symptoms
of polyneuritis. Be
9:00 a.m. 4 ec. residue given by mouth
2:00 p.m. No results; 2 cc. residue by mouth
5:00 p.m. No improvement; 2 ce. residue by mouth; 4 ce. extract
given by mouth
March 2. 9:00 a.m. Bird living and crouched on legs; 2 ce. extract given
11:00 a.m. 2 ce. extract given
2:00 p.m. Bird improved but totters about cage
5:00 p.m. 2 ec. extract given
March 3. 9:00-a.m. Bird tottering about cage;. decidedly improved but
not normal; 4 ec. extract given
2:00 p.m. Bird walking normally; 2 cc. extract used.

Note. This bird never regained its normal vigor. It continued to improve
and walked and flew about its cage but much of the time remained crouched
down. After ten days it was put back on normal food, but even then showed
less vitality than the other birds.

Conclusions

From. the above experiment with polyneuritic pigeons it was demon-
strated. that the water extract from the thyroid glands made by 95
per cent alcohol made 0.8 per cent acid with HCl possessed the same
curative property which has been attributed to water-soluble vitamines,

‘es

Lon a
Brey

Ih ean. * § & ped en

NUTRITIONAL VALUE OF ALCOHOL EXTRACT OF THYROID 105

Il. EFFECT OF ACID ALCOHOLIC THYROID EXTRACT ON METAMORPHOSIS
OF TADPOLES

It has been found by Gudernatsch (3) and others that the meta-
morphosis of young tadpoles is hastened by feeding thyroid glands.
Gudernatsch found that feeding the whole gland produced the hind
legs in nine days after feeding, and the fore legs two days later. When
these thyroid-fed tadpoles put out their anterior limbs and began to
shorten their tails, they were eighteen to twenty days old. Swingle
(4) obtained similar results by feeding iodoform, potassium iodide and
iodine crystals. His most rapid results were obtained from the iodine
crystals, limb buds appearing ‘‘in a few days.’’ More tardy results
were obtained with iodoform and potassium iodide.

It is claimed by Swingle and other workers that iodine is the active
principle which hastens the metamorphosis of tadpoles, and this theory
is generally accepted. In the following experiments done in this
laboratory with the acid alcoholic extract, controls were made with
iodine erystals, potassium iodide, thyroid nucleoprotein and the thy-
roid “‘residue”’ in amounts with corresponding iodine content.

Rana pipiens, approximately 15 mm. in length, were used and kept
in tap water which was changed every day. To the fresh water were
added the various substances in doses corresponding to 0.10 mg. of
iodine a day, Each jar contained fifteen or twenty tadpoles and the
experiments were repeated four times. A total of not less than sixty
tadpoles was used for each substance, but out of this number at least
fifteen died before complete metamorphosis. By far the most rapid
results were obtained with the thyroid extract prepared by extracting
the glands with 95 per cent alcohol made 0.8 per cent acid with HCl,
as described in part I, and which proved to have the property of water-
soluble vitamines. The limb buds appeared within eight hours, and
complete metamorphosis with the bulging eyes and disappearance of
the tail within four days. By the time complete metamorphosis had
occurred in these specimens, only slight signs of limb buds appeared
in the specimens treated with the iodine crystals, and no change oc-
curred in the specimens fed the other substances. Negative results
were obtained with the nucleoprotein and ‘“‘residue,” after continuing
the feeding nearly three weeks. As the ‘‘residue” and alcoholic extraet
contained the same amount of iodine (1 cc. containing 0.05 mg.
iodine) and no results were obtained with the residue or nucleoproteins,
it is evident that the iodine content alone is not the only factor. It is

7

Fig. 5. Four days
Fig. 6. Five days
Fig. 7. Control

Fig. 1. Less than twenty-four hours
Fig. 2. Thirty-six hours
Fig, 3. Forty-eight hours

Fig. 4. Three days
106

NUTRITIONAL VALUE OF ALCOHOL EXTRACT OF THYROID 107

very probable that the iodine of the thyroid gland persists in more

than one combination. The alcoholic extract may have contained,
and according to Kendall (5) did contain, a substance not present in

the residue, but Kendall did not obtain any such rapid results in the

metamorphosis of tadpoles after feeding them ‘‘thyroxin”’ as took place

with this alcoholic extract.
The following table represents the condition of the specimens when
the experiment was performed on tadpoles approximately ten days

after development from the egg. All the specimens did not develop

in exact uniformity of time, there being a lag of four to six hours in

_ some, but all showed complete metamorphosis by the end of the fourth

day.
Table of results
=
ee | 38 =
HOURS CONTROL . CRYSTALS @ : a : a ACID ALCOHOLIC EXTRACT
, o" | eee 8
12 Appearance of limb buds
36 Growth of legs
48 : Emaciation begun.
Growth stopped
72 No increase Tail beginning to disap-
in growth pear
96 | Growth | Limb buds Complete metamorphosis
See figures. |

Effect of age on metamorphosis

The above experiment was repeated with four sets of tadpoles-of the
same age but hatched from eggs procured from different stores.» The
results were the same in each experiment.

When the experiment was repeated two weeks later, when the
tadpoles were a month old, the development was slightly retarded,
complete metamorphosis occurring not later than six days. In the
experiment with tadpoles two months after they had been hatched,
complete metamorphosis took place within ten days. Fewer specimens
died when the older tadpoles were used.

GENERAL CONCLUSIONS

It has been found that the thyroid gland, as well as other tissues of
the body, contains water-soluble vitamines, the property being demon-
strated by the curative action on polyneuritic pigeons.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 1

108 EMILY C. SEAMAN

It was also found that the acid alcoholic extract had a marked —
accelerating action on the metamorphosis of tadpoles, the most rapid
- development occurring in specimens two weeks after they had been _
hatched, and slightly slower development as the age increased.

BIBLIOGRAPHY

(1) Eppy: Journ. Biol. Chem., 1916, xxvii, 113.

(2) OsBORNE AND MENDEL: Journ. Biol. Chem., 1917, xxxii, 309. .
(3) GupERNATscH: Amer. Journ. Anat., 1913-14, xv, 431.

(4) Swinete: Endocrinology, 1918, ii, 283.

(5) Kenpauu: Lecture delivered before the Harvey Society, 1919.

se

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a a ee ee ey

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THE ALKALI RESERVE IN EXPERIMENTAL SURGICAL
SHOCK

BERNARD RAYMUND
From the Hull Physiological Laboratory, University of Chicago

Received for publication April 26, 1920
INTRODUCTION

The work herein reported was taken up in response to suggestions
made by the Sub-Committee on Shock of the Committee on Physi-

ology, National Research Council. Since the question of acidosis as a

factor in shock became of such general interest, it seemed desirable to
devote attention first to this, and leave for further consideration other
factors, perhaps of equal importance. Hence this paper will be con-
fined to the presentation of such data as have been accumulated in
this laboratory with regard to the alkali reserve in shock.

_ Attention was first directed to the possible réle of acidosis in the
condition of shock by the claim of Spiro (1) in 1902 that the admin-
istration of hydrochloric acid to rabbits, or the intravenous injection
of sodium hydrogen phosphate in dogs, was followed by “shock”
effects. Howell (2) stated in 1903 that the intravenous injection of a
0.5 per cent sodium carbonate solution had a beneficial effect upon
animals in shock but took the view (3) that this was due primarily to
its action upon the heart. Apparently the question of its neutralizing
effects did not enter into consideration. Dawson (4), using sodium
bicarbonate after hemorrhage, reached the same conclusion. Seelig,
Tierney and Rodenbaugh (5) using repeated small injections of sodium
acid phosphate, stated that they were able to bring about a condition
of rather profound shock in dogs, as judged by blood pressure readings.
On the other hand, intravenous injections of sodium bicarbonate proved
of decided benefit to animals in shock, but the authors judged that it .
acted directly upon the heart muscle. Yandell Henderson in 1910 (6)
stated it to be his belief that the fatally rapid transudation of fluid
from the veins in shock was due to the action of acidosis bodies upon
the proteins of the tissues, causing them to imbibe water. He admitted

109

110 BERNARD RAYMUND

that the process was probably very complex. Crile attempted to show
in 1915 (7) by histological methods that the effects of the injection of
sodium acid phosphate upon brain, liver and adrenal cells were in all
respects similar to those found after death from surgical shock, and
that such post-operative lesions could be prevented by the previous
injection of sodium bicarbonate. He declared (8) that the bases of

the body are in many cases exhausted by acid products developed

during operations and recovery thereby rendered impossible. Corbett
however (9) was unable to demonstrate any increase in urinary ammonia
during traumatic shock, nor any increase in the hydrogen ion concen-
tration of the blood, and concluded that the relation of acidosis to shock
was not clear.

At the time this work was begun, Cannon (10) had stated in an in-
formal memorandum to the Committee on Physiology of the National
Research Council that ‘it is probable that the acidosis is causally re-
lated to the clinical condition presented inshock. . . Arterial pres-
sure falls (vascular walls relax and cardiac contractions become less
vigorous) when acid is injected experimentally into the blood vessels.”

Furthermore ‘‘a fairly close inverse relation exists between the degree

of acidosis and the height of the arterial blood pressure—the more
marked the acidosis the lower the pressure” and ‘‘as the degree of
acidosis increases the general condition the patient becomes worse
—the greatest degree is found near death.

Early in the next year Cannon (11) reported work undertaken with
Bayliss which seemed to show that “if the alkaline reserve is dimin-
ished by very slow injection of acid (N/2 HCl) into a vein, there is a

fall of blood pressure to the shock level after the alkali reserve reaches -

a certain critical point (88 per cent in the cat). . . . Bruising or
mashing the muscles of the hind legs is followed by a progressive fall
in alkali reserve, and when the critical point is reached the blood pres-
sure falls to the shock level.’”’ However in attempting to repeat the

experiments with acid Bayliss (12) was unable to obtain the fall —

“except in cats obviously unhealthy.”

Basing his assumptions upon this previous work Cannon (13) sug-
gested that an excessive production after wounds of some acid metab-
olite and its distribution through the circulation might cause dilata-
tion of the blood vessels, excessive lymph formation in all parts of the

body and thus bring on exemia or shock. Similarly Henderson, Prince -

and Haggard (14) found that the carbon dioxide capacity of the whole
blood could be greatly reduced by shock procedures and ventured the

eee ee

ALKALI RESERVE IN SURGICAL SHOCK 11]

opinion that the acidosis of shock and of ether anesthesia is compensa-
tory to, or a result of, the acapnia produced by hyperpnoea. In his
first paper in 1918 Cannon (15) while still emphasizing the importance —
of acidosis in shock does not assign to it a causative réle, merely stating
that the drop in alkali reserve under anesthesia is greater the less the
original margin of safety in the individual, and that such cases showed
an ominous fall of blood pressure when operated, especially if at the
time in a state bordering upon shock. In subsequent papers (16),
(17) he stated that ‘‘as the blood pressure falls there is a loss of the
alkali reserve of the blood (acidosis) roughly corresponding to the drop
in pressure.” In other words, acidosis had.been relegated to a position
of secondary importance, although Cannon still maintained that the
injection of sodium bicarbonate was of prophylactic value in cases of
shock. »

Practically the same conclusion as to the importance of acidosis in
shock had been reached independently by Guthrie (18) and McEllroy
(19), although Henderson and Haggard (20), (21) stated that if in dogs
under ether anesthesia ‘‘the level of CQ. and alkali is reduced below
the critical value lying between 33 and 36 volumes per cent of COs,
“a condition of general depression of all functions results. . . . If
the marked resistance to further depletion of the carbon dioxide capa-
city, which occurs at the critical level, is broken down and the CO,
capacity further reduced, the result is a condition of vital depression
from which the subject does not spontaneously recover. This condi-
tion may be termed acapnial shock.’”’ A low carbon dioxide capacity
they regard as one of the definite criteria of shock. Subsequently
Gesell] (22) has shown that after either hemorrhage or visceral trauma,
the reduction in the alkali reserve of the blood tends to be compensated
for by a transfer of alkali from the tissues, and that the injection of
N/2 HCl into normal animals is followed by indications of cardiac
failure following a rise in arterial blood pressure. In this case also
there is a passage of alkali from the tissues into the blood, so that suc-
cessive doses of acid become less efficient in lowering the alkali reserve.
Likewise Gasser and Erlanger (23) state that the alkali reserve does
not show a sharp decline until after the animal is in a relatively advanced
stage of shock, and that the reduced CO, capacity is theresult, and not
the cause of the fall in the arterial pressure.

tig BERNARD RAYMUND

CRITERIA OF SURGICAL SHOCK

Although the criteria for surgical shock are well established and

quite generally recognized, most of the workers. in the problem have
shown a tendency to set some certain arterial blood pressure as the
shock level, so-called, and to use this as a basis for determining at
what time the animal passes into a state of shock, and to what degree.
That the level of arterial pressure may be quite high when an animal
is in true shock, and that the converse may just as readily be true, was
pointed out by Meltzer (24) in 1908. This conception is given addi-
tional emphasis by Mann (25). He proposes as criteria of shock the
following signs, which have become generally adopted: a, loss of sen-
sibility; b, pallor of the mucous membranes; c, small, weak pulse; d, ir-
regular, rapid, shallow or gasping respiration; and e, markedly lowered
blood pressure (one-third to one-fourth of original level).

In view of the uncertainty as to what really constitutes a shock level
of blood pressure, it seemed desirable on the whole to adopt these
eriteria as being the most satisfactory we possess, and in the experi-
- ments: described in this paper they were constantly used in deciding

whether or not the animal was in true shock. More emphasis was

placed upon the first sign than upon the others, and in addition it was
found that Guthrie’s respiratory sign (26) often proved of value... This
consists in a markediy prolonged inspiratory pause, and while not
always present, is, when it appears, a reliable index, marking the begin-
ning of the terminal respiratory paralysis.

EXPERIMENTAL PROCEDURE

Itther was used exclusively as the anesthetic, being given intratrach-
eally and at such rate that the lid reflex was not abolished at any time
during the experiment. In some experiments the patellar reflex was
also used as an index of the depth of narcosis, although it was evident
that it is neither as sensitive to the effects of ether nor, in the con-
scious dog, to those of prolonged low blood pressure, as the lid reflex.
Thus in one experiment without ether and under local anesthesia, it
was elicited after the corneal reflex had disappeared and when the
heart was only beating with each occasional inspiration. That with
the ordinary equipment it was possible in this way to keep the effects
of the anesthetic at a minimum is shown in the protracted control
experiments to be described later.

——

ALKALI RESERVE IN SURGICAL SHOCK 113

In order to obtain uniform results the dogs were not fed for eighteen
hours previous to the experiment. Such exceptions as occur are noted.
Determinations of the alkali reserve were made by Van Slyke’s direct
method (27). The blood was drawn either from the saphenous vein
or, when the dogs were under ether anesthesia, from the femoral or
carotid artery, oxalated, and immediately centrifuged for ten minutes,
usually in a stoppered tube. Precautions to prevent the escape of
carbon dioxide, such as the use of paraffin oil, were not taken. Peters
(28) has stated that if the blood is centrifuged within fifteen minutes,

the shifting of the carbonate into the erythrocytes does not occur, when
: the surface of the blood exposed to the air is small. In every instance
the blood used in these determinations was centrifuged not more than
ten minutes after being drawn. The plasma was saturated with alveo-
lar air and analyzed in the usual manner. In a few cases it was allowed
to stand over night in the refrigerator, but the analyses were checked
by preserving a sample that had been analyzed beforehand and rede-

termining the alkali reserve the next morning. In no case could any

change be detected. The results of the analyses, made in duplicate,

were calculated to volumes per cent carbon dioxide at 0°, 760 mm.
__ pressure, on the basis of saturation at 37°C., according to Van Slyke’s
_ formula. When parallel determinations were made on the whole blood
the sample was divided into two portions, and while one was being
centrifuged the other was saturated and analyzed. The reading so
obtained was corrected for oxygen, etc., by the use of a few drops of 10
per cent sodium hydroxide solution.

Blood pressure and respiration were recorded in the usual manner.
In most of the experiments shock was induced by a uniform technic,
essentially that adopted by all workers in the field. After the blood
pressure had reached a fairly stationary level, the abdomen was opened,
the ether discontinued, the intestines removed and handled gently and
continuously for fifteen minutes. Whereupon the guts were at once
replaced and the abdomen closed securely with hemostats. From this
point on the animal was kept warm, either by an electric light or by a
heating pad, in order to eliminate the factor of lowered body tempera-
ture. If after a certain lapse of time, preferably an hour, the blood
pressure showed no permanent lowering, another fifteen-minute period
of trauma was resorted to as before. Occasionally the trauma had to
be repeated several times before the animal showed any indication of
shock; but except in one or two of the earlier experiments, the intes-
tines were always replaced and not allowed to lie exposed to the air:

114 BERNARD RAYMUND

By closing the abdomen the degree of trauma can be much more accu-
rately gauged, and there is furthermore no interference with the muscles
of respiration. :

In highly resistant animals there was sometimes resort to traction on
the kidney, trauma to the liver or to the urinary bladder. These
measures, especially the first, always caused a great disturbance in
blood pressure. Such measures as clamping the inferior vena cava,
occlusion of the portal system or of the abdominal aorta (29), (30),
(31), seemed unnecessarily severe and were not used. Moderate trauma
to the intestines in the manner described is sufficient to produce what
appear to be the main clinical signs of shock, and the method can be
controlled with fair accuracy.

In five of the experiments the procedure described above was followed,
except that the entire gut was inflated with moist air at 40°C. to a pres-
sure somewhat above the systolic blood pressure for a period of ten
minutes. Five minutes were allowed for tying off the stomach and
rectum, inserting the cannula in the caecum, and for deflating the gut
and replacing it in the abdominal cavity. The guts were kept covered
with hot towels throughout the entire period. This method is by far
the most satisfactory of any that were tried, and makes it possible to
control the degree of trauma with almost quantitative accuracy. By
abolishing the circulation in the gut, asphyxia of the vessel walls and
probably of the smooth muscle cells is set up, yet on the other hand
the trauma is not so severe as to abolish the myenteric reflex. This is
a very desirable feature, since it is doubtful if operative procedures in
man ever completely do away with the reflex.

In seven experiments the method of Mann (32) was followed in in-
ducing shock. All the structures in each limb with the exception of
the supplying artery were tied off with iron wire ligatures. In this
method the trauma is continuous throughout the experiment, but is
possibly less severe than that involved in the foregoing procedures.
Hence it is easier to follow the course of shock, and in this respect the
method possesses certain advantages.

TYPES OF SHOCK

Naturally, if it is desired to compare the susceptibility of animals to
trauma under various conditions, some certain basis for comparison
must be found. By many workers in the problem the shock level of
arterial blood pressure has been set at 50 mm. of mercury. But since

“i tee ale ce MST a See a eA * 7 ios Nm “a
ete ee nr ee er ae < * ; he .
4 : 2 : ee es By pi Bois mm 2 = » -_, Yo <a, e :
; ea a a St) Se eed Yee ee oN
= Se eee Pe

ALKALI RESERVE IN SURGICAL SHOCK 115

this figure is admittedly arbitrary, and since its adoption is not uni-
versal, it seemed inadvisable to compare the resistance of animals to
trauma on the basis of the length of time, after beginning shock proce-
dures, that the blood pressure remained above this level. As the only
alternative it was necessary to select as a criterion of susceptibility the
survival time of the animal, that is, the length of time it remained alive
under the conditions of the experiment, after beginning traumatization.

Using this asa basis it was found that the animals studied fell into
four general classes, that is, there were in all four types of shock, so-
called. These were as follows:

Type I. With one or two exceptions, in the dogs showing this type
of shock, only one fifteen-minute period of trauma was needed to set
up a serious condition. Of a total of thirty-one dogs, five, or 16.1 per
cent, showed type I.

SRY PUPVOWOL UIC, nk. sk a ee ew dane ee 2 hours, 10 minutes
Minimum survival time................... Pat Fo diene 0 hour, 52 minutes
Mean....... PUP. vis es ea: he Ue Uae air Meee ects 1 hour, 27 minutes

Type II. Dogs showing this type of shock had to be given two
fifteen-minute periods of trauma, but not more. Ideally these two

periods should have been an hour apart, but this rule could not be

followed very closely. When shock was produced by Mann’s method
(see above) the trauma was of course continuous. However I have
included two.of the dogs so traumatized in this class, since the survival
time of each was found to lie within the proper limits. Of a total of
thirty-one dogs, eight, or 25.9 per cent, showed type II.

Peretti) BUEVIVG! CIC. 0... 0... ws es cs eee ene db 2 hours, 47 minutes
PEI MULE AW EE CINE vans ce ck as os the nee ce 1 hour, 20 minutes
ER SEAS. tah OR a ea a at ka 2 hours, 9 minutes

It will be seen that type I and type II overlap somewhat and that
the distinction between the two is in a measure arbitrary. This how-
ever is not the case with the last two types, which are clearly set off
from the first two and from each other.

Type III. In this type are included the dogs which required as a
rule more than two periods of trauma to seriously alter their condition.
Of a total of thirty-one dogs, thirteen, or 41.9 per cent, showed type
III. This includes three dogs traumatized by ligating the four limbs.
All of these dogs died in true shock except one (exper. 90) which died
of accidental hemorrhage. However this dog showed the same rate

116. BERNARD RAYMUND

of fall of blood pressure as the others in this class, previous to hemor-
rhage, and so it is included with them.

Maximum survivah time... : 222) cme en ceils 5 hours, 32 minutes
Minimiush survival tame... i. 0. «aan eae wae ey 2 hours, 56 minutes

Mean oh STA ore wo BG eee eee ae 4 hours, 15 minutes

Type IV. Since the dogs in this class did not show typical shock,
it is perhaps a misnomer to characterize this as one of the four types.
However for the purpose of convenience this misnomer will be allowed
to stand. Of a total of thirty-one dogs, five, or 16.1 per cent, showed
type IV. This includes two dogs traumatized by Mann’s method.

Maximum survival time. . ,...00/. 65 aie.c Suche + + wanes owe 7 hours, 52 minutes
Minimunt survival time... 5 2c ah ve cna cei 6 hours, 46 minutes
Mean coe As oe os ee RE ee 7 hours, 15 minutes

All of the dogs showing the first two types of shock gave a profound
fall in blood pressure within thirty minutes after beginning the initial
trauma. I had at first thought of using the degree of such fall as a
criterion of susceptibility, and of determining the types of shock upon
this basis. But the fall varied between such wide limits, 2 mm. to 102
mm., that this was impossible. Of the dogs exhibiting type III shock,
all but three showed a fall in blood pressure (2 mm. to 49 mm.) thirty
minutes after beginning initial trauma, while these three gave a rise
in pressure (2 mm. to 36 mm.). However on classifying the data it
was found that, without exception, every dog showing type IV shock
gave a rise in pressure (3 mm. to 21 mm.) at the end of the thirty-min-
ute period. This seems very significant, even granting that in type
III shock such a rise of pressure may be seen. It shows that some
notion of the course of shock may be obtained thus early in the experi-
ment. This time rather than some other was chosen at which to make
the observations on blood pressure, for the reason that at this time
the wide fluctuations in blood pressure set up by the trauma have
disappeared, while general and widespread damage to the circulation
presumably has not had opportunity to occur.

The data summarized above are shown in graphical form in figure 1.-

In the figure the heavily shaded areas represent periods wherein the ©
intestines were traumatized. In the experiments in which the four
limbs were tied off the block representing the survival time is left
unshaded. In three such experiments where the ligatures were removed
before the close of the experiment (indicated by the letter A in the

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118 7 BERNARD RAYMUND

figure) the period after removal is indicated by cross-shading. Further,
in these experiments trauma was considered to have been begun when
both hind-legs had been ligatured off and before the fore-legs had
been ligatured. In three early experiments in which the guts were
allowed to lie exposed, such periods of exposure are indicated in the
figure by the letter Z. Prolonged stimulation of the central end
of the vagus is indicated by the letter V, either in conjunction ©
with trauma to the viscera, as in experiment 69, or alone. In experi-
ment 75 where the dog was killed by such stimulation while the arterial
blood pressure was still 123 mm., the fact is indicated by the letters VX.
The letter X, experiment 66, indicates that the dog was killed with
ether in order to bring the experiment to a close. It will be seen in
column 4 ofthe figure that the length of the preliminary etherization
period varied from one hour or less to more than four hours and fifteen
minutes in one experiment. However it is improbable that this had
much effect upon the survival of the animal (see control experiments,
table 1, below). : |

Five experiments were performed with Dr. A. C. Ivy under local
anesthesia, without the use of ether, aseptic precautions being observed.
Blood pressure and respiration were recorded in three of these experi-
ments; in all the plasma alkalies, red cell count, hemoglobin and cor-
puscle volume were determined at intervals. Only one of the dogs was
strictly normal. The others had had the vagi and splanchnies severed
‘above the diaphragm and the coeliac ganglion extirpated under asepsis
some two or three weeks previously. However these with one excep-
tion have been included in the analysis given above. Since the experi-
ments were few in number it is not proposed here to draw any conclu-
sions from them save as regards survival time.

THE ALKALI RESERVE IN NORMAL DOGS NOT SUBJECT TO ETHER

Sixty-eight determinations of the plasma alkalies were made on forty |
dogs not subjected to ether. The results may be summarized as follows:

Maximum alkali reserve............ 59.5 volumes per cent (exper. 68)
Minimum alkali reserve............. 32.4 per cent (expers. 4 and 20)
WIGBR oiice oo och ces oa oe 43.4 per cent (68 determinations)
Mean D6aGrt TARO ok. ication 105 -

Mean respiration, .. 0... secs s00 sees 6 31

al ae

ALKALI RESERVE IN SURGICAL SHOCK 119

The dog showing the maximum alkali reserve had been fed.
In fourteen dogs, eighteen determinations were made of the alkali

reserve of the whole blood, as follows:

Maximum alkali reserve..................... 55.6 per cent (exper. 101)
Minimum alkali reserve..........0.......... 36.6 per cent (exper. 92)
Mean, whole blood.......................... 47.0 volumes per cent

RT. PALO TEs as HV a 108
MMU TMOITSUION . 00. ses ceca cee cece tcc ees 23

None of these dogs had been fed. What effect feeding might have
on the alkali reserve was not definitely determined, but from the data
in hand there would seem to be no intimate relationship: that is, the

_ alkali reserve did not invariably increase after feeding. Of course this

would depend largely on the character of the food.

The figures given above agree with those found by Morriss (33) for
plasma, but not with those cited by McEllroy (19). Henderson,
Prince and Haggard (14) give 48 volumes per cent as the normal

reserve of the whole blood in the dog.

THE ALKALI RESERVE IN DOGS UNDER CONTINUOUS ETHER ANESTHESIA

In determining the effect of the anesthetic upon the alkali reserve,
arterial plasma was used, except in the first two experiments where the
analysis was made upon the plasma of venous blood throughout. Al-
though comparison is made here between the alkali reserve of the normal
venous plasma and that of the arterial plasma after etherization, the
error introduced thereby is probably slight and no doubt constant.
The results are given in table 1.

For the sake of convenience the etherization may be divided into
four periods of unequal length, viz.: period 1; first 45 minutes of ether
anesthesia; period 2, 45 minutes to 2 hours; period 3, 2 to 4 hours;
period 4, beyond 4 hours. On this basis the data presented in the
following table may be summarized briefly as follows: |

Period 1. First 45 minutes of ether

Number of determinations............ ae
Maximum alkali reserve........ epee ae 41.9 per cent (experiment 43)
Minimum alkali reserve.............. 19.8 per cent (experiment 54)
NES aia oh caw a gh 6 oun 33.9 volumes per cent

Maximum fall from normal........... 25.1 per cent (experiment 54)

120 BERNARD RAYMUND
Minimunt falls o Feu avis eae 1.3 per cent (experiment 46)
Megn fall. is cts avai serie Pen. 10.5 per cent ©
Mean heart rate: ois os eee Pi 189
Mean increase in same............... 91
Mean respiration..... ia Siecd-4.> Coa ane 64
Mean increase in same............... 31
;
Period 2. Ether, 45 minutes to 2 hours

ee

Number of determinations............ 18
Maximum alkali reserve.............. 35.3 per cent (exper. 30)
Minimum alkali reserve.............. 16.4 per cent (see note 2, above)
PROBL «cg ones ies as sae soa SURED ON cami 28.0 volumes per cent
Maximum fall from normal........... 34.4 per cent (see note 3, above)
Minimum fall from normal....... .... 4.0 per cent (exper. 30)

Mean fabbii cin. daccuie seed RD 15.8 volumes per cent.

Mean heart rate. ....... cic +054 $a : 151
Mean increase in same............... 45
Mean respiration............. cee tune | 69

Mean increase in same............... 26

Maximum alkali reserve.............. 31.9 per cent (exper. 96)
Minimum alkali reserve.............. 19.5 per cent (exper. 68)

DROOL PUTIN PES RT GE 26.1 volumes per cent

Maximum fall from normal........... 40.0 per cent (exper. 68)
Minimum fall from normal........... 13.8 per cent (exper. 96)

PB OTT CR oe oon aed 4s ss he ha racant rt 22.5 volumes per cent

WIOGN HOATE PALO... Sowa dy as cesaves 159
Mean increase in same............... 53
Mean respiration..............++- peeve 62
Mean increase in same............... 31

Period 4, Ether, 4 hours plus

(See data for experiments 9f, 92, 96, table 1)

THE ALKALI RESERVE IN DOGS AFTER TRAUMA UNDER LOCAL ANESTHESIA.
NO ETHER

The effects of visceral trauma upon the plasma alkalies when only
local anesthesia was used are summarized below in table 2. These
experiments were performed by Dr. A. C. Ivy and the writer upon one

ALKALI RESERVE IN SURGICAL SHOCK 121

TABLE 1

Showing the effects of ether anesthesia upon the alkali reserve of the plasma in dogs

; . sane
endear aukant | FatuFRom|> °°") gearr | ResPrRa-
Wah ni SO EtON RESERVE | NORMAL RATE TION
Dias- :
tolic |Systolic
per cent per cent mm, mm,
y {| Normal 50.7 90 28
\| 15 min. ether 37.6 13.1 210 70
10 Normal 47.5 ° 90 23
15 min. ether 40.4 7.1 264 60
W* Normal 45.7 98 28
{| 15 min. ether 38.1 7.6 248 60
46 {| Normal 38.5 104 16 |
20 min. ether 37 .2 1.3 168 48
‘a (| Normal 45 Sit: 72 12
|| 25 min. ether 35.6 | 10.2 132 60
19* Normal 44.3 102 24
35 min. ether 38.1 6.2 244 40
oh {| Normal 40.9 80 6
{ 38 min. ether 30.5 10.4 146 | 155} 140 56
70 Normal 51.0 60 16
45 min. ether 36 .2 14.8 140 150 132 56 ©
egg {| Normal 44.9 | | 120 24
50 min. ether 30.0 14.9 123 131 150 64
4 29 Normal 36.6 75 24
55 min. ether 23.3 13.3 102 109 96 72
66 Normal 51.6
55 min. ether 34.2 17.4 126 | 188 122 66
5G Normal 37.6 78 16
56 min. ether 27.1 10.5 111 118 150 96
3] '}| Normal, 49.4 96 15
75 min. ether 28.7 20.7 94 104 132 44

122 BERNARD RAYMUND
TABLE 1—Concluded
ARTERIAL <
aS BLOOD PRESSURE
“Mans | companies” | cseees | nanan = | ae
ie: Dias- :
tolin Systolic
per cent per cent mm. mm.
(| Normal
55¢ 4| 30 min. ether 27.7 204 44
|| 70 min. ether 16.4 60 174 84
Normal 39.3 132 56
30 5 min. ether . 30.0 9.3 228 66
75 min. ether 3D.0 4.0 180 38
Normal 44.9 108 20
54 25 min. ether 19.8 25.1 180 52
90 min. ether 28 .2 16.7 115 124 144 76
Normal 43 .0 72 56
38 45 min. ether 31.5 11.5 186 72
90 min. ether 24.0 19.0 147} 153 180 72
Normal 40.0 130 80
60 50 min. ether 34.3 5.7 124 | 130] +144 108
85 min. ether 29.6 10.4 118 | 125 126 104
Normal 39.3 108 174
40 10 min. ether 31.9 7.4 144 68
109 min. ether 28 .2 bk ge | 151 | 154 -141 102
Normal 44.9 144 48
43 45 min. ether 41.9 3.0 144 48
150 min. ether 32.8 12:1 134 | 1388 192 76
Normal 40.4 120 20
89 43 min. ether 31.9 8.5 109 | 121 140 72
110 min. ether 26 .2 14.2 109 | 125 150 68
Normal 45.3 102 20
14* 7 min. ether 28.1 17.2 192 92
56 min. ether 31.9 13.4 162 88
113 min. ether 29.0 16.3 168 76
Normal 41.9 | 132 35°
67 58 min. ether 29.3 12:8 92 96 132 56
120 min. ether 23 7 18.2 94 97 180 38
180 min. ether 21.4 20.5 55 59 180 64

——

ALKALI RESERVE IN SURGICAL SHOCK 123

TABLE 1—Continued

i : ARTERIAL
Pe eres | ee ee |
a jt Systolic
| per cent per cent mm. mm,

Normal 59.5 84 32

cae 40 min. ether 35.7 23.8 98 104
68i {| 88 min. ether 27.1 34.4 SQ) 604: tae 84
| 145 min. ether 19.5 40.0 54} 58] 180 52
| 205 min. ether 22.3 37.2 26 30 102 6
‘| Normal 51.0 88 16
50 min. ether a ef 17.3 128: |. 131 124 40
112 min. ether |. 30.5 20.5 119 | 122] 176 40
ie 171 min. ether 29.0 22.0 | 119] 121 148 64
91 = 4| 230 min. ether 31.5 19.5 124] 126) 144 60
295 min. ether 35.3 15.7 126} 128] 106 48
350 min. ether 33.4°|. 17.6 119 | 122] 120 58
490 min. ether 26.3 24.7 77 79 148 48

|| 595 min. ether | 25.4 25.6 37 | 38] Dying

‘| Normal 45.7 : 108 44
93 min. ether 30.0 15.7 108 |} 118 120 40
213 min. ether 287, 16.0 | 82] 102] 128 52
92§ {| 349 min. ether 24.0 pa We 94} 100 198) | 4g Be
422 min. ether | 26.8 18.9 86| 90] 164 — 52
530 min. ether 26.8 18.9 68 | 72] 160 68
| 650 min. ether 30.9 14.8 58 | 62| 176 72
‘| Normal 45.7 120 28
60 min. ether 34.7 Bh hy 144 60
120 min. ether 33.8 11.9 128 48
240 min. ether 31.9 13.8 160 76
96§ | 300 min. ether 33.8 11.9 156 76
360 min. ether 35.7 10.0 160 88
420 min. ether 36.07 10.0 168 84
480 min. ether 35.7 10.0 184 92
600 min. ether 31.9 13.8 160 120

_ * Experiments 9, 10, 11, 12 and 14 performed on the same dog.

+ Effects of over-etherization shown in experiment 55.

t Accidental hemorrhage in experiment 68.

§ Experiments 92 and 96 were aseptic ether controls performed with Mr. C.
F. G. Brown assisting. Both dogs lived 12 hours after concluding the experiment.

THE AMERICAN JOURNAL OF PHYSIOLOGY, vor 53, No. 1

124 BERNARD RAYMUND

normal dog and upon four dogs that had had the splanchnics and vagi

sectioned in the thorax and the coeliac ganglion evulsated. The
operations were performed aseptically two to three weeks previous to
the time at which the experiments were conducted, and the dogs had
recovered completely.

TABLE 2

Showing the effects of visceral trauma upon the alkali reserve of the plasma in dogs
Local anesthetic. No ether

vans, ¢ [SETS mane
EXPERIMENT CONDITION ALKALI FROM BLOOD HEART RESPI- TEMPER-
NUMBER ie RESERVE NORMAL Grn ee: RATE RATION ATURE
per cent | per cent mm. "Cs
78 {| Normal 41.9 96 186 16 | 37.0
Typel || 46 minutes* | 32.4 9.5 60T 60 12 39.1
Normal 40.9 116 32 39.6
, tI 57 minutes* 35.3 5.6 162 26 39.7
o 120 minutes* | 23.0 |. 17.9 a 156 20 | 39.0
Normal 40.0 124 80 14] 39.1
T am 53 minutes* 25.8 14.2 48 144 34 38.7
os 93 minutes* | 22.1 | 17.9 | 42+ | 124 | 20 | 38.6
Normal 37.2 164 140 12 38.3
33 minutes* 30.9 6.3 48 170 28 38.1
Te 1 69 minutes* 29.0 8.2 42 134 32
135 minutes* 22.3 14.9 477 140 32 37.0
76 | Normal 35.3 144 44 38.9
rt 7 55 minutes* 30.9 4.4 162 32 $7:
ape 99 minutes* | 24.2 | 11.1 t 80 12 | 36.4

* Lapse of time after beginning initial trauma.
+ Animal in shock at time of observation.

Analyses were made on the plasma of blood drawn by syringe from
the inferior vena cava or external jugular vein. The normal dog
(exper. 78) was in a state of profound shock at the end of the first forty-
five minutes after beginning the initial, and only, period of trauma.
As shown in the table the alkali reserve of the plasma had fallen only
9.5 volumes per cent and was in fact no lower than the minimal value

for the normal dog. This was a very clear case of type I shock. Of

a oS ae > Mahe . i er ate ;
as aa ee elias NT

ALKALI RESERVE IN SURGICAL SHOCK 125

f 7 the other four dogs, determinations made before the signs of shock

appeared, gave a maximum fall from normal of 14.2 volumes per cent
(exper. 79), and a minimum of 4.4 volumes per cent (exper. 76). The
average fall for the four, within the first hour after beginning the initial

_ trauma, was 8.1 volumes per cent. As will be seen, the reading in

experiment 77 was quite above the normal minimum for the normal
dog, namely 32.4 volumes per cent, while the other three gave readings

not far below this point.

When we come to the final determinations, however, when all the
dogs showed definite signs of shock, matters are somewhat altered.
Thus two dogs show a fall from the normal alkali reserve of 17.9 vol-

umes per cent (expers. 77 and 79). The average fall for the four is 15.2

volumes per cent. All show readings below the mean value found
after two to four hours of continuous ether anesthesia (26.1 volumes
per cent). There appears to be no relation between the type of shock
and the degree of alkali depletion. However the data give an indication
of what trauma, uncomplicated by ether anesthesia, will accomplish
toward setting up a state of acidosis in the dog. It is seen further that
until shock actually appears, the alkali reserve remains well above what
may be called the limit of safety. The acidosis is certainly not marked.
The changes in heart rate and respiration are neither marked nor con-
sistent. As many dogs showed a decrease in both as showed an increase.

It should be emphasized that the marked fall in alkali reserve occurred
only after the dogs were in a state of shock. The slight fall observed
in the first hour, then, was due to local changes set up by the trauma
and not to any circulatory failure. That the onset of acidosis is gradual
is revealed in experiment 78, where the dog died from shock before the
alkali reserve had fallen even below the minimum normal value.

THE ALKALI RESERVE IN THE ETHERIZED DOG AFTER TRAUMA

The fall in alkali reserve in anesthetized dogs following trauma can
best be illustrated by summarizing representative experiments, The
data for four such experiments, illustrating the four types of shock, are
given in table 3. 2

The results given in the table above are shown graphically in figures,
2, 3, 4, 5A and 5B.

The data in the above table agree satisfactorily with those witadindd
in table 2. As before, the alkali reserve of the plasma remained prac-

126 BERNARD RAYMUND
TABLE 3
Showing the alkali reserve after trauma in etherized dogs
ALKALI advigrens Semoun BODY
TE | Ncommiemoms | aeeg. eam Sane | Ranon| TEMPEE-
MAL Dias- , ;
tlic Systolic
per cent| per cent 4 "C’
(| Normal 43.8 132 | 28
99 62 min. ether | 20.2 | 23.6] 153 | 165 | 188] 64 41.0
Type I Obs.* 30 min. | 21.1 | 22.7] 96! 103] 160 | 64 42.0
|| Obs.* 95 min. | 20.2 | 23.6 30} 42.0
‘| Normal 36.6 75 | 24
55 min. ether | 23.3 | 13.3 | 102] 109] 96] 72 |
29 Obs.* 55 min.| 23.3 | 13.3] 64] 78] 180] 48 38.9
Type II ) Obs.* 95 min.| 17.8 | 18.8 47 51} 180] 80 36.8
Obs.* 127 min.| 16.6 | 20.0| 50} 53] 144] 52 35 .6
Obs.* 170 min.| 15.7 | 20.9] 29] 32t|-108| 32°
‘| Normal 44.9 120 | 24
50 min. ether | 30.0] 14.9] 123] 131] 150] 64
Obs.* 20 min.| 27.1] 17.8 | 104] 107] 180] 68
69 Obs.* 78 min.| 27.1 | 17.8] 94] 98] 168] 40
Type III || Obs.* 135 min.| 26.2 | 18.7] 67] 74] 120} 36
Obs.* 200 min.| 15.9 | 29.0] 55} 61] 126] 40
Obs.* 255 min.| 13.0 | 31.9] 51 58 | 132 | 32
Obs.* 325 min.| 17.8 | 27.1 20t 52 Si:
(| Normal 51.6
55 min. ether | 34.2] 17.4 | 126] 138] 122] 66
Obs.* 35 min.| 29.4 | 22.2] 126] 141] 120] 72 ,
66 Obs.* 65 min.| 30.3] 21.3} 96] 111 | 150] 76
Type IV Obs.* 115 min.| 29.4 | 22.2] 77} 84] 144] 72
Obs.* 175 min.| 26.5 | 25.1 78 | 84] 150] 78
Obs.* 242 min.| 22.6 | 29.0 79 96 | 192] 60
Obs.* 310 min.| 23.6 | 28.0} 46] 53
Obs.* 382 min.| 32.3] 19.3] 58] 67] 126! 30 | Noshock

* Observation taken so many minutes after beginning the initial trauma.
t Observation taken when the animal was in shock.

tically unaffected in this case at the level to which it was brought
by etherization, until the blood pressure had fallen; and only showed
a significant reduction after the condition of the dog had become serious.

ALKALI RESERVE IN SURGICAL SHOCK 127

a Alkah Reserve |
“a of Plasma, Vols.o%
| iss i
™ 1. Normal Alnali Reserve this Expt 34.6 _
100 |
. qo; 15,
90°
9° 4
Gh 120
So
Ye 5
%e lt
20
lo
i 7 Trauma
a1 La a
° det Dk ee 7
afi VE er Tneesetnes Ex posed->

Fig. 2. Experiment 29. Showing the arterial blood pressure and the alkali
reserve in a case of type II shock.

Mean Are. B p. i
veal | Aureos
xy Vols of,
ie: --+/ erve,thisExbes yng 0
ito (2 luo
ito
too |
i ie i | 30
80 |

‘7 "to |
q
j igh °
So |
A 4 |
‘ aa lo
4o |
Ps
Trauma Ta
‘ 5 Rm.
Hours under 7 3 4 ss

es her off?

_ Fig. 3. Showing arterial blood pressure and alkali reserve mm experiment 69.
Type III shock.

128 BERNARD RAYMUND

Naturally there were exceptions. In experiment 90 (not shown) for
instance, the alkali reserve of the plasma fell to 14.3 volumes per cent
while the blood pressure was still 83 to 90 mm. The data also show
that as the condition of the dog becomes worse, the heart rate decreases.

I have never seen in the dog a case of cardiac shock, as described by’

Howell (2), (3), result from visceral trauma. At the same time the

Kati
serve

‘yo

--—

| 30

(Killed)

jo

20

Trayma T. Ts. TO} hot
— a : _ £8 2. wire . =

: > é 8
oe ‘ > Lneest ines Exposed

7

Fig. 4. Showing arterial blood pressure and alkali reserve in experiment 66.
Type IV shock.

respiration rate usually decreased. Hence the terminal rise frequently
observed in the alkali reserve of the plasma.

Although the data tend to show a correlation between the type, 7.e.,
the severity of shock and the fall in the alkali reserve, it appears doubt-
ful, considering the experiments as a whole, whether this is at all close.

True, the dogs exhibiting type IV shock were characterized by a remark-

to

, 20

ALKALI RESERVE IN SURGICAL SHOCK 129

4 ably constant alkali reserve. In these dogs compensation, such as by
'_ a decrease in the rate of respiration, etc., was no doubt very perfect,

_ even when the blood pressure had fallen markedly. On the other hand,

. as regards types II and III, the distinction between the two could not

q be drawn upon the basis of the alkali reserve. Thus the lowest level

| to which the plasma alkalies were carried by this method, namely 13

volumes per cent (exper. 69, above) was in a case of type III shock.

Aliali Res.
Myo t------- Normal A ital Reserve, Plasma= 49.5
| __ _Normat Alkali Reserve, Whole Blood=44.8
Yo
hole
zo loo do ~~
co wae
‘Plasma ©----____ OL a i
~ at ‘g : ais
20 a
lo |
F , ; :
Hours under 2 3 4 5 6
Erher

Fig. 5a. Showing alkali reserve of plasma and of whole blood. Aseptie
ether control for experiment 99. October 30, 1919.

Again, selecting readings in the first hour after beginning the initial
trauma, the alkali reserve in experiment 38 was 18.5 volumes per cent,
as compared with 23.3 volumes per cent in experiment 29, although
the type of shock in the first case was of the third order while in the
second the dog showed type II shock. The blood pressure readings
taken at the same time were 94-101 and 64-78 mm. respectively.
However, it would be impossible on the basis of the data in hand to
assign to any given range of arterial pressures definite values for the

130 BERNARD RAYMUND

‘plasma alkalies. The venture was made and proved utterly hopeless. —
Nor.is there any critical level of blood pressure at which a marked
decline of the alkali reserve is to be expected.. The condition of the
animal, in short, cannot be gauged by its alkali reserve alone.

7 Mesh Are B bx

Alkali Res.

| _Nermal Alk\Res., Whe leBieotmysh

1 yo
30
40 “Plasmao- ae Oe Yi Fram 190

10

Trauma

Hoursunser 2 , 4
Ether

Fig. 5b. Showing arterial blood pressure, alkali reserve of plasma and of |
- whole blood in experiment 99. Type I shock. November 6.

THE ALKALI RESERVE IN CONTROLLED SHOCK EXPERIMENTS

Tlie accuracy of the conclusions reached above can be best borne
out by presenting the protocol of one of a series of controlled shock
experiments performed with the assistance of Mr. C. F. G. Brown.
Dogs were kept under ether anesthesia for considerable periods of time, —
the ‘anesthetic being administered by intubation. Blood pressure
tracings were taken in only one experiment, but blood was drawn from
the femoral artery with aseptic precautions at regular and rather fre-
‘quent intervals. At the close of the experiment the dogs were sewed

ALKALI RESERVE IN SURGICAL SHOCK 131

s up and allowed to recover. At the end of a week shock was instituted

in the customary manner.

Protocol. Experiment 95, October 31, 1919. Aseptic ether control. Dog 6&6,

male. 15 kilos. Fed. .
LKALI RESERVE
TIME ; vet: Maca 7% Etat PROCEDURE
Blood Plasma , et bees .
a.m, apt’ Pings *C.
8:40 41.0 40.9 96 16 Sample, left saphenous
9:05 Ether, intubated, heat
10:05 28.3 26.8 140 40 Sample, rt. fem. art.
Knee jerk positive
11:05 27.4 24.9 140 44 Knee jerk positive
p.m. |
12:05 25.6 25.8 142 36 Knee jerk positive
12:30 Vomited
1:05 | 28.3 22.1 136 68 Very excited
2:05 28.5 23.0 156 40 Lid reflex negative
3:05 27.5 24.0 152 60 Lid reflex positive’
4:05 25.8 22.3 152 36 Lid reflex positive
4:35 ; 140 48 35 Lid reflex positive
5:05 25.7 18.3 156 36 Lid reflex negative
5:35 160 64 Lid reflex positive
6:20 37 Sewed up, ether off

The lid reflex was positive except where otherwise indicated. The
dog was under ether 9 hours and 25 minutes. Recovery was good and
the dog ate the next morning. After the lapse of a week shock was
instituted as shown in the protocol of experiment 100.

The lid reflex was positive throughout the experiment. The ether
was disconnected during each period of trauma. On section the heart
was found contracted, a further proof, if any were necessary, that in
spite of the dog’s great excitability the ether had been kept at a mini-
mum level. The total survival time after beginning the initial trauma
was three hours and forty-five minutes. Hence according to the classi-
fication adopted this was a case of type III shock, there being two
periods of trauma, two hours and thirty minutes apart.

It should be noted that the lowest point to which the alkali reserve
of the plasma fell, namely 17.7 volumes per cent, was only 0.6 volume
per cent below the lowest point reached in the control experiment, and
that this value, 18.3 volumes per cent, was perfectly compatible with

132 BERNARD RAYMUND

Protocol. Experiment 100, November 6, 1919. Shock experiment. Dog 56,
male. 10.1 kilos. Not fed.

BLOOD ALKALI
PRESSURE RESERVE Solis we tte peri
idacane RATE | RATION] PER- pera
— Systole} Blood | Plasma ATURE
m “an hale vol. vol, °C
sab ql * | per cent| per cent pln
9:10 49.4 | 42.8 80 | 12 Normal venous sample
9:35 Ether, intubated, heat

10:28 | 109 | 127 | 31.8 | 31.5 | 140] 44 | 39.5 | Sample, left fem. artery
11:10 | 101 | 121 | 30.8 | 23.0} 144] 68 | 39.0

11:15 94 | 113 Trauma, 15 min.
11:30 66 89 Closed up, heat
11:55 84 | 100 160 | 68 | 38.0

p.m.

12:10 78 90 | 29.9 | 26.8 | 184] 80 | 38.5
1:10 78 94 | 30.7 | 28.7 | 156] 48 | 393
1:45 89 | 109 39.5*| Trauma, 15 min.

2:00 61 79 160 | 32 | 39.8 | Closed up, heat

. Myenteric reflex positive

2:15 49 63 | 28.9 | 17.7 | 160} 36 | 39.8

2:25 53 67 176 | 28 | 39.0 | Ether disconnected,

breathing with diffi-

culty, in shock

2:45 | - 26 34 | 19.7 | 18.0 96 | 24 | 38.5 | Respiration spasmodic,

‘“‘shivery”’ °
3:00 Dead

life. At the time, however, that the low reading was obtained in the
shock experiment, the dog was already moribund, with an arterial
blood pressure of 49 to 63 mm. Obviously this fact could never have
been ascertained by a consideration of the plasma alkali alone. Fur-
thermore the alkali reserve of the whole blood at this time was even
slightly higher than in the control experiment at the same hour, namely
four hours, forty minutes after beginning ether anesthesia. Bayliss
(34) has shown that in cats a reduction of the alkali reserve of the plasma
to a level as low as 5 volumes per cent by the intravenous injection of
acid does not prevent the quick and apparently complete recovery of
the animal. Thus the conclusion is inescapable that there is no critical
level of alkali reserve, at least in the dog and cat.

‘Nip Ala

eee ees

es Ls “a Mi fe, ear - ‘. i _ 7
ee ee Ay OW Ses eae ge ‘3 Se Oe ae = . a Ba : Sarr —
= bs teat To ESSE ARs. 2) Ce SO Ce Pe ct a ea ce : Jabs at eee . So ee ae
: 33 o— ee eS ee et ee Ro A 3
: : — 7 A SO os 5
. ie yh ee a oe PD = sae

ALKALI RESERVE IN SURGICAL SHOCK 133

_ ATTEMPTS TO REPRODUCE TRAUMATIC SHOCK BY INTRAVENOUS INJECTION

OF ACIDS AND ACID SALTS

Since the claim put forward by Cannon (11) that the blood pressure
in cats can be brought to the shock level by the intravenous injection
of N/2 HCl has been effectually disproved (34), it seems unnecessary
to give an extended account of the experiments conducted along this
line in connection with the present paper.

Intravenous injections of sodium acid phosphate in concentrations
of 0.15 to 0.25 molar were made in thirteen dogs and two cats. Five
cats and three dogs were given intravenous injections of N/4 HCl.
While the injection volume was larger than if half-normal acid had been
used, still it is doubtful if there was any significant elevation of blood
pressure in consequence of this. Since the acid phosphate was quite
ineffective in lowering the alkali reserve of the plasma to any extent,
only the results obtained with hydrochloric acid will be given here.

The injection volume used in dogs varied between 6.9 and 42.3 cc.
per kilo. All the cats were given 10 ce. per kilo. The lowest point

to which the alkali reserve of the plasma was brought in the dog was

11.0 volumes per cent, from an initial normal value of 44.9 volumes
per cent. Of this fall 16.7 volumes per cent were due to etherization,
and the acid caused a further fall of 17.2 volumes per cent. There
were signs of cardiac failure before death and on section the lungs were |
found to be intensely edematous. Signs of shock were conspicuous for
their absence. The blood pressure on beginning the injection was 118-
125 mm. After the alkali reserve had fallen to its lowest point the
blood pressure remained above 90 mm. for forty minutes and at death
was still57 mm. Fourteen and one-tenth cubic centimeters of N/4 HCl
were given per kilo.

The lowest point to which the plasma alkalies fell in the cat was 10.7
volumes per cent, from a value of 34.3 obtained an hour and three
quarters after etherization. Of this fall 15.0 volumes per cent were
due to the ether. The dose of N/4 HCl was 10 ce. per kilo. The

blood pressure was fluctuating but rose from a level of 78-80 mm.

at the time the last blood sample was drawn to 81-95 mm. and then
fell rapidly to zero. There were no signs of cardiac failure; death oc-
curred in apnoea. There was no hyperpnoea at any time nor any signs

_ of shock, but the possibility of intravascular clotting and embolism was

not excluded. Observe that in this cat the alkali reserve was at all
times below the critical level of 38 volumes per cent cited by Cannon
(11). |

134 — BERNARD RAYMUND

Summarizing, it can be said that none of the animals showed any _
signs of shock; in fact up to the point of death they were all unusually
active. There were strong evidences of cardiac failure in the dogs,
_ but whether it occurred in the cats is doubtful. That the oxygenation
of the blood was markedly interfered with is shown in experiment 60.
After the alkali reserve had been lowered from an initial normal value
of 40 to 11.5 volumes per cent, the blood pressure was still 111-126
mm. ‘The injection of acid was continued until the dog had been given
42.3 cc. per kilo of N/4 HCl. The blood pressure fell rapidly from
98-105 to about zero. On drawing a sample of blood from the heart
it was found to be quite black and could not be oxygenated at all. The
spectroscope revealed reduced hemoglobin only; there was no methemo-
globin nor acid hematin. The alkali reserve of the plasma was negli-
gible. In one cat in which death occurred very suddenly on injecting
acid, the blood in the right’ ventricle was found to be coagulated im-
mediately at the close of the experiment.

SUMMARY AND CONCLUSION

1. The dogs used in these experiments are classified on the basis of
the length of survival after beginning the initial trauma. This makes
unnecessary the arbitrary designation of any given arterial pressure as
_ the shock level. |

2. On this basis four general types of shock were made out, ranging
from the more severe characterized by sudden onset and death, to that
in which few or none of the cardinal signs of shock were observed.

3. The larger number of animals showed the intermediate types of
shock and lived on the average from two hours, nine minutes, to four
‘hours, fifteen minutes (types II and III) after beginning the initial —
trauma. | ;

4. The average normal alkali reserve of the venous plasma in the
dog was found to be 43.4 volumes per cent, sixty-eight determinations.
The values ranged from 32.4 volumes per cent to 59.5. The average
for whole blood was 47.0 volumes per cent, eighteen determinations.
The maximum reading was 55.6, the minimum 36.6 volumes per cent.

5. In dogs under ether anesthesia the mean value fell to 33.9 volumes
per cent. As anesthesia was protracted the mean alkali reserve fell
to 28.0 volumes per cent (forty-five minutes to two hours) and finally
to 26.1 volumes per cent (two to four hours). As etherization was
continued beyond this point the mean fallin alkali reserve per unit of
time was seen to decrease.

ALKALI RESERVE IN SURGICAL SHOCK 135

6. Five dogs traumatized under local anesthesia alone showed no

- striking fall in the alkali reserve of the plasma until their condition

- had become quite serious. No level of blood pressure could be set as
critical in this respect. Under the conditions of the experiments the
average fall from the normal reading was 15.2 volumes per cent.

7. Substantially the same was shown by eleven dogs which were
traumatized under ether anesthesia. When shock ensued suddenly the
fall in the alkali reserve of the plasma was relatively insignificant.
_ When shock was late in appearing, the plasma might show a high alkali
4 reserve for some time after the animal had become practically mori-
bund. There was apparently no correlation between the type of shock
and the degree of alkali depletion. The condition of the animal could
not be gauged by its alkali reserve.

8. In several experiments it was found that the alkali reserve of the
plasma and of the whole blood fell no lower in profound shock than in
aseptic ether controls performed on the same dogs a week previous to
the experiment, and from which they recovered rapidly and com-
pletely. If there is a critical level of alkali reserve it was not discovered.

9. Intravenous injections of N/4 HCl and of isotonic acid phosphate

solutions did not produce shock or anything resen Bling this condition

in either dogs or cats.

The writer desires to thank Dr. A. J. Carlson, Dr. A. B. Luckhardt
and Dr. A. C. Ivy for their kind advice and many suggestions, and Mr.
C. F. G. Brown for his assistance in the laboratory.

BIBLIOGRAPHY

(1) Sprro: Hofmeister’s Beitr. 1902, i, 269.
(2) Howe: Contributions to medical research, 1903, 57.
(3) Howreuu: Amer. Med., 1904, vii, 482.
(4) Dawson: Journ. Exper. Med., vii, 1.
_ (5) Sgpiic, TIERNEY AND RopeNsBAuGH: Amer. Journ. Med. Sci., 1913, exlvi,
195.
(6) HenpErson: This Journal, 1910, xxvii, 152.
(7) Crite: Ann. Surg., 1915, lxii, 257.
(8) Crize: Can. Med. Assoc. Journ., 1915, v, 1.
(9) Consett: Journ. Amer. Med. Assoc., 1915, lxv, 380.
(10) Cannon: Memorandum to the Sub-committee on Shock, Committee on
Physiology, National Research Council, August 25, 1917.
(11) Cannon: Ibid., February 25, 1918.
(12) Bayuiss: Intravenous injections in wound shock, London, 1918, 60.
(13) Cannon: Memorandum to the Sub-committee on Shock, Committee on
Physiology, National Research Council, March, 25, 1918.

136 : BERNARD RAYMUND

(14) HENDERSON, PRINCE AND Hacaarp: Journ. Amer. Med. Assoc., 1917, Ixix,
965.

(15) Cannon: Journ. Amer. Med. Assoc., 1918, Ixx, 531.

(16) Cannon: Journ. Amer. Med. Assoc., 1918, Ixx, 611.

(17) Cannon: This Journal, 1918, xlv, 544.

(18) Guturie: This Journal, 1918, xlv, 544; Arch. Int. Med., 1918, xxii, 1. — p

(19) McEuuroy: Journ. Amer. Med. Assoc., 1918, Ixx, 846. 1

(20) HENDERSON AND Haaaarp: Journ. Biol. Chem., 1918, xxxili, 345.

(21) HENDERSON AND Haaearp: Journ. Biol. Chem., 1918, xxxiti, 365. ‘

(22) GrsELL: This Journal, 1919, xlvii, 468. ;

(23) GASSER AND ERLANGER: This Journal, 1919, 1, 104. +

(24) Metrzer: Arch. Int. Med., 1908, i, 571.

(25) Mann: Johns Hopkins Hosp. Bull., 1914, xxv, 205. |

(26) GuTurRiE: Journ. Amer. Med. Assoc., 1917, Ixix, 1394.

(27) Van SLYKE: Journ. Biol. Chem., 1917, xxx, 347.

(28) Peters: This Journal, 1917, xliv, 84.

(29) JANEWAY AND Jackson: Proc. Soc. Exper. Biol. and Med., 1915, xii, 193.

(30) ERLANGER AND Woopyatt: Journ. Amer. Med. Assoc., 1917, Ixix, 1410.

(31) ERLANGER, GESELL, GASSER AND Exitiott: Journ. Amer. Med. Assoc., 1917,
Ixix, 2089.

(82) Mann: Journ: Amer. Med. Assoc., 1918, Ixxi, 1184.

(83) Morriss: Journ. Amer. Med. Assoc., 1917, Ixvili, 1391.

(84) Bayttss: Op. cit., 60.

ee <<

PHYSICO-CHEMICAL STUDIES ON BIOLUMINESCENCE

Il. Tue Propuction or Licut sy Lucioua VITTICOLLIS IS AN
OXIDATION!

SAKYO KANDA

From the Marine Biological Laboratory, Kyushu Imperial University, Tsuyazakt
(Fukuoka), Japan

Received for publication May 22, 1920

INTRODUCTION

The problem whether the production of light by a fire-fly is an
oxidation is an old one, having been raised as long ago as 1783 (2, p.
355). Unfortunately, different investigators offer varying results.
Spallanzani, for instance, found that the light produced by Lampyrises
disappeared in Ne, He and COs, but it appeared again at the admission
of air or, better still, Qo. It is doubtful how exact his method was,
because it was a work of 1796. On the other hand, according to
Macartney, the Lampyris produced a brilliant light without O2, and it
neither became stronger in O» nor weaker in He (2, p. 356).

Mangold well points out, therefore, the status of this problem and
states:

1 In his paper in this Journal, January, 1920, Doctor Kanda stated that the
production of light by Cypridina is not an oxidation. I think it will be admitted
that his results were convincing to the extent of showing either that no oxygen
was required or very litile (as much as might, in spite of his elaborate precautions,
have been present as an impurity). In the interest of clear discussion I believe
it should be known that in a personal letter to me, Doctor Kanda now adopts the
second alternative. The paper referred to is, I take it, to be interpreted as prov-
ing that the luminous material in Cypridina is very rapidly destroyed, without
proportional light production, if any considerable amount of oxygen is present;
and that the long-continued strong light production which he observed was due
to the very small amount of oxygen present as an impurity in the gases used.
As Doctor Kanda forwarded his manuscript for publicetion in English through
me, I am venturing to add this note.—E. P. Lyon.

137

138 SAKYO KANDA

Die Frage nach der Bedeutung des Sauerstoffes fiir die Lumineszenz ist hier
aber noch nicht . . . . zu einem endgiiltigen und véllig klaren Abschluss
gekommen, zumal noch die letzten Arbeiten iiber Leuchtkifer zu scheinbar
entgegengesetzten Ergebnissen gefiihrt haben. Die Methodik spielt hier ja eine
besonders grosse Rolle, zumal es fiir einwandfreie Versuche erforerlich ist, so
geringe Mengen freien Sauerstoffes auszuschliessen . . . . (2, p. 355).

In short, the problem of oxidation in question is by no means settled.

The writer, therefore, made an attempt to settle this problem with
new methods and apparatus devised for ‘‘einwandfreie Versuche,” as
Mangold puts it; and he found that the results of these experiments
were quite decisive. In this paper, therefore, he will report these
results together with the description of methods and apparatus, which
were new as far as he is aware. The work was carried out at the
Science Department of the Kyushu Imperial University. The writer’s
thanks are due to Dr. Tsuneya Marusawa, the professor of physical
chemistry, for his generous help and suggestions, and also to Mr. Tetsuzo
Hagiwara, Doctor Marusawa’s assistant, who assisted the writer all
the way through the work. The writer appreciates Prof. Ayao Ku-
waki’s kindness for the privilege of the use of the laboratory. |

MATERFAL

The material used for all the following experiments was a Japanese
fire-fly, Luciola vitticollis. The luminous organ of this species differs
according to the sex. The luminous organ of the male, which is smaller
in size than the female, consists of the last two segments of its abdo-
men, while that of the latter consists of only one segment next to the
last.

The luminous organs of the male which were used for all the follow-
ing experiments, except one series, were carefully cut off from the rest
of the body of the live animals. The luminous organs of the female
were specially prepared for one series of experiments which were carried
out to determine the quantity of oxygen to be consumed by the organs
in the oxidation for the production of light. This will be mentioned
later on.

THE PREPARATION OF PURE GASES
At first the writer used Hy, No and CO: gases which were prepared by

ordinary methods and also O2 from a bomb. The intensity of light
produced by the luminous organs of the animals was always strongest

PHYSICO-CHEMICAL STUDIES ON BIOLUMINESCENCE 139

and lasted longest in oxygen. The production of light, however, also
resulted, though only for a short time, when He, Ne and COs gases.
were used. On the other hand, no light was produced in vacuum.
These peculiar results led the writer to doubt whether the gases used
were in reality pure, though they were prepared with special care.
The careful analysis of H. and Ny» gases with an Orsat’s apparatus
revealed that they were impure. Their impurity extended even to
1-5 per cent, due to the mixture of O2. The writer has become con-

Fic. 1

vinced since then that gas washers which are used in an ordinary
method of gas preparation could by no means purify a gas passed
through them, though the air in them was evacuated beforehand.

An exact method, therefore, was to be planned to obviate the fail-
ures mentioned above. It was thought that if gas purified by an
analytical method could be used, it would serve for this purpose. Soa
new gas holder was devised, as shown in figure 1. In the first place,
the flask, F, was filled with a gas-absorbing solution, i.e., 150 cc. of

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 1

140 SAKYO KANDA

20 per cent Cs Hs; (OH); + 800 cc. of saturated KOH for On, for ex-
ample. The solution was drawn in the glass tubes, A and B, and a
separating funnel, C, up to the stopcocks, a, b and c: The tube B

was connected to one of the arms of the Orsat’s apparatus by means of

a rubber tube. All air in the rubber tube and in the rest of the glass
tube B, from the stopcock b, was evacuated before the connection with
the Orsat’s was made. A receiving bottle, R, which was connected by
means of a rubber tube to the glass tube A was evacuated and sealed up.
Now 100 cc. of a gas, either H, or Ne, which was carefully prepared

in the laboratory, were introduced into the burette of the Orsat’s

apparatus. Oxygen gas which was always mixed as an impurity with

H. and Ne gases was repeatedly absorbed by the alkaline pyrogallol
solution in a pipette of the apparatus until the volume of the gas in
the burette became constant. The gas thus purified was to be pre-
served in the flask F of figure 1. In order that the gas might be drawn
into the flask, a stopcock of the arm of the Orsat’s, say O, to which the
flask was connected was to be opened. Thus the rubber tube in the
vacuum was now filled with the gas. Then the stopcock b was opened.
And lastly, the stopcock a was opened with careful regulation not to
draw the solution in the bottle R from the flask F too fast. When
about all the gas in the burette of the Orsat’s was drawn out, all stop-
cocks just mentioned above were again closed. This same procedure
was repeated several times until a desirable amount of the gas had
filled the flask F. The virtue of this method was that if any traces of
O. gas were left with the gas in the flask F, though hardly possible,

the solution in the flask would absorb O, during the period of preser-

vation. It is believed that gas absolutely free from even a trace of O2
gas was available for use by this method.

The oxygen gas taken out from the bomb was also preserved in a
flask with a saturated KOH solution in the same way as above after
its analysis, although no trace of CO. was detected. It was found,
however, that the O2 gas from the bomb was impure to the extent of
2 to 3.25 per cent. Presumably the impurity was nitrogen. No
attempt was made to remove the mixed gas or gases from Oz, except
CO.. Carbon dioxide was not used for the experiments of these series
because of the difficulty of freeing it from Os.

PHYSICO-CHEMICAL STUDIES ON BIOLUMINESCENCE 141

METHOD

_ In the first place, ten isolated luminous organs of the male were
_ placed in the experiment bottle, H (fig. 2). The bottle was fitted with
_ a tight rubber stopper in which two glass tubes, G and H, with one
_ stop-cock for each, were inserted. It was then fixed on an iron stand.
’ The glass tube G was connected to the glass tube A and the glass tube
a HT to one of the arms of a T-shaped glass tube, J, as illustrated in

figure 2. The experiment bottle, £1, was also connected to the glass
tube, A;, of the flask, 71, in the same way as just mentioned above.
And one of the arms of the T-shaped glass tube J was connected to a
Gaede’s oil vacuum pump, P, through a manometer, M... As.it. was
essential to exclude all air from without, melted paraffin was put all
over the rubber stoppers of the experiment bottles H and Fi, especially
those places where the stopper and glass were in contact.

‘The vacuum pump was then started to evacuate air in the experi-
ment bottle EZ or E,;. Meanwhile the stopcocks g and H were opened.
When the manometer reached the zero point, the stopcock H was closed

142 SAKYO KANDA

and the pump was stopped. Of course, some air was still to be left in
the bottle and connections? because the pressure was not an absolute
zero. Nevertheless, the material in the bottle produced no light.
In order to make the vacuum of the experiment bottle and other spaces
complete, the procedure of evacuation was repeated two or three
times after filling the bottle with the pure gas which was to be used.
For this purpose, the stopcocks a and c® were opened and the bottle E
was filled with the gas. After this filling was complete the stopcocks
a and c were closed and the pump was again started to work. ‘This
procedure was repeated two or three times. After the last evacuation
of the gas, the stopcocks g and h were closed, and the gas admitted up
to the stopcock g, opening the stopcocks a and c. The last procedure
of this experimentation was simply to introduce the gas into the bottle
E to see whether any light was produced by the material or not. But
this should be done together with the bottle E, which was prepared in
the same way.

As Os: gas was to be used in the bottle, Ei, it was thought that two
complete evacuations might be enough. After the last evacuation,
the stopcocks g; and fh; were closed and the stopcocks a, and ¢, were
opened. In so doing, the gas was admitted up to the stopcock 4.
Now both the bottles, H and E,; were ready for the experiment. ‘The
room was then darkened. The stopcock g; with the left hand and the
stopcock g with the right were opened at the same time. In this way,
O. gas into the bottle H; and Hz or Ne gas, as the case may be, into the
bottle H, were admitted at the same time. Thus the production of
light by the isolated luminous organs of fire-flies in the two gases was
observed simultaneously.

The O, experiment was always carried out in comparison with any
other experiment as a control of special kind, besides a second control
for which air was used. It was thought that the evacuation of air in
the experiment bottle might have some effect on the production of light

2 As the Gaede’s pump is capable of developing a vacuum 0.001 mm. of Hg.,

1
the air left i ly
e air left is only 0,

said that there were originally about 12.6 cc. of O2 in the experimental bottle
of capacity of 60 cc., as the amount of Og in air is about 21 per cent. There is,
therefore, only about 0.0000165 cc. of Og left after one evacuation.

3 The alkaline pyrogallol solution was always poured into the funnel, C, by
means of a rubber tube connected to the stopcock, N, of the bottle, R, which
was already disconnected from the flask, F.

of one atmosphere after one evacuation. It may be

LT Saat Oe ee eT

PHYSICO-CHEMICAL STUDIES ON BIOLUMINESCENCE 143

as the result of taking water content away from the material or of
some stimulation and other change. Besides the control using air,
some experiments were performed by introducing air from a glass gas-
holder into the experiment bottle, just the same as the other gases were

q introduced, after the evacuation of air. These experiments were also

compared with those of Oz, as well as with the controls in which air was
used without any treatment. It should be remarked that the intensity

of light produced by the material was much stronger in air which was

introduced after evacuation than in air without any treatment. The
writer will try to explain this fact later.

EXPERIMENTAL

As already stated, the purpose of this investigation was to determine
whether the production of light by the fire-fly is an oxidation, as is

generally assumed, or not. The results obtained prove what most

previous authors believed. These experiments were, of course, re-
peated several times with no exception, when conditions were properly
controlled. If there was any exception, it was found that either the
gas used was impure, or the method or apparatus imperfect.

The production of light by the luminous organs of the fire-fly in varying
gas atmosphere and condition: The methods of these experiments were
very simple, as already described in the previous section. Table 1 is
the summary of the results of these experiments. The figures in the
table simply show a comparative intensity of light produced by the
isolated luminous organs of the male in a given gas atmosphere. The

figure “4”, for example, means that the most intense light was pro-

duced by the material in one of the four gases, including air.
As table 1 has shown, no light was produced by the luminous organs

in Hz and Ne atmospheres or in a vacuum. But the admission of air or

better still, O. gas, resulted in a brilliant light. Fortunately, once

the writer had Ne gas in which 1 per cent of O2 gas was mixed. He

therefore tried to see whether the material would produce light or not.
It was found then that the material produced intenser light in this
mixture than in air. The light in the former continued for about 12
hours, while the control in air lasted for about 70 hours at about 20°C.
About 3 hours after the extinction of light, the material produced
light at the admission of air. These results will convince any unprej-
udiced minds that the production of light by the material is an oxida-
tion. Furthermore, it is evident that free oxygen is absolutely neces-

144 | SAKYO KANDA

sary for an oxidation of this sort, as the material produced no light in
vacuum. And it seems therefore probable that even though some oxy-
-gen supplier or carrier is assumed to exist in the cells or tissues of the
‘material, it plays no réle by itself alone in this process of oxidation for
the production of light.

The writer stated in the second paper of this series that the produc-
tion of light by dried crushed Cypridinas was not an oxidation. Some
Japanese critics thought that this statement was dogmatic beyond
the facts actually found: Their reason was that the writer showed
only that no oxygen in the medium was necessary for the production
of light by the material, but he did not show at all that oxygen con-
tained in the cells or tissues of the material was not used. The writer
could make no answer to this objection. Now it may be answered

TABLE 1

| The production of light by the isolated luminous organs of Luciolas in varying gas
atmosphere and condition

na fo] NX ‘ Nn na
65 [a8 ).de ag | 23
62 | 58 | 588 BE | 83
ZP Dp Bb Dp Dp
Sy) Zo %°o Zo Zo
o< Of | oma 2 o< o<
GAS AND CONDITION 5 5 B a = a e ° = e 5 .
Paw Pw pe ~ =)
ce | 38 ava| & | 8H | ee | @
= a a o a i=)
ek BZ = e F B E D | 8
=) ~ 4 < - on i
Comparison of intensity of light.| 4 3 2 1 0 0 0
Readmission of air............. 4 2h 242) 1 3 3 3

that it is not probable that any oxygen in the cells, or tissues of the
dried crushed Cypridinas is used for the production of light by them,
because no oxygen in the cells or tissues of the luminous organs of the

fire-flies seems to be used for their production of light, even though

this process is certainly an oxidation.

As already stated, the light produced by the material in air admitted
after one evacuation was much stronger than that produced in air
without any treatment. This fact could not be explained on a mere
basis of the volume of oxygen contained in air. But as the cells and
tissues of the material were alive and some nerve ganglia seemed to be

located in the tissues, it seemed possible that stimulation by mechan- —

ical agitation occurred when air was admitted after an evacuation.
This might be the same phenomenon as in the case of the fire-flies
producing a stronger light when water is sprinkled on the cage contain-

se.

iz
F
i
os
>
‘
4
iy,
i
i
,
4
7
F
g
:

attempt was made to estimate the amount of O. converted

for this experimentation.

PHYSICO-CHEMICAL STUDIES ON BIOLUMINESCENCE 145

ing them, or when the cage is shaken. Besides such biological factor,

the surface of contact may also act. Evacuation may. increase the
-contact-surface of the material for the readmitted oxygen gas and in

consequence the rate of oxidation may increase. Whatever the reason,

the result was a stronger light when air was readmitted after evacua-
tion. The fact that the intensity of light produced by the material in

the gaseous mixture of Nz with 1 per cent of O2 admitted after evacua-
tion was stronger than that of light produced in air with no evacuation
may also be explained in the same way. That the light produced by
the material in air which contains about 21 per cent of O. is weaker
than that in the mixture of Nz with 1 per cent of O2 is incomprehensible
if considered merely from the viewpoint of an oxidation. But it is not
necessarily so if the biological and physico-chemical factors, mechan-
ical agitation and surface action just mentioned above, are considered.

An estimation of oxygen consumed by the luminous organs: An

into CQ, in the production of light by the isolated luminous
organs of the female. A preliminary experiment showed that
quite a large amount of CO, was given off during the produc-
tion of light by the luminous organs. This encouraged the
writer to undertake further careful experiments. The method
of this series of experiments was a little different from others,
though quite simple. The bottle shown in figure 3 was used

In the first place, the experiment material was isolated as

carefully as possible to minimize the admission of other sub- yg. 3
stances, which might in some way obscure the results. For ‘

this purpose the luminous organ of the female was more suitable than

that of the male, because the female luminous organ of this species
consists of only one abdominal segment next to the last, as already

stated. If the thoracico-abdominal regions were pressed by the fingers
of the left hand, the last two or three segments of the abdomen were
stretched out. Then the last segment was carefully cut off by means of
sharp scissors and the thoracico-abdominal regions were again pressed
hard. In doing so,:eggs and other matter contained in the abdomen
were pressed out from the cut. They were all cleaned off with special
care. After this cleaning, the luminous segments of sixty females thus
isolated were placed in the experiment bottle E shown in figure 3. The
bottle H was tightly fitted with a rubber stopper in which two capillary

glass tubes, A and B, were inserted. The tube B was connected by

146 SAKYO KANDA

means of a rubber tube to one of the arms of the Orsat’s apparatus for
gas analysis. The tube A which had one stopcock, a, was connected to
the vacuum pump through a manometer. After all connections were
thus made, melted paraffin was put all over the stopper of the bottle
E as usual. Now the stopcock of the arm of the Orsat’s, say 0, to
which the tube B was connected, was closed. The pump was started
to evacuate the air in the bottle H. After complete evacuation, the
stopcock a was closed.

Exactly 100 ce. of O2 gas, which contained about 2.14 per cent Ne
but was absolutely free from CO, and was held in the burette of the
Orsat’s, were ready to be sent into the bottle E at any time. Now the
stopcock O was opened. Oxygen gas was thus introduced into the
bottle H from the burette. After this filling was over, the stopcock O
was closed. The exact volume of the gas introduced into the bottle
was read and the rest of the gas left in the burette.was blown out. At
the same time the barometer and temperature of the laboratory were
read. For certain hours the material thus treated was allowed to use
O, for the production of light, the exact volume of which was already
known.

Twenty-five hours from the time of treatment the material was still
producing light. But it was thought that further delay might com-
plicate the results by increasing CO, gas which might be given off due
to the decay of the material, or to other causes. Therefore the tube
A was connected to a syphon by means of which distilled water was
caused to run into the bottle H. The stopcocks O and a were opened
in the order mentioned. The gas contained in the bottle was thus
displaced by the water and returned to the burette. When the water
reached the stopcock O, the latter was closed. The volume of the
returned gas and the temperature and barometer were read at the
same time. The carbon dioxide mixed with the O. gas was absorbed
by a concentrated KOH solution until the gas volume became constant. _
The O». was also absorbed by the alkaline pyrogallol solution after an
addition to it of 16 ce. of Ne. The results of the experiments calculated
by reducing to the normal conditions are given in table 2.

The amount of O. consumed in the production of light by the mate-
rial was found to be 6.01 ce., as shown in table 2. But the methods of
the experiment were not beyond criticisms, which may be mentioned
as follows: a, The gas in the burette of the Orsat’s apparatus was dis-
placed by distilled water. It was certain that the water in the burette
would dissolve CO, which was sent for analysis from the experiment

hie
eS

ae nS eg We a cl

i

PHYSICO-CHEMICAL STUDIES ON BIOLUMINESCENCE 147
bottle into the burette, after the luminous organs had used Oz for the
production of light. An error would thus be introduced into the
results. In other experiments, therefore, the water in question was
replaced by mercury before the gaseous mixture of O2 and CO, was
sent back into the burette. Unfortunately, however, a certain tech-
nical fault was found in the process of analysis. So these results have
been ignored and the experiments made with water displacement are
published. 6, It was not at all certain whether the whole amount
of Ons, i.e., 6.01 ec., was exclusively used up for the production of light
by the isolated luminous segments, as some of it might have been used
for oxidation of substances of the segments independent of the process
of the light production.

The writer was anxious to remove as far as possible these errors and
objections just mentioned. It was, however, late in the season and
the material could not longer be secured. He therefore reports the
result as it was, though unfinished.

TABLE 2
An estimation of O2 consumed by the isolated luminous organs of Luciolas for 25
hours
Oz ORIGINALLY O2+CO2zarreR | DEVELOPED CO CONSUMED O
rs *'33 nonRs younp | 02 REMAINING FOUND
cc. cc. cc. cc. cc.
58.52 58.17 5.66 52.51 6.01

A relation of the production of light to water: It was supposed that if
the production of light by the material was an oxidation, the material
might be preserved longer in vacuum than in air. So the following
experiments were tried. Seventeen glass tubes of the capacity of
about 25 cc. were drawn narrow at one end and sealed up at the other
end. Isolated luminous organs of ten males were placed in each tube.
Three of these tubes were sealed up at a certain time. These were
the controls of one kind. Another three tubes which were thoroughly
evacuated once were sealed up after readmitting air. These were
controls of another kind. The material in the controls of these two
kinds was of course producing light. The rest of the tubes were sealed
up while the process of evacuation was going on. The material in
these tubes was not producing light. At an interval of 2 or 3 hours,
one of these tubes was opened in the dark room to see whether the
material produced light or not. In doing so it was thought that the

148 SAKYO KANDA

exact time might be detected at which the material no longer pro-
duced light. But as table 3 has shown, the results were contradictory
and were quite contrary to the writer’s expectation. That is to say,
the material was preserved longer in the air than in the vacuum. :
The material of the controls of the second kind which were evacuated
and were sealed up after admission of air, seemed to furnish an explana-
tion to the riddle. There should be no difference in the durability of
light between the first and second -controls because the volume of air
in both was practically equal. But perhaps the material which was
temporarily exposed to a vacuum might have lost some water during
the process. The loss of water might perhaps have shortened the —

TABLE 3

The production of light by the isolated luminous organs in air admitted after the
enclosure in vacuum

PRODUCTION OF
PRODUCTION OF siete stor OR PRODUCTION OF LIGHT
CONDITION LIGHT BY THE ISOLATED LIGHT ’ BY THE ISOLATED
TIME BY THE ISOLATED LUMINOUS ORGANS BY THE ISOLATED LUMINOUS ORGANS —
IN HOURS LUMINOUS ORGANS SEALED IN AIR LUMINOUS ORGANS | SEALED IN VACUUM
SEALED IN AIR AFTER EVACUATION SEALED IN VACUUM AT THE
‘ ADMISSION OF AIR °.
1 +: + - +
5 + “ - +
10 + - ~ -.
15 = - -
20 “ -- ~ ~
DB - “ - +
30 + + ~ -
35 - - - -—
40 + — - ~
45 + ~ ~ -
50 - ods is as

endurance of the light-producing substance. This view may also
explain the failure of experiments in which all the material was sealed
up for a long time in the vacuum tubes. It may be asserted as prob-
able, therefore, that water is necessary for the production of. light by
the fire-fly. } bhai: |

This view is strengthened if the following fact is considered. That
is to say, dried crushed luminous organs of the fire-fly produce light, —
though faint, if moistened. The fire-fly seems, however, to be quite
different from Cypridinas. The more dried the longer the latter is
_preserved, while the dried fire-fly or its luminous organ is preserved
only 5 or 6 days. That is to say, the dried luminous organ of the

ee
a

5 Sees

PHYSICO-CHEMICAL STUDIES ON BIOLUMINESCENCE 149

fire-fly did not produce light after 5 or 6 days even though moistened.
A question arose whether the luminous organs of fire-flies produced
light when they were dead if moistened or not. And absolute dryness
of the organs in question might be one of the causes of death. This
idea was tested but no decisive results were obtained.

The effect of temperature: Harvey states that “Luciola photogenin is
destroyed at about 42°, while the photophelein is still active after ten
minutes boiling” (1, p. 348). The writer found that the light produced
by the isolated luminous organs of Luciola vitticollis disappeared when
heated at 50°C., but it returned again when cooled. The return of
light took place after about 5 or 10 minutes and it was very faint.

SUMMARY AND CONCLUSION

1. The material used for experiments was a Japanese fire-fly, Luciola
vitticollis.

2. The gases used for experiments were Ho, Ne and On.

3. New methods and apparatus were contrived to purify and manip-

ulate the gases to fit the purposes of this investigation.

4. The isolated luminous organs of the animals produced no light
in H, and Nez or in vacuum. The oxygen of the cells or tissues of the
organs, therefore, seemed not to be used for the production of light.

5. The intensity of light produced by the isolated luminous organs
was greatest in O2 atmosphere, next in air which was introduced after
evacuation, then in N2 mixed with 1 per cent of O2 and last in air.

6. The isolated luminous organs of sixty females which were placed
in 58.52 cc. of O2 gave off 5.66 cc. CO. in 25 hours. The amount of
‘O2. consumed was 6.01 ce. |

7. Water seemed to be necessary for the production of light by the
isolated luminous organs.

8. The light produced by the isolatedluminous organs disappeared
when heated to 50°C., but it appeared again when cooled..

The principal conclusion on the basis of the experimental results
mentioned above is that the production of light by Luciola vitticollis
is an oxidation.

BIBLIOGRAPHY

(1) Harvey: This Journal, 1917, xlii, 318.
(2) Mancotp: Winterstein’s Handb. verg. Physiol., 1910, iii, 225.

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THE AMERICAN
JOURNAL OF PHY SIOLOGY

VOL. 53 SEPTEMBER 1, 1920 No. 2

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA
I. Mrxep Diet REAcTION

G. H. WHIPPLE, C. W. HOOPER anp F. 8. ROBSCHEIT

From the George Williams Hooper Foundation for Medical Research, University of
California Medical School, San Francisco

Received for publication April 3, 1920

This series of papers deals with the regeneration of red cells and
hemoglobin following simple anemia and the influence of diet factors
upon this reconstruction. It will be shown that the curve of hemoglobin
regeneration can be influenced at will by various diet factors. We be-
lieve that it is desirable to mention at least two lines of investigation
which are being followed in this laboratory. To determine the value
of various food factors when given alone or combined with other sub-
stances. To determine further the few or many substances which
promote speedy regeneration of hemoglobin and red cells or to ascer-
tain the optimum food combinations which will give a maximum blood
regeneration following simple anemia. Inorganic substances and cer-
tain drugs are being investigated and this work will be presented in its
proper place. Some experiments will deal with splenectomized dogs
as well as bile fistula dogs but we prefer to present at a later time the
bulk of our work on splenectomized animals which deals’ with the rela-
tion of splenectomy to blood regeneration under fixed experimental
conditions. A preliminary report covering a part of this anemia work
has been published elsewhere (1).

This work on blood pigment regeneration forms an essential part in
any study of “pigment metabolism of the body.” It is obviously
closely related to a study of bile pigment excretion which was first taken
up in our work and has been reported in part in earlier publications (2),

(3). It will be recalled that the excretion of bile pigments may be

151 . | ws

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 2

152 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

influenced or modified by various diet factors. For example, meat will
cause increased flow of bile but a decided drop in total bile pigments.
Carbohydrate, on the contrary, will reduce the flow of bile but increase
the total bile pigment output. Decreased functional activity of the
liver is associated with a decided fall in bile pigment elimination. We
have assumed on the basis of much experimental evidence that the liver
plays a constructive réle in the bile pigment output. We hope to show
the same relationship on the part of the liver to the constructive mechan-
ism of blood regeneration.

/ We wish to emphasize that a curve of blood pigment regeneration
cannot be established without accurate determination of at least two
factors,—hemoglobin and blood volume. With a knowledge of these
two factors we can estimate the total volume of hemoglobin pigment
in the body circulation—the ‘pigment volume.” Also with the hemat-
ocrit values we are able to compute the total volume of red cells in the
body circulation. Reasonably accurate methods for the determina-
tion of circulatory blood volume are of recent development. A critical
review of many factors in this blood volume work and comparison of
various methods have been published from this laboratory very
recently (4). The method used in this laboratory for accurate determi-
nations of hemoglobin has been recently described by F. S. Robscheit
(5).

Practically all these anemia blood regeneration experiments were
performed upon dogs born and raised in our kennels—a bull dog cross
which gives a very active, vigorous and healthy laboratory animal.
Unless otherwise noted, the dogs were in fine normal condition during
the entire experiment. These dogs will eat the food mixtures as a rule
without any delay or wastage. They have been immunized against
distemper and are kept in a separate room to obviate any cross infec-
tions by transient animals.

METHODS

The blood volume method used in these experiments has been de-
scribed in detail elsewhere (4). In some of the earlier experiments dry
oxalate was used for blood collection instead of isotonic fluid oxalate
and in these experiments the calculated blood volume is too high. A
note will be made in all such experiments as a correction cannot be
introduced because the amount of solid oxalate and the corresponding

shrinkage of cells was an unknown variable.
¥

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 153

It may be stated in a word that the blood volume method consists
in the introduction of a measured amount of.a dye ‘‘brilliant vital red”’
- into the blood stream. After a four minute period the dilution of the

7 dye in the plasma is colorimetrically determined. “Brilliant vital red”
q has been furnished us through the courtesy of Dr. H. M. Evans of the

Department of Anatomy and we wish to acknowledge many favors and
_ valuable advice given us by Doctor Evans. The red cell hematocrit
is read in an accurately calibrated centrifuge tube into which blood has
been drawn, using an isotonic sodium oxalate solution. It is then a
simple matter to calculate the plasma volume, red cell volume and total
blood volume. This method can be quickly and accurately performed.
_ It causes the dog a minimal degree of inconvenience, only that due to a
_ hypodermic needle puncture of a vein, and the loss of only 35 ce. of
whole blood. This is such a small amount of blood removed from the
large circulating blood volume that we do not include it in our calcu- —
lations and feel that no secondary anemia factors are added to compli-
cate the reaction curve following the initial bleeding.

The hemoglobin determinations are made by means of a modification
of Palmer’s method, recently described in detail by one of us (5). This
insures an accurate measure of the hemoglobin, as relatively large
amounts of packed red cells are used. In some of the earlier experi-
ments the Sahli hemoglobin tubes were used.and in some instances
these tubes had faded, giving hemoglobin values which were too high.
The base line of any experiment although too high will not disturb the
fairly accurate curve of regeneration which is more important. A
footnote will be appended to all experimentsin which the Sahli reading
are given.

Red and white cell counts are made in the routine manner. © Identical
counts have been repeatedly obtained by venous puncture and from a
freely bleeding ear puncture. The former is now our routine procedure.

The simple anemia is produced in the following manner: A simple
_ blood volume is performed. The next day the dog is bled one-fourth
the determined blood volume. /This is easily done by inserting a needle
into the jugular vein and aspirating the blood into a calibrated flask
containing oxalate. “The following day the same amount of blood. is
aspirated in exactly the same manner. During these two bleeding days
and the next resting day the dogs are on a bread and milk diet. Fol-
lowing the resting day asecond blood volume is done to determine the
actual amountof anemia produced. The calculated and actual figures do
not correspond but too many factors enter this equation to permit a

154 G. H. WHIPPLE, C. W. HOOPER AND F. §. ROBSCHEIT

discussion at this time: for example, the reserve of blood cells thrown
in from the marrow, to mention only one. At times a third bleeding is
necessary if the reserve has been too great. A third blood volume is
then done. The dog is then placed upon a fixed diet and complete
blood volume, hemoglobin and blood cell determinations are done once
each week thereafter. Special care is taken to insure a sufficient food
ingestion based on the number of calories and nitrogen intake. The
weight curve is a good index of the general nutrition.

The dogs are kept in individul cages which are comfortable and of
suitable size to permit of much exercise. The cages are cleaned once
daily by the attendant, but all feeding is done by the person in charge
. of the experiment. The mixed foods are usually eaten at once when
placed each morning in the cage. Fluids are usually given by stomach
tube. Water is furnished in the cage at all times except in metabolism
experiments when it is usually given by stomach tube. It should be
emphasized again that these dogs were raised in the laboratory and
are therefore healthy, vigorous and very active at all times. The lab-
oratory routine disturbs them not at all and the performance of the
blood volume requires only a few minutes. They will eat all manner
of food mixtures with ‘relish and alacrity. Unless otherwise noted,
these dogs are in their usual healthy, active condition throughout the
entire experiment. ‘‘Mixed diet’? in these experiments indicates a
mixture of food materials obtained from the University Hospital, con-
sisting of bones, bread, cooked meat, potato, rice, macaroni and general
table scraps. .

EXPERIMENTAL OBSERVATIONS

The dogs used in most of the experiments tabulated below are young
animals in the active growth period,—one year of age or less. Such
animals are increasing in size, weight and strength and the demand for
tissue building or growth factors is acute. It is therefore of interest to
keep this fact in mind during our analysis of the subsequent experi-
ments. It might be assumed that the reconstruction of red cells in a
rapidly growing animal might be handicapped by the tissue demand
for normal growth factors. On the other hand, it may be argued that
the growth capacity of the younger organisms might be greater as re-
gards the construction of tissue cells, including the red cells. When
we review the experiments to be submitted in subsequent communica-
_ tions it may be stated that the difference between the adult and the
young dog is not great as regards the capacity of the animal to regener-

9/25] 1726] 1444] 681 | 748 | 51.8] 120 | 0.69] 8,7 | 12,6/15.5 | 93

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 155

ate new red blood cells. This statement applies to young dogs between
6 and 12 months of age and takes into consideration the individual
variations which are met with in different dogs. It is possible that
this statement may not hold for pups less than 6 months of age. In

general we may be safe in stating that if there is any difference between

young and adult dogs, there is a slight difference in favor of the adult

TABLE 1
Blood regeneration—mizxed diet. Dog 19-93. Bull pup, female, age 13 months
7 ? ? g
12 |
az : :
ses 8 o
Bes] aw = ra 5 Zz fe)
3 Zbl S a 5 eB at ° 3
88] 8 5 5 = fa 3 m REMARKS
= abl 6 5 5 a 5 | a
Ss Bal > “ - a 4 a ; ee M4
; (esse | ale|s ef-e | She |i
Soper 6 | 4 | 8 |] epee) a foe | 8] a 8 .
a |e Bk hit Oi) eee oe POR Re
cc cc ce bse tea kgm ce
8/11} 1780) 1280} 580 | 688 | 53.7) 139 | 0.91) 7,6 | 16,2) 12.6} 101

8/12) Diet: Bread and milk

- 8/12) Bled 320 ce. Slight distress. Injection 100 ec. N /1 salt solution

8/13} Bled 320 cc. No distress

$/15| 614| 986] 732 | 249 | 25. 3| 62 | 0. 97| 3,

13. J 76 | Normal

8 /15| Diet: Mixed diet

g/21| 658] 1023] 716 | 292 | 28.5] 64| 0.80] 40| 6,2/13.25| 77 | Diet poor
. : in meat .
8/28} 962) 1210) 728 | 465 | 38.4 79 | 0.69] 5,7 | 10,8/13.3 91
9 /4 | 1280) 1308} 724 | 572 | 43.7) 98 | 0.82) 6,0 | 10,0/14.1 93 | More meat
in food

9/11) 1538} 1420) 673 | 733 | 51.6] 108 | 0.64) 8 4 8,8}14.3 99
9/18] 1612) 1355} 644 | 700 | 51.6) 119 | 0.68) 8,7 | 10,4/14.5 94

10 /2 | 1830) 1490} 675 | 793 | 53.2) 123 | 0.72| 8,5 | 12,0]/15.7 | 95

dogs who at times seem to show a slightly shorter period of blood
regeneration under similar circumstances.

The first three experiments (tables 1, 2 and 3) were performed upon
three young dogs of the same litter, all of the same weight, activity and
general appearance. It will be observed that there are individual dif-
ferences even under these favorable conditions. It is possible that
some of this variation may be explained by individual preferences of the

156 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

different dogs for various food factors. They were all given an excess
of mixed food from which of course they could pick out the bits of food
which they preferred. During much of this diet experiment the “‘mixed
food” was poorer than usual in meat and bones. This explains the fact
that the period of blood regeneration appears somewhat longer than
in some of the subsequent experiments. The explanation for this fact
is found in paper IV of this series, which shows the remarkable efficiency
of a meat diet in promoting blood regeneration.

TABLE 1-3

Experimental history. Dog 19-93

BLOOD
REGENERATION
EXPERIMENT DIET WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
kgm.
Begin 1/16/19 | Rice, potato, | 14385 126 10.95 | Born July 11,1918 —
Bled 590 ce. milk 594 86 10.25
End 3/7 /19 1098 113 10.35 | Maximum regenera-
tion 5 weeks
Begin 3/17/19 | Rice, potato, | 1040 89 11.35 | Table 42
Bled 502 ec. milk (re- 566 82 11.15
End 4/30 /19 peat) 1313 107 11.15 | Complete regenera-
tion 4 weeks
Begin 8/11/19 | Mixed diet 1780 101 12.6 | Table 1
Bled 640 cc. 614 76 13.0
End 10 /2 /19 1830 95 15.7

It is to be noted that all these three dogs increased markedly in body
weight. It should be stated that this was a general growth with increase
in size, length of limb and body, not merely a deposit of fat. One is not
surprised to note a gradual increase in blood and plasma volume during
the experiment. The total volume of red cells, hematocrit reading of
red cells and hemoglobin follow curves which are parallel. The color
index is almost constantly between 0.65 and 0.85 and there is not a very
strong tendency in these experiments for the color index to drop much
below normal in the first two weeks following the hemorrhage. We
shall not attempt at this time to discuss the fluctuations in white blood
cells which are tabulated. |

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 157

The ‘‘pigment volume” is a convenient and expressive term which
indicates the total volume of circulating or effective blood pigment in
the blood stream at the time of estimation of blood volume and hemo-
globin. The pigment volume is the product of blood volume times per
cent hemoglobin. In all tables the pigment volume is the first value

TABLE 2
Blood regeneration—mizxzed diet. Dog 19-94. Bull pup, female, age 13 months
I! =
Bas S = 3 Z 8
eae} # | 2] 2] & ‘yw | 8 :
ae Mart 3 8 2 2 A 3 2 REMARKS
Spas eal - < F z . . ; SI Pa
ge /ese 8 | eis] § So Rolly | ea eae
B } a a : 9 a = ro)
8 a a a ee Wf
| cc. ce. ce. paul cag kgm. | ce.
8 /11| 1705) 1242) 572 | 658 | 53.0} 137 | 0.78) 8,8 7,6| 11.0) 113
8/11] Diet: Bread and milk :
8/12} Bled 310 ce.
8/13} Bled 310 ec. No distress
8/15} 540| 1000| 761 | 230 | 23.0| 54 | 0.84] 3,2 | 6,4|13.5 | 74 | Normal
8/15} Diet: Mixed diet
8 /21| 686] 1090] 768 | 306 | 28.1) 63 | 0.67] 4,7 | 8,6|13.65| 80] Diet poor
in meat
8/28} 800) 1194) 812 | 370 | 31.0} 67 | 0.78) 4,3 | 6,0|13.4] 89
9/4 | 972) 1292} 843 | 443 | 34.3) 75 | 0.66) 5,7 | 12,0|13.9 | 93 | More meat
in diet
9/11} 1032} 1328) 836 | 489 | 36.8) 78 | 0.67| 5,8 | 8,2|14.0] 95
9/18} 1062} 1205} 736 | 465 | 38.5} 88 | 0.69) 6,4 | 6,2/13.8 | 87
9 /25| 1226} 1295} 760 | 524 | 40.4; 95 | 0.68) 7,0 | 7,8 |14.45} 90
10 /2 | 1298; 1276) 710 | 554 | 43.4) 102 | 0.74| 6,9 | 17,4 14.55) 88 | More meat
in diet
10/9 | 1520) 1463} 827 | 624 | 42.6) 104 | 0.73] 7,1 | 13,6 115.5 | 95
10 /23} 1550) 1463) 823 | 620 | 42.4) 106 | 0.74) 7,2 | 8,8 |16.25) 90

given as we believe it gives the best general index of the curve of blood
regeneration.

Table 2, dog 19-94 is an experiment with a dog presenting some un-
This dog at times shows an eosinophilia yet
only an occasional parasite egg can be demonstrated in the feces.

known abnormal factor.

158 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

Treatment by oil of chenopodium and santonin has yielded no results.
The dog is not as well nourished as the others of this litter and at times
presents a slight relative degree of anemia. This fact is to be consid-
ered in a study of the abnormally slow blood regeneration in this dog.
In spite of this the dog gained about eleven pounds during the course
of the experiment.

Table 3 at the start of the experiment shows the remarkably high
- figure (152 per cent hemoglobin) which may be observed in normal dogs.
Red blood counts of 7 to 9 millions are the rule.

TABLE 2-3
| Experimental history. Dog 19-94

BLOOD
REGENERATION
EXPERIMENT DIET : WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
kgm. |. .
Begin 1/16/19 | Bread, 300] 1250 126 10.65 | Born July 11, 1918
Bled 620 ce. grams, milk 462 84 10.00
End 3 /7 /19 500 cc. 899 94 11.70 | Maximum regenera-
tion 5 weeks
Begin 3/17/19 | Bread, 300] 1146 93 13.05 | Table 27
Bled 608 cc. grams, milk 542 78 12.40
End 4/30/19 500 cc. (re-| 1081 99 13.20 | Maximum regenera-
peat) tion 5 weeks |
Begin 8/11/19 | Mixed diet 1705 113 | 11.00 | Table 2
Bled 620 cc. B40) a TR 13.5
End 10 /23 /19 1550 90 16.25

October 9. Two doses of oil of chenopodium, 48 hours apart. Few ova found. —

No worms expelled. Last dose followed by santonin mixture. No effect.

Table 4 shows an experiment upon a young dog (6 months) in which
one week’s diet of rice, potatoes and milk followed the bleeding—then
the usual mixed diet. The dog did not eat the rice, potato and milk
diet and lost much weight. Also she developed signs of mild distem-
per, but this soon cleared up after being put on a liberal mixed diet.
There was then a rapid gain in weight as well as in blood regeneration.
The leucocytosis is probably to be explained by the mild infection with
distemper. The total regeneration of red cells is complete in one
month.

TABLE 3

Bull pup, female, age 13 months

Blood regeneration—mizxed diet. Dog 19-95.
I 5
ae tela| i :
sas @ bp 5 < 4 4 i
MeL Sel s 8 a FI A 3 6 REMARKS
a ap 5 > ° | =} =) a
Mabe el. Fe hg S . <1 a ; es 4
ieee le |e.) o. |S Bde bl el!
Seen os) ah) se ee pe eo} we phe be |g
a |. See we ew le tT ele a te Eee Le
cc. ce. cc. teen ‘at kgm. ce
8/11] 2088] 1369} 568 | 794 | 58.0} 152 | 0.88) 8,6 | 10,4 |13.2 | 104
8/11] Diet: Bread and milk
8 /12| Bled 342 cc.
8/13} Bled 342 cc. No distress
8/15| 750| 1005| 700 | 300 | 29.8] 74 | 1.03} 3,6 | 8,4 l13.65| 74 | Normal
8 /15| Diet: Mixed diet
8/21) 843} 1106] 728 | 367 | 33.2} 76 | 0.93] 4,1 | 17,8 /14.2 | 78 Diet poor
; in meat
8/28} 829) 1066] 747 | 414 | 35.5} 78 | 0.75) 5,2 | 10,2 |14.0 | 838 :
9/4 | 954| 1243) 814 | 423 | 34.0} 77 | 0.71) 5,4 | 19,0 |14.65| 95 |{More meat
9/11] 1313) 1338} 726 | 600 | 44.8) 98 | 0.65] 7,5 | 13,2 |14.5 | 92 in diet
9/18) 1325] 1243} 674 | 552 | 44.4) 107 | 0.74) 7,2 | 16,0 }14.65) 85
9 /25| 1730| 1412} 678 | 720 | 51.0) 122 | 0.74) 8,2 | 13,0 }15.25) 93
10 /2| 1874) 1410|.618 | 779 | 55.2) 1383 | 0.75) 8,8 | 16,415.45} 91
10/9 | 1655) 1343] 654 | 676 | 50.3) 123 | 0.66) 9,0 | 14,4 15.70} 86
TABLE 3-3
Experimental history. Dog 19-95
BLOOD
REGENERATION
EXPERIMENT DIET WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
kgm.
Begin 1/16/19 | Rice, potato, | 1232 100 11.35 | Born July 11, 1918
Bled 564 cc. milk 560 75 11.00
End 3/7 /19 983 90 11.20 | Complete regenera-
tion 5 weeks
Begin 3/17/19 | Rice, potato, | 1092 88 12.80 Table 41
Bled 566 cc. milk (re- 570 75 12.45
End 4/30/19 peat) 1237 98 11.95 | Complete regenera-
tion 5 weeks
Begin 8/11/19 | Mixed diet 2088 104 13.20 | Table 3
Bled 684 ce. 750 74 13 .65
End 10/9/19 1655 86 15.70 | Maximum regenera-
tion 7 weeks

159

be TABLE 4 : a

Blood regeneration—mized diet (following rice, potatoes and milk). Dog 19-96.

. Bull pup, female, age 6 months

12 =
i o

a nee z 8 3 a a a = REMARKS
bens HAE > > iq a = : Ay
ye etna =e aes aie Sti ot | Sod taaee
gd | S53) 9 i : : § . es 9. |*o
g {oma & $ . oe ee = a 3 o
a | fm Fy ta es x S a z z a
ce. ce. ce. Deus se kgm. | ce.

1/16} 1200} 1025) 488 | 540 | 52.3) 117 | 0.58) 10,1 | 20,8 |10.15} 101

1/16} Diet: Crackermeal and milk

1/17| Bled 256 cc.

1/18} Bled 256 cc. No distress

1/20 514| 778| 574 | 196 | 25.2| 66 | 1.10 3,0 16,0 | 9.75 80 | Normal

1/20} Diet: Boiled rice, 200 grams; potatoes, 200 grams, milk, 500 ce. .

1/27} 422) 826) 604 | 218 | 26.4) 51 | 0.48) 5,3] 12,4} 8.60) 96] Mild dis-

| temper

1 /27| Changed to mixed diet*

2/3 | 765| 915) 554 | 357 | 39.0} 84 | 0.67} 6,3) 15,8 /10.1 | 91 | Recovered
from dis-
temper

2 /12| 1013) 1080) 574 | 500 | 46.3) 94 | 0.65] 7,2] 12,0 /11.35) 95

2/19} 1243) 1243) 646 | 584 | 47.0) 100 | 0.71) 7,0} 12,8 |11.80) 105

2 /28| 1288} 1108] 537 | 554 | 50.0) 116 | 0.73} 8,0] 9,2 414.95} 98

* Developed mild case of distemper. Refused to eat rice and potatoes. :

TABLE 4-3

Experimental history. Dog 19-96

BLOOD
REGENERATION
EXPERIMENT DIET WEIGHT REMARES
Pigment | Blood per
volume | kilogram
kgm.
Begin 1/16/19 | Rice, potato, | 1200 101 10.15 | Born July 14, 1918
Bled 512 ce. milk for 1 514 80 9.75
End 2 /28 /19 week. Mixed | 1288 93 11.95 | Table 4
diet for 4 weeks
- Begin 3/17/19 | Rice, potato, | 1520 92 13.50 | Table 44
Bled 620 ce. milk 527 72 12.40
_ End 4/30/19 778 95 9.90 | Maximum regen-
eration weeks
Begin 8/11/19 | Mixed diet 1750 98 | 13.65 | Table 5
Bled 668 ce. 650 72 13 .35
End 10 /2 /19 2012 98 15.45

160

x
as
a
i baer

Ve a ee

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 161

Table 5 shows a second experiment on the same dog, 19-96, given in
table 4. The first experiment was done at the age of 6 months and this
experiment at the age of 13 months. The period of blood regeneration
is longer in this experiment as we believe the correct explanation is the
low meat content of the mixed diet during this period. This was also

TABLE 5
Blood regeneration—mizxed diet. Dog 19-96. Bull pup, female, age 13 months

i] E es F
bE {2 | § :
Sam Ss) a loa [ue 6 8
OgA 5 i) p < ‘al g |

a > oe > 5 3 3 a 5 3 e REMARKS
S |EBa ¢ > > i Z F ‘y

onl ho <q : a al ‘ .
ie Aso] 8 Ss ts) is) @ ° ° a a
] s2S3} o a . f : ra) : a g °
& | otal 8 < eS " Fr) 2 “ ; eI 8
a | & a a ni ea ee 5 a ES Fe
per | per
ce. cc. | ce. jeter ES 1 Bay kgm. | cc
8/11} 1750) 1335} 658 | 670 | 50.2) 131 | 0.90} 7,3} 14,6 |13.65) 98

8/11) Diet: Bread and milk

8 /12| Bled 334 ce.
8/13} Bled 334 cc. No distress.

8/15] 650| 964] 684 | 275 | 28.5| 67 | 0.90] 3,7 | 21,6 [13.35] 72 | Normal

8/15) Diet: Mixed diet

8 /21| 660} 993] 686 | 297 | 29.9} 66 | 0.80} 4,1| 12,0/13.9 |. 71 | Diet poor
in meat
8/28} 801} 1155) 770 | 368 | 31.9) 69 | 0.66] 5,2| 12,4|14.05] 82 ;
9/4 | 1054| 1340] 852 | 474 | 35.4| 7910.72] 5,5] 16,2|14.7 | 91 Pee meat
9/11} 1255] 1340| 742 | 596 | 43.7| 94 | 0.64] 7,3] 18,2|14.65} 91 |{ in diet*
9/18] 1348] 1225] 639 | 575 | 46.9] 110 | 0.65] 8,5] 10,8 |14.85] 83
9/25] 1511) 1280] 634 | 630 | 49.2] 118 | 0.59] 10,0| 9,6 |14.90| 86
10/2 | 2012} 1517] 665 | 846 | 55.7) 133 | 0.68] 9,8 | 14,0 |15.45} 98

* Poikilocytosis of red cells of moderate degree. Experimental history, table
4-b.

noted in tables 1, 2 and 3, which experiments were all done at the same
time. Our experiments are usually carried out in groups of four.
Table 6 illustrates several points. This dog had been under obser-
vation for some time under a variety of dietary conditions (see experi-
mental history—table 6-b). A period of sugar feeding gave no blood
regeneration (refer to table 16 in the following paper) and 2 weeks of
meat feeding had caused a considerable rise in the pigment volume.

162 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

The increase in pigment volume brought this figure back to normal in
4. weeks of mixed diet. The blood volume figures are based on deter-
minations made with dry oxalate. This procedure, as has been pointed
out, causes a shrinkage of cells and a dilution of the dye which gives
blood volume figures abnormally high. Note that the blood volume
figures per kilo are 100 to 136 cc. This fact may account for some of the
fluctuations in the estimated plasma volume.

TABLE 6

Blood regeneration—mized diet (following metabolism experiment). Dog 17-28.
Bull dog, female, adult

Q
ll S gs |
BH [e=] &
sha 52 3 o
PE S| ga = a fe) Z g
WAP s = a ° =)
OBR! 6 c} 5 < 5 rr}
POO] RP 3 a s a] 4 ¢ REMARKS
= ae a) > S) a A 5 a
b e “1 8 > < é oe Z x ° i] Ry
« a5) 8 si iS) co) ¢ 3 ¥ ee A
/ a 32 5 ° Q . : . ° 5 9 M4 8
E eta) Slog ai] | ck) Bo] ed Gil fay og
aA |e a ey a a = 5 a z B a
per | per
cc. -|. CC. cc Raent Sal RirsaicoPets BE kgm. | ce.
1/19) 1620} 1500} 600 | 900 | 60 | 108 | 0.76} 7,1 | 7,4 |11.60) 129

Diet: Sugar diet, January 19 to February 23. Lean meat diet, February
23 to March 10. Mixed diet, March 10 to May 7. (Refer to table 16)

3 /16| 1010) 1246) 735 | 511 | 411] 81 | 0.64] 6,3 | 7,2 |10.40) 119 | Good con- —
dition
3 /23|' 973] 1158) 660 | 498 | 43 | 84 | 0.64] 6,5 | 10,6) 9.90) 116
4/13} 1910} 1645) 757 | 888 | 54 | 116 | 0.71} 8,2 | 22,0 |12.30} 134
4 /20| 1385] 1260] 630 | 630 | 50 | 110 | 0.66] 8,3 | 9,8 12.60} 100
5/7 | 1895) 1709} 769 | 940 | 55 | 111 | 0.66) 8,4 | 13,2 |12.50} 1386 | Good con--
dition

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

Table 7 is placed in this paper to give a comparison between the mixed
diet experiments and fasting periods. It will be noted that during
this fasting period the blood regeneration is very slight. This point —
will be discussed at length in the next paper.

DISCUSSION

From a review of the tables it is at once obvious that the pigment
volume and total red cell volume after hemorrhage are below the expected
values. There are several obscure factors in this reaction which call

i? ee ae See

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA |

TABLE 6-8

Experimental history. Dog 17-28

163

BLOOD
REGENERATION
EXPERIMENT DIET WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
kgm.
Begin 9/13/16 | Bread and milk 8.4 | No blood volume
. data given
Bled 450 ce. 8.4
End 10/25 /16 8.8 | Hb. and R.B.C.
back to normal
Begin 1/19/17 | Sugarandmetab-| 1620 129 11.6 | Table 16
Bled 750 ce. olism 549 87 10.4 | Lean beef diet 2
weeks followed
by mixed diet.
. Table 6
End 2 /23 /17 541 117 8.0
Begin 5/7/17. | Sugar+ R.B.C., | 1895 136 12.5 | Table 76
’ Bled 854 cc. metabolism 594 76 ie 0 T:
End 6 /18 /17 1085 154 8.3 | Put on bread and
milk diet
Begin 9/11/17 | Sugar 2 weeks 1650 113 | 14.0
Bled 794 cc. Sugar 2 weeks + 751 84 13.5
End 10/19 /17 diamino-acid 1040 106 10.0 | Beef heart 3
of gelatin weeks, mixed
diet 3 weeks
Begin 6/3/18 .| Desiccated kid- | 1739 73 17.65
Bled 949 cc. ney, bread and 792 62 16.50 | (8 bleedings)
End 6 /28 /18 milk 1300 74 16.10
Begin 8/9/18 | Sugar and Hb. | 2417 93 17.00 | Table 80
Bled 1076 cc. intraperitone- 756 66 15.90 | (3 bleedings)
End 11 /13 /18 ally 2000 106 15.05
Begin 12/2/18 | Lean meat and | 2118 105 15.70 | Table 51
Bled 1140 ce. gelatin 708 82 15.05 | (8 bleedings)
End 1 /22 /19 1380 99 14.65
Begin 5/1/19 | Fasting 2018 97 15.90 | Table 14
Bled 980 ce. 763 71 14.75 | (8 bleedings)
End 5 /21 /19 1007 86 11.50 | Found dead.
Renal calculi

164 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

for discussion. Let us take a type experiment with simple values. A
dog with a hemoglobin of 100 per cent, hematocrit of 50 per cent and
blood volume of 600 cc. will have a pigment volume of 600. On two
successive days the dog is bled one-fourth of the estimated blood vol-
ume. Allowing for replacement of plasma which is known to take

place, we should expect after these bleedings a hemoglobin of 55 per

TABLE 7 :
Blood regeneration—fasting. Dog 17-27. White bull mongrel, female, adult
ug 2
ty 2 a
2e5 a a } Z S
Br ee 2 ae ae vis : |
BOO! RB 3 a = = 3 - REMARKS
cs) ep 5 > ° a A. s a
S> |HRal 5 > ty Z s : My
_ ZHO < : . ed x 3 EA
- H-5| & s is) is) 8 s) 3 = a
<3) sO Q ° n : : ¥ ° : Q 5 °
Per Re ae Bl eee oe. ae
A |e ) iy rd 8 q i) a E E =)
cc ce ce ae poe’ kgm. | ce.
3/11) 2100) 1500) 735 | 766 | 51 | 140 | 0.99 7,1 10,4| 15.8} 95
3/13} 1627| 1122} 561 | 561 | 50] 145 | 1.03] 7,0 | 8,0| 15.7} 72 ’
3/13| Diet: Bread and milk
3/13} Bled 375 ce. No distress
3/14; Bled 375 ce. No distress
3 /16 1005| 1116] 770 | 346 | 31 | 90 | 1..05| 43 [12,4| 15.5) 72] *
3 /16| Fasting begun
3./22) 1165) 1153) 726 | 427 | 37 | 101 | 0.90) 5,6 | 6,8) 18.8) 7% 2
3 /29| 1086} 1075) 656 | 419 | 39 | 101 | 0.67| 7,5 | 6,2] 12.3) 88 x
4/4 | 1213} 1054) 622 | 483 | 41 | 115 | 0.65) 8,8 | 6,8] 11.6) 91 ”
4/10} 1240) 1000} 620 | 380 | 38 | 124 | 0.95} 6,7 | 6,2] 10.7} 93 | Excellent
condition

* Slight anisocytosis.

Blood volume with dry oxalate. Hemoglobin by Sahli tubes. Experimental
history, table 15-b. See below.

cent and total volume of red cells 165 cc. The plasma is constant under
such conditions and will be as normal, 300 cc. volume. The blood
volume therefore is 465 cc. and pigment volume 256 (465 0.55) which
is 43 per cent of the pigment volume at the beginning of the experi-
ment. If there is no reserve red cell influx we should expect a red cell
volume of 55 per cent and a pigment volume of 43 per cent normal fol-
lowing two bleedings of one-fourth the total blood volume. The tables

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 165

q show figures which are consistently below the expected values. This

calls for a broad discussion of the various methods of blood volume de-
termination and possible errors inherent in the various methods. We

- do not wish to review all these points at this time but hope soon to do so
- in connection with experiments dealing with the actual technique of

blood volume determination.

Two possibilities are to be mentioned, however, in this discussion.
First the blood volume values may be too high, calculated by the dye
method, and the actual bleedings represent more than 50 per cent of
the total blood volume. Second, the possibility that the body has the
power to modify the ratio of cells to plasma in different parts of the body

_ under normal or abnormal conditions. It is easy to mention these two

possibilities but very difficult to adduce experiments which are conclu-
sive. For example in the abnormal condition of shock it seems clear
that there is a remarkable disturbance in the ratio of cells to plasma in
different parts of the body.

_ It may not be wise to pursue this question further but best merely to
call the reader’s attention to the fact that there is a discrepancy be-
tween the expected and actual values for red cell volume and pigment
volume after unit hemorrhages. This point will be taken up again.
The pigment volume figures, however, give a clear-cut ings of the curve

of blood regeneration.

SUMMARY

The term pigment volume used in these papers is equivalent to blood
volume times per cent hemoglobin. This means the volume of avail-
able red cell pigment circulating in the body at the time of blood volume
determination.

Given a uniform degree of anemia we may observe the curve of red
blood cell regeneration as influenced by a variety of diet factors. Ane-
mia is produced by bleeding the dog one-fourth of the determined blood
volume on each of two successive days.

Under these experimental conditions a diet of ue table scraps
will effect complete blood regeneration to normal in a period of 4 to
7 weeks. ri

Under similar experimental conditions there will be little blood
regeneration during a fasting period—mainly a maintenance factor
equivalent to the normal daily wastage of red cells.

’

(1) ‘Hooper AND SW beta

_ (2) Hoorzr anv Wurppie: This Toit
(3) WHIPPLE AND moyen: This i”

A .
¢ &
By
s .
. i
‘ 2 P
i
i?
. : .
rf
{
.
.

‘
F
f
j

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA

II. Fasting CoMPARED WITH SuGAR FEEDING

Analysis of ‘Sparing Action of Carbohydrates’
G. H. WHIPPLE, C. W. HOOPER anp F. 8. ROBSCHEIT

From the George Williams Hooper Foundation for Medical Research, University of
California Medical School, San Francisco

Received for publication April 3, 1920

That there is a distinct difference between the blood regeneration
during fasting periods as compared with sugar diet periods comes out
from an analysis of the experiments given below (table 25-a). This
difference in blood regeneration is not great but is distinctly in favor of
the fasting condition,—in other words, a dog will form more red cells
and hemoglobin during a fasting period than during a similar period of
sugar feeding. In neither case is any nitrogenous material taken into
the body, so whatever hemoglobin and red cell stroma may be formed
must be constructed in the body from body protein or protein split
products. The well known “sparing action of carbohydrates” must be
considered in the analysis of these experiments given below. It appears
that these experiments can be explained most satisfactorily on the basis
of a certain protection or sparing of body protein on the part of the
sugar, associated with a definite amount of conservation of protein split
products.

Having established the curve of blood regeneration which is the result
of a mixed diet subsequent to the anemia period, we wish to present —
experiments to show the type reaction associated with fasting or sugar
feeding. In making any analysis of results it is necessary to know how
much reserve capacity the normal dog possesses—how much regenera-
tion of red cells or hemoglobin can be effected during periods of fasting
or sugar feeding. We must take into consideration too the daily wear
and tear of the red cells which is not an accurately established factor.
We do not know the life history of the red cell in the normal or anemic
dog. We do know the duration of life of the red cell in the normal
human being (1), but this life cycle may be different in disease. The

167

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 2

168 G. H. WHIPPLE, C. W. HOOPER AND F. 8. ROBSCHEIT

dog’s red cells are extremely fragile and this may or may not indicate a
shorter life cycle for dog than the established 30-day period for normal
human beings. Data on this point are very much to bedesired but in
their absence we must postulate an unknown factor of red cell or hemo-
globin replacement which is present in all our tables.

Any diet, therefore, which is capable of giving a rising curve of blood

regeneration following simple anemia is doing two things. The diet is —

responsible for a replacement of the red cells (3 to 5 per cent per day)
which are worn out day by day, as well as the rise in general level of red
cell volume above the anemia level. For the sake of analyis we may
assume that the replacement value for human beings and dogs is the
same—about 3 per cent per day or complete replacement of the total
volume of red cells in approximately 30 days. Under most favorable
conditions we may see the volume of red cells regenerate from an ane-
mia level of one-third or one-half normal back to 100 per cent within 4
or 5 weeks. To supply a deficit of 50 to 65 per cent of its red cell volume
the body requires 30 days or more over and above its maintenance of
red cell wastage. This wastage (wear and tear of red cells) in human
beings may be 100 per cent in 30 days. This indicates the importance
of this replacement factor and further emphasizes the fact that our
curves show the reaction of the body in excess of this i. or replace-
-ment value.

. When we note a falling curve of hemoglobin and red cells after a ina
diet period we need not hastily postulate hemolysis or blood destruction

from some hypothetical toxin. It may be safer to consider the possi- —

bility that the body can form no more hemoglobin or red cells to repair
the daily wastage. Even the replacement fraction is not being supplied
and the curve subsides gradually depending upon the life cycle of the
red cells remaining.

EXPERIMENTAL OBSERVATIONS

The experimental procedures have been described in detail in the
preceding communication. The majority of these dogs have been un-
der observation in the laboratory since birth and we have studied their
reaction to simple anemia following hemorrhage as influenced by a
variety of diets. Some of these observations precede and others follow
these tabulated fasting or sugar feeding experiments. The experimental
histories given with each animal give a review of the many experiments
done on the same dog. The value of comparison under these conditions

tts
a ee SS le
eee

:

7

:
1)
;
A
‘

;

;
y.
:
iy
i

:

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 169

i is greater than obtains in a series of isolated experiments. The dogs
- were all very fat and well nourished at the beginning of these experi-
_ ments and were able to tolerate the fasting or sugar periods without
_ disturbance of health or activity.

During the metabolism experiments the dogs were kept in standard

: metabolism cages constructed with sharp pitch of the cage bottom to
_ insure rapid and complete drainage of urine. The dogs were catheter-
ized every 24 hours and the catheterized specimen, bladder washings

TABLE 8

Blood _regeneration—fasting—metabolism—s plenectomy. Dog 17-37. White bull

mongrel, female, age 10 months

=
' g = <
AR S ie<}
a8 fea] 4 S S
bee a = a } 4 Me
=] z p s = & ° 5
OBA R b < ra i i

BOO 2 1 | = a
» > 2 ° ) eI a =| 8
— = ° > <3)
S a8 . < F . z . ; -
ol a-8 =) 3 3 S) 8 'S) bg = ra)
io} S25 ° m : . e fo) : x i) °
& od a S < 9 ® 2 a . . a 3
) ey E Fy 0 a ss 5 Fe B E3
ce ce. cc per cent|per cent kgm. ec.

3 /26 927 | 850 | 459) 391 46 109 | 0.80 | 6,8 9,4 | 10.60} 80

3/26 | Diet: Bread and milk

3/28 | Bled 212 ee.

3/29 | Bled 212 ce.

3/30 | 313 | 626 | 451| 175 | 28 | 50 | 0.68 | 3,7 | 15,4 | 10.40 60
3/30 | Fasting begun

4 /2 403 | 651 | 475 | 176 | 27 62 | 0.74| 4,2 | 6,4} 9.50) 70

4/9 492 | 769 | 500} 269] 35 64 | 0.64 | 5,0 | 7,2 | 8.50} 90
4/16 | 532 | 729 | 474] 255 | 35 73 |0.66| 55 | 5,0] 7.70} 96
4/23 | 540 | 772} 502 | 270} 35 70 | 0.66 | 5,3.| 7,8 | 6.90) 112

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

and cage urine were made up to a uniform volume. A mixed speci-

men was preserved and duplicate Kjeldahl analyses made. A few cubic
centimeters of acetic acid in the cage collection bottle insured an acid
reaction in the urine. With care a male or female dog may be catheter--
ized daily without causing any cystitis. After the catheterization the
water or sugar solution was given by stomach tube.

Fasting experiments. The two preceding experiments (tables 8 and
9) are to be compared as the experiments were done at the same time.

s
TABLE 8-a
Total urinary nitrogen—fasting. Dog 17-37
DATE, 1917 Stites palee de eocaet WEIGHT REMARKS
grams ce. pounds
March 31 2.80 650 21.5 0 feces
April 1 2.30 405 21.1 Trace of feces
2 2.58 433 20.8 0 feces
3 3.19 345 20.6 0 feces
4 2.97 395 20.3 0 feces
5 3.56 418 19.9 Slight diarrhea |
6 2.83 460 19.6 0 feces :
7 2.88 19.3 0 feces
8 2.49 427 19.0 0 feces
9 2.80 385 - 18.8 0 feces.
10 2.91 473 18.3 0 feces
11 2.69 359 18.3": 0 feces
12 2.91 423 17.9 Trace of feces
13 2.60 371 17.6 0 feces
14 2.60 429 17.5 0 feces
15 2.85 411 17:3 0 feces
16 2.86 414 ge | 0 feces
17 2.60 447 16.8 0 feces
18 ae ff 458 16.5 0 feces —
19 3.19 396 16.3 0 feces
20 3.02 421 16.1 Trace of feces.
21 3.42 416 15.8 0 feces
22 3.56 445 Ey f 0 feces
23 3.86 471 pe | 0 feces

Dog given 400 ce. of water daily by stomach tube.

TABLE 8-3
Experimenal history—dog 17-37—splenectomy
BLOOD
REGENERATION
EXPERIMENT DIET WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
kgm,

Begin 12/15/16 | Sugar + gela- 630 66 10.5

Bled 350 ce. tin 10.2

End 1 /11 /17 544 84 8.1 | Maximum regenera-
tion 2 weeks, then
lean meat diet 3
weeks. Mixed
diet

Begin 3/26/17 | Fastingmetab-| 927 80 10.6 | Table 8

Bled 424 cc. olism 313 60 10.4 | Table 73

End 4/23 /17 540 112 6.9 | Bread, milk, Blaud’s
pills, 11 weeks.
Slight regenera-
tion

Splenectomy 10 /23 /16.

170

a ees "

= ee ee

> PSR.) ee a ee ye ee ee

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 171

The dogs are of the same litter but one had been splenectomized. We
can make out no difference in the reaction of the splenectomized dog

as compared with the control under these experimental conditions.

During the fasting period there is a steady rise in hemoglobin, red cell
hematocrit, red cell count, total red cell volume and pigment volume.
The rise is most pronounced during the first week, asa rule. It is noted
that the plasma volume remains constant or decreases slightly. This

TABLE 9

Blood regeneration—fasting—metabolism. Dog 17-38. White bull mongrel, female,
age 10 months

I =
Bee) 2 | 8 |e : :
ofa a p 5 < 4 5 i
a poo 3 a ic 2 a 3 Po
= mm 3 > 9° a a 5 a
= Ha = a ies z a " e &
oe Pee ee 3 3 3 3 3 . 2 a
eee eS a eed | we | So) mid We gg | 8
a ie 2 By Fs Fs ss 8 ei Fs B Fr
cc. ce. cc per cent|per cent kgm. ce.
3/26 | 1395 | 1090 | 490 | 600); 55 128 | 0.80 | 8,0 | 15,8 9.8; 111
3/26 | Diet: Bread and milk
3/28 | Bled 272 ce.
3/29 | Bled 272 cc.
3/30 | 443| 738| 531| 207| 28 | 60|0.73| 41 |122| 9.3| 79
3/30 | Fasting begun
4 /2 532 | 819 | 573 | 246] 30 65 | 0.74 | 4,4 | 10,6 8.6} 95
4/9 491 | 723 | 441 | 282); 39 68 | 0.65 | 5,2 9,6 7.5 96
4/16 585 | 770 | 4389 | 33811] 48 76 | 0.62 | 6,1 | 12,0 6.8 | 113
4/20 | 585 | 750| 420] 330] 44 78 | 0.51 | 7,6 | 11,4 6.3 119

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
_ Experimental history, see table 20-b.

fact accounts for the increase in blood per kilo, due to loss of body
weight.

The nitrogen figures for total urine are of interest. The daily output
is fairly constant until we reach the last 4 days of the experiment which
show a distinct increase above normal. It is possible that this increase .
represents the body protein disintegration which may be observed after
a long period of fasting, giving rise to the premortal rise in nitrogen.
Both these dogs recovered the lost weight promptly when placed upon

172 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

a bread and milk diet. There is no evidence for an increase in body
protein katabolism to supply the essentials for hemoglobin construc-
tion. The same remarks apply to the following experiment (table 10).

The above experiment (table 10) shows a considerable increase in
red cells from 5,200,000 to 7,000,000 with an unchanged red cell hemato-
crit. Poikilocytosis is marked during this period and there is evidence

TABLE 9-a

Total urinary nitrogen—fasting. Dog 17-38

TOTAL
DATE, 1917 \ ie a aa bbs pein WEIGHT REMARKS
grams ce. pounds
March 31 3 a2. 720 19.4 0 feces
April 1 2.16 412 19.1 0 feces
2 2.18 471 18.8 Trace of feces. Vomited
3 2.77 236 18.6 Moderate feces |
4 2.74 18.1 0 feces
5 2 .94 430 17.9 0 feces
6 2.86 442 17.7 0 feces
7 8:25 428 17.3 Slight diarrhea
8 2 .86 422 ay 0 feces
9 2 .66 432 16.7 -0 feces
10 2.97 - 403 16.3 Trace of feces
11 2.46 413 16.2 0 feces
12 2.83 434 15.9 Slight diarrhea
13 2.72 883 15.7 0 feces
14 £72 443 15.5 | 0 feces
15 3.05 446 15.1 0 feces
16 3.58 418 14.9 0 feces
17 3.42 408 14.7 0 feces
18 3.25 441 - 14.4 0 feces
19 4°14 441 14.1 0 feces
20 4.09 726 13.8 0 feces
21 4.17 517 14.3 Slight diarrhea

Dog given 400 cc. water daily by stomach tube.

for a certain amount of fragmentation of the red cells in this and other
experiments of similar nature. In some instances there is evidence for
a faulty construction of red cells during periods of stress when red cell
regeneration is being accomplished with difficulty on a limited diet.
It will be possible with the accumulation of much data to ascertain
which diets favor stroma construction and which diet factors accelerate
hemoglobin construction. It is evident at present that these two fac-

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 173

tors do not always run parallel under experimental conditions, as well
as in disease.

TABLE 10
Blood regeneration—fasting—metabolism. Dog 16-160. Bull mongrel, female,
~ age 2 years
ig a
Bes a 5 S 3 3 7
eee} 2) a | 2 | § atl 3 :
boo; 8 3 a = a | jp REMARKS
hc} a > io) hed ° ‘a oS io
S |eae] > < . mn a a L ey Ey
« Q -8 a s o o a o e a
a so 3 ° D ‘ * ‘ ° ff Py o fo)
> om Q ° < = = ie) =| ioe) a °
a |& PP Rb he a | Sh ot LP Ree
ce. ce. ce. ras Rat kgm. cc.
5/3 1241] 633 | 590 | 47.6 10.45] 119
5/3 | Diet: Bread and milk
5/6 45.2} 90 | 10.00
5 /20 | 1316] 1125) 523 | 591 | 52.5) 117 | 0.70) 8,3 | 14,2 |10.60) 106 | R.B.C.frag-
: . ment+ +

5 /21 | Bled 281 cc.
5 /22 | Bled 281 ce.

5/24) 482| 752| 492 | 250 | 33.2| 64 | 0.55] 5,8 | 14,8 '10..20| 74 |

5/25 | Fasting begun. Metabolism

5/31 | 585| 688] 455 | 226 | 32.8] 85 | 0.82/ 5,2 | 6,0| 9.10] 76 | * Poik.
6/5 | 591| 672| 437 | 225 | 33.5] 88 | 0.72] 6,1 | 6,8| 8.40] 80] * Poik.++
6/12 | 636] 707| 457 | 248 | 34.3] 90 |-0.64| 7,0 | 8,2| 7.55} 94 | * Poik.t++

6/12 | Diet: 200 grams bread and 300 cc. milk. Metabolism discontinued

6/18 | 696} 791| 510 | 274] 34.6] 88 | 0.881 5,0 | 5,4] 8.15} 97 | * Poik.++
6/26 | 451| 550] 360 | 184 | 33.5} 82 | 0.681 6,0 | 9,0| 8.20] 67 | * Slight

. ’ poik.
‘

6/26 | Diet: Changed to mixed diet

* Poikilocytosis of red cells.
Experimental history, see table 18-b.

| This second group of experiments (tables 11, 12 and 13) presents
several factors in common. During the fasting period the blood regen-
eration was notable in two experiments during the first week following
the bleeding. In fact this level was scarcely increased during the sub-

174 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

sequent fasting period. This rapid increase during the first week may
be explained by some reserve factor which is called in during the emer-

gency period. The following weeks show little increase in pigment —

volume because the body can only supply the material needed to re-
place the daily wear and tear on the red cells. The third experiment
(table 12) shows a more gradual rise in the pigment volume, hematocrit
and hemoglobin during the entire fasting period.

TABLE 10-4
Total urinary nitrogen—fasting. Dog 16-160

TOTAL
DATH,-1918 eo aceen ben rete dyee WEIGHT REMARKS
grams ce. pounds
May 26 1.76 270 21.8° | Some water vomited
27 1.90 120 21.4 About 50 ce. vomited; 0
feces ;
28 1.85 145 20.8 No vomiting. 0 feces; dog
very active
29 2.07 HS. 20.6 No vomiting. 0 feces
30 2.02 155 20.3 0 feces
ie 2.10 188 20.1 0 feces
June 1 2 .02 199 19.8 0 feces
2 1.96 134 19.3 0 feces. 300 cc. water
3 2.44 167 19.1 0 feces. Good condition
4 2.16 206 18.9 0 feces
5 2.41 180 18.5 Solid feces
6 ya Bs! 207 18.3 Little feces
7 2.02 >) a 17.9 0 feces
8 1.79 191 17.6 0 feces
9 1.90 186 17.4 0 feces
10 2.02 181 17.3 0 feces
11 1.90 178 17.0 0 feces
12 2.30 190 16.6 Feces + in urine. Dog in
excellent condition
13 1.73 * | 205 16.4 Soft feces

Dog given 200 cc. water daily by stomach tube.

All three experiments show a rapidly developing ‘‘dietary deficiency
disease’ which develops after a period of bread and milk feeding sub-
sequent to the fasting period. This dietary deficiency condition is
characterized by ulcerated mucous membranes and much gastro-intes-
tinal disturbance. We are inclined to the opinion that this condition
is analogous to scurvy in human beings. In the near future we hope

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 175

. TABLE 11

_ to report a series of experiments bearing upon this point, making clear
_ the factors concerned in the development of this abnormal condition
as well as its cure and prevention in the dog.

Blood regeneration—fasting. Dog 18-103. Brindle bull, female, age 1 year

ll =
a ; :
Pier
st Q 5 % °
eas} 2 | 8 | 2 | § n | 8 :
POO! 8 3 3 = el | 2 REMARKS
4 a a) > Qo. = ss 3 2)
= ons > < m “ 4 : s e Ay
* 8:26 a s io) oO @ ie) e q a
Segeeike | 2 | acl | gd Bow] A els
a |& a|e|/a@}] a |h] &§ | & |] &] e] a
ce. ce. Ce. il poi kgm. ce.
‘8/28 1540] 1115] 552 | 547 | 49.0] 138 | 0.97 7,1 | 16,0 |15.45) 72
8/28} Diet: Crackermeal and milk
8/28) Bled 279 cc.
8/29} Bled 279 cc.
8/31} 810, 965| 650 | 311 | 32.2] 85| | | — [14.05] 65 |
4 8 /31| Bled 241 cc.
9/3 | 590| 955] 70s | 242 | 25.3] 62 | 0.941 3,3 | 26,0 [14.35] 65 | * Poik.
9/3 | Fasting begun :
d 9/10} 946} 1076} 692 | 358 | 33.3} 88 | 0.72] 6,1 | 13,0 |138.15| 82
| 9/16} 852} 882) 552 | 321 | 36.4] 97 | 0.88) 5,5 | 11,4 |12.15] 73
9/25| 745) 774) 484 | 275 | 35.5} 96 | 0.69] 7,0 | 7,2 |10.90) 71
9 /30| 700) 784) 485 | 295 | 37.7) 89 | 0.57] 7,8 | 13,2 10.30) 76 | Good con-
dition
9/31) Diet: 250 grams crackermeal, 300 cc. milk
10/11} 699] 889) 572 | 311 | 35.0} 79 11.30} 79
10/18} 692} 822] 544 | 274 | 33.3] 84 |0.74 | 5,7 | 4,8|10.25| 76] * Poik.+
10/18} Diet: Mixed diet
10/22) Death from dietary deficiency disease

* Poikilocytosis of red cells.
No previous anemia experiments on this dog.

176 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

‘TABLE 12

ei
ie 2)
4

.j

=

“

:

e

}.

:

4

J

-

§

‘

Blood regeneration—fasting (followed by rice, bread and milk, then yeast). Dog
18-114. White bull, female, adult bees

. :
: = ey q ;
f<3] ‘| mH [a= ®
SHA 8 C) '
PE 3] a - a ro) Za 8 4
Hab S 5 = -e ° = ‘
Ofna! § p B < K 5 i
o |"os| 8 ° 2 a A 4 a REMARKS |
G > (o) > ° = a =) a : ;
& |eBRal > < e * q a : 4 ay |
| Me She 8 5 3 3 2% 3 3 td a 1
Q yO pe | ° nm . ° x fo) vi Py fo} ra)
& | ota) °g < ma ma ze = a : a °
A | a cy ei a | & 8 i | - E a
per | per
ce. ce. ce. sont | cee kgm ce
5/1 | 2550) 1830) 698 | 1115} 60.9} 189 | 0.75] 9,3 | 12,6 |13.95) 131

5/1 | Diet: Bread and milk

5/2 | Bled 458 cc.
5/3 | Bled 458 ec. No distress

5/5 620| 1000

——_

5/6 | Fasting begun

690 | 301| 30.1] 62 | 0.86| 3,6| 14,0 [12.60] 79 |

5 /14| 890] 1043} 619 | 394) 37.8) 85 | 0.76) 5,6] 8,0 /11.30) 92 | * Poik.
5/21) 899] 968] 598 | 375] 38.7} 93 | 0.86] 5,4] 6,0/10.45| 93 | * Poik.
5 /28| 1103} 1077; 588 | 478) 44.4) 102 | 0.69] 7,4] 6,4] 9.55] 108

5 /28| Diet: 200 grams bread, 300 grams rice, 500 ec. milk

6/4 | 1166| 1166| 654 | 502| 43.1| 100 | 0.62| 8,1| 6,6 lto.45| 115 | * Poik. ++

6/5 | Diet: 30 grams compressed yeast, 100 grams bread, 500 cc. milk

6 /11| 1070] 1084] 609 | 464] 42.8) 99 | 0.56) 8,9] 7,410.35] 105 | * Poik++
6 /18| 1232| 1109] 572 | 526] 47.4] 111 | 0.57] 9,8] 6,0 |10.30| 107 | * Poik.+

6 /25| 1167] 1094] 547 | 537] 49.1] 107 | 0.54] 10,5 | 10,4 |10.25] 107 |
7/1 | 1148] 1125] 594 | 516] 45.9] 102 | 0.65] 7,8| 11,6 |10.00| 112

7/1.| Diet: Changed to mixed diet. Extra food. Recovery—4 days

* Poikilocytosis of red cells. : ’
+ June 16: Mucous colitis. No yeast given; milk boiled.
June 17: No mucus; 10 grams yeast given; milk boiled.
June 18: No mucus; 20 grams yeast given; 300 cc. fresh milk.
t Beginning ulceration of mucous membrane of mouth. Beginning salivation.

-
'
,

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA ¥ is

Autopsy (see table 14)

Dog 17-28. White bull, female. See experimental history, table 6-b. )
May 22, 1919. Found dead and cold. Blood clotted. Left ventricle slightly

_ hypertrophied. Lungs and pleurae negative. Slight hypostatic congestion in

right lower lobe. Spleen soft and flabby; normal size; pale pink-gray and cellu-

lar. Peritoneal cavity is normal. No pigmentation and no adhesions. Gas-.

tro-intestinal tract not opened, but superficially not abnormal. Liver normal
size and color; indefinite hazy patches in which lobules look washed out (0.5 to

TABLE 12-3
Experimental history. Dog 18-114

BLOOD
REGENERATION

EXPERIMENT DIET WEIGHT REMARKS

Pigment | Blood per
volume | kilogram

kgm.
Begin 4/24/18 | Cooked liver, 553 94 7.85 | Slight anemia
Bled 279 ce. bread, and 435 105 7.65 | Table 63
End 5 /29 /18 milk 791 84 9.60 | Maximum regenera-
: tion 2 weeks.
Mixed diet
Begin 8/14/18 | Powdered liv-| 1773 106 12.20
Bled 816 cc. er, cracker- 770 95 11.75 | (3 bleedings)
End 9 /27 /18 meal andJ| 2190 112 12.90
milk
Begin 11/14/18 | Cooked liver | 1640 105 13.50 | Table 61
Bled 976 cc. only 592 81 12.50 | (8 bleedings)
End 12/11/18 1902 111 13.65 | Complete regenera-
tion 3 weeks
Begin 5 /1 /19 Fasting .. 2550 131 13.95 | Table 12
Bled 916 ce. 620 79 | 12.60
End 5/28/19 — 1103 108 9.55

2cm.in diameter). Bladder contains a little syrupy pus-like urine; slight cystitis.
Left kidney large and hard; few cysts on surface; large stone fills pelvis (2 x 10
em. +). Right kidney shrunken to small size (2 x 3.x 5 cm.); stone in pelvis.
Marrow of femur almost all fat. Marrow of rib is normal. Pancreas is small
and warty looking—chronic change involves all of lower arm and head, all but
distal fourth of upper arm. No evidence of acute process. Ovaries negative.
Uterus shows sites of placental attachment. Urine: obtained from bladder at
post-mortem. Small amount, mostly pus. No sugar. Few hyaline casts.
Many pus cells and small round epithelial cells, few partially destroyed red cells.
Bacteria abundant.

178 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

The experiment given in table 14 is complicated by nephritis, pye-
litis and bilateral renal calculi. The blood regeneration during the
two weeks preceding death was normal in every way. If anything, the

TABLE 13

Blood regeneration—fasting (followed by rice and bread and yeast vitamine). Dog
18-116. White bull, female, adult

1 2 rs

ae £ z

she ps 8 5
pHs a 5 4 fe) Zz 5
mZ2b5| s = ° =

PO Ri gting sea 5 3 Fe a 3 - REMARKS
ear a> ° S ° a a =) ‘ a
Sieael| > < r " < y ; & “
oe iI -% a s ro) '<) & S) ~ - a
iS} 32 Q ° mM : . " ° . 9 o °
& | otal 8 < a ® oO 3 a , a 8
a | re) ry a a jan) 5 ea 3 z re)
per per
ce. ce. ce. cent 1 cand kgm. |. ec
5/1 | 2620) 1930). 852 | 1050} 54.4) 133 | 0.85) 7,8 | 8,0 |18.75} 103

5/1 | Diet: Bread and milk

5/2 | Bled 483 ce.
5/3 | Bled 483 ec. No distress

5/5 | 778| 1245] 884 | 355] 28.5| 62 | 0.79| 3,9 | 12,8 [17.45] 71 |

5/6 | Fasting begun

5/14 | 1178] 1357| 818 | 525] 38.7] 87 | 0.84] 5,2 | 14,0 115.95] 85 | * Poik.
5/21 | 1025] 1205] 742 | 434] 36.0] 85 | 0.75] 5,7 | 9,4 [14.85] 81 | * Poik.++

5/28 | 1176] 1233] 720 | 519] 40.7] 95 | 0.73] 6,5 | 7,6 |13.85| 89 | * Poik.++

5/28 | Diet: 300 grams rice, 200 grams bread

6/4 | 1109] 1265| 798 | 462| 36.5| 88 | 0.62| 7,1 | 9,8 l14.75| 86 | * Poik.++

6/5 | Diet: 1 gram yeast vitamine,t 100 grams bread, 500 ec. milk

6/11 | 1065] 1232| 761 | 458| 37.2! 86 | 0.70| 6,1 | 8,2 [14.65] 84 | * Poik.+
6/18 | 1368] 1323] 730 | 580] 43.8] 103 | 0.54] 9,6 | 10,2 [14.60] 91 | * Poik.+
6 /25 | 1280] 1380] 780 | 594] 43.0] 93 | 0.51].9,2 | 5,6 [14.05] 98 | * Poik.++

6/25 | Diet: Changed to mixed diet. Developed dietary deficiency disease
6/30 | Death

* Poikilocytosis of red cells.
+ Vitamine prepared according to Seidell’s method (2).

blood pigment production was above the average. This is of some
interest because of the frequent occurrence of anemia in human beings
associated with advanced chronic nephritis. This dog’s death was

eit: Anna

~~ eee oe ——_.

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 179

certainly due to renal injury and insufficiency, but the picture was not
that of a primary essential chronic nephritis. In this single instance
the development of a subacute renal disease did not impair the function
of the organs of the body which are responsible for blood and hemoglobin
regeneration.

Sugar feeding experiments. The first three experiments are similar
and give strong evidence to show that there is very little regeneration
of red cells and hemoglobin during a sugar feeding period. The reac-

TABLE 13-3 é
Expermiental history. Dog 18-116
BLOOD
REGENERATION
EXPERIMENT DIET WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
: kgm.

Begin 4/24/18 | Meat extract, 903 99 9.75 | Table 65

Bled 540 ce. bread and 422 89 | 9.65 | (8 bleedings)

End 5 /29 /18 milk 865 89 10.85 | Mixed diet 2 weeks.
Complete regener-

ation

Begin 8/14/18 | Thyroid, 1842 95 14.20

Bled 944 ec. crackermeal| 1055 98 14.25 | (3 bleedings) _
End 10/24/18 and milk 1640 114 11.85 | Complete regenera-

tion 7 weeks

Begin 12/2/18 | Beef heart 2120 115 | 14.90 Table 52

Bled 1160 ee. and liver _ 776 90 13.95 | (3 bleedings)

End 12/30/18 2270 106 15.50

Begin 5/1/19 | Fasting 2620 103 18.75 | Table 13

Bled 966 ce. 778 71 17.45

End 5-28-19 1176 89 13.85

tion is constantly in favor of the fasting period during which time the
dog can make a definite gain in red cells and hemoglobin, in excess of
the maintenance requirements. In the first two experiments (tables
15 and 16) we have control observations in the same dogs during fast-
ing periods (tables 7 and 14). The regeneration is distinctly more
during fasting. In all three experiments the gain in pigment volume
is present in the first week and we may wish to assume some emergency
reserve to account for this reaction. The following weeks show little

180 G. H. WHIPPLE, C. W. HOOPER AND F. 8S. ROBSCHEIT

or no subsequent gain and may even show a falling off which indicates
that the body cannot fabricate sufficient hemoglobin and red cells for
its daily needs.

Splenectomy (table 17) dois some time before this decetianulll does
not appear to modify the reaction of the red cells and hemoglobin under
the conditions of these experiments.

TABLE 14

Blood regeneration—fasting—nephritis and renal calculi. Dog 17-28. White bull,
female, adult —

la =
az es z
Bes a = a 8 a 8
ele el eilal [fla
Be, a>| #8 rs) 3 a a a REMARKS
—. HA S > > x Zz Ss ic)
fay a, 8 H ° H a
=. 18 af. 2 s 3 3 re 8 ye a R
3a if) ae oe ee ee ee ee eee
a lk a Fy é | @ | 8 oj 2 2 a
cc. C6 ce. pf ae? kgm. | ce
5/1 2018] 1550) 706 | 829 | 53.5) 130 | 0.76) 8,6. | 7,0 [15.9 | 97
5/2 | Diet: Bread and milk |
5/2 | Bled 390 ce.
5/3 | Bled 390 cc. No distress
5/5 | 763| 1040| 686 | 349 | 33.5] 73 | 1.14] 3,2 | 9,4 |14.75| 71 |
5/6 | Bled 200 cc. No distress
5/8 | 631] 956 648 | 295 | 30.8] 66 | 0.94] 3,5 | 8,2 [14.25] 67 |
5/8 | Fasting begun : |
5/14 | 903} 1041) 643 | 415 | 39.9] 87 | 0.79) 5,5 | 7,8 | 18.0} 80
5/21 | 1007| 988] 535 | 482 | 43.8} 102 | 0.74) 6,9 | 8,2 | 11.5) 86 "
5/22 | Found dead. Autopsy given below

* Dog is sick, weak and thirsty; distended abdomen.
Experimental history, see table 6-b.

The three metabolism tables (tables 15-a, 16-a and 17-a) in general
show the characteristic reaction in urinary nitrogen. In every instance —
at the beginning when the anemia reaction is most intense we note the
expected drop in urinary nitrogen when sugar is administered. The
“sparing action of carbohydrate” is not disturbed by the presence of
this degree of secondary anemia. The first and second experiments

1/24 598 950| 627 | 323 | 34.0] 63 | 0.73] 4,3 | 8,2 |10.50| 91

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 181

_ show a distinct rise in urinary nitrogen associated with the bleeding
_ periods. These dogs showed considerable distress during the second
_ bleeding period and it is highly probable that more than one-fourth of
_ the total blood volume was removed on these two days (note the high
_ figure, 130 cc. per kgm., for blood volume as determined by the dry
_ oxalate method). We are inclined to attribute this rise in urinary
_ nitrogen to the shock of the bleeding—to the tissue injury produced by

TABLE 15

Blood regeneration—sugar feeding—metabolism. Dog 17-27. White bull mongrel,
female, adult

12 5
ae 5 ei
258 * 8 . g
ee ae ie 6 a
o8y Rp Dp < K 4 }
rife, 2 a | = a | REMARKS
S |eRal & 3 e fi - a 4 a
eee} A s 3 3 ee S) ° te a
i] 52 5 ° D ° ‘ . ‘o) 4 Py Oo fo)
IER FG Gere iiouae ar ae eae Ci Rete
aA lm QW a a rs an) 3 8 E z
per | per
cc cc. cc leond cost kgm. ce.
1/19 | 1630} 1507| 603 | 904 | 60.0) 108 | 0.71) 7,6 | 9,0 |11.60) 130 | Fasting

1/20 | Bled 375 ce.
1/22 | Bled 375 cc.

1/24 | Diet: 50 grams cane sugar, 25 grams glucose

2 [2 806} 1203) 698 | 506 | 42.0) 67 | 0.56] 6,0 | 6,0 | 9.6 | 125 | * Anis.++

2/9 763| 1090} 643 | 447 | 41.0} 70 | 0.57/ 6,1 | 8,8 | 9.0 | 121 | * Anis.++.

2/14 | 750) 1028) 596 | 482 | 42.0} 73 | 0.70) 5,2 | 7,6 | 8.2 | 125 | Dog refuses
f sugar

* Anisocytosis of red cells
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
Compare fasting period, table 7.

the hemorrhage, and this observation is in harmony with those of Buell
(3) working with pigs. Our third experiment (table 17-a) shows little
or no increase in urinary nitrogen, no clinical reaction and a low esti-
mated blood volume (80 cc. per kgm.).

After the first exhibition of sugar we note a uniform low level of urin-
ary nitrogen excretion. This low level is constant until the end of
the experiments except in the first (table 15-a). Here we note a rise
in urinary nitrogen during the last five days. It is fair to say that this

182 G. H. WHIPPLE, C. W. HOOPER AND F. 8S. ROBSCHEIT

TABLE 15-a

Total urinary nitrogen—sugar. Dog 17-27

DATE, 1917 cnn 24 nouns Tae WEIGHT - ‘REMARKS
grams ce. pounds
January 17 3.25 467 25.9 Fasting
18 2.58 411 26 .0
19. 2.58 461 25.6
20 2.86 422 25.1 Bled 375 ce.
21 3.75 381 24.1 0 feces
22 4.09 330 24.0 Bled 375 ce. Very weak
January 23 | Anemia period begun—Diet: 50 grams sugar, 25 grams glucose
23 3 3D 537 23.8 0 feces
24 3.39 511 23.1 Diarrhea
25 1.99 427 22.8 0 feces
26 1.74 367 22.6 0 feces
27 2.02 390 22.3 Slight diarrhea
28 2 .69 430 22:2 _0 feces
29 2.38 435 21.9 0 feces
30 2 .63 404 21.9 0 feces
31 1.74 401 21.7 0 feces
February 1 1.62 417 21.6 0 feces
2 1.46 401 21.2 0 fece:
3 1.62 459 20.9 0 feces
4 1.96 375 0.8 Feces+
5 1.76 377 20.6 0 feces
6 2.07 400 20.5 0 feces
r lost 20.4 0 feces
8 1.60 520 20 .2 0 feces
9 1.90 475 19.8 Diarrhea |
10 2\35 482 19.3 Diarrhea
11 3.86 720 18.4 0 feces. Vomitus +. Dog
is sick
12 4.76 568 17.7 Vomitus +. Refuses sugar
. solution
13 4.76 361 18.1 0 feces
14 3.67 541 * 18.0 0 feces
15 4.20 505 17.8 Diarrhea +. Milk diet

Dog given 400 cc. water daily by stomach tube.

dog was sick, vomited the sugar solution and had diarrhea. This
period is properly considered as an interval of intoxication in which
little sugar was retained and perhaps this amount favored the intestinal
irritation which was conspicuous.

ee Sa ee ee ae

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA

TABLE 15-3

Experimental history. Dog 17-27

183

EXPERIMENT

BLOOD REGENER-
ATION

DIET WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
kgm
Begin 9/13/16 | Lean meat 8.3
Bled 450 ce. 8.9 | (3 bleedings)
‘End 10 /24 /16 11.4 | Complete regenera-
tion of Hb. and
R.B.C.,
Begin 1/19/17 | Sugar, metab-| 1628 130 11.6 | Table 15
Bled 350 cc. olism 598 91 10.5
End 2 /16 /17 646 119 8.6 | Bread and milk 5
. months. Slight
regeneration
Begin 10/17/17 | Sugar, metab-| 2150 123 13.4
olism
Bled 826 cc. Sugar, gela- 718 84 13.2
End 11 /24 /17 tin, metab- 915 108 10.1 | Sugar, gelatin, crack-
olism ermeal, lard and
butter, 3 weeks.
Dietary. deficiency
disease. Recovery
2 weeks
Begin 3/13/18 | Fasting 1626 Ci: 15.7 | Table7
Bled 750 ce. 1004 72 15.5
End 4/10/18 1240 93 10.7
Begin 6 /3 /18 Cooked thy-| 1939 87 16.35
Bled 1022 ce. mus, bread 723, 70 15.75 | (3 bleedings)
End 6 /28 /18 and milk 1245 78 15.70
Begin 8 /9 /18 Hemoglobin 1914 90 16.15 | Table 79
Bled 988 ce. intravenous-| 817 67 | 15.45 | (8 bleedings)
End 8 /30 /18 ly. Sugar 1155 80 13.75 | Killed September 3

Tables 18 and 18-b deserve special mention as they show the results
of several experiments done under controlled conditions on the same
dog. The experimental history refers to table 10, which shows the
blood regeneration on this dog during a fasting period. There is a dis-

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 2

184 G. H. WHIPPLE, C. W. HOOPER AND F..S. ROBSCHEIT

tinct increase in pigment volume, hematocrit and hemoglobin during the
fasting period. We see in table 18 the same dog under identical condi-
tions on a sugar diet. During the same interval we note practically the

same value for pigment volume, hematocrit and hemoglobin at the be- -

ginning and end of the sugar feeding. There is a trifling rise during the
first week but this is subsequently lost with return to the initial level.
One of the tables to follow (table 21) shows the same dog on a sugar and

TABLE 16
Blood regeneration—sugar feeding—metabolism. Dog 17-28. White bull, female,
adult
R
II = ; E
ao (e=}
s He Q sy o
pee} 2 | 2] 8 | & 3 3
S68) k a = se +} 4 ca
> s 5 i) = 4 REMARKS
~ i ro) > ° & a = 8
S |eBal = 4 > Hi g a : = =
ee e|e|e] 8/8
e | smz = < a = i 3 a . a =
a |e a Fy ei i | 8 0 ae fe a
per per
cc. ce. cc. odnt th bend kgm. | cc
1/19 | 1620} 1500} 600 | 900 | 60.0) 108 | 0.76) 7,1 | 7,4 |11.60} 129 | Fasting

1/20 | Bled 375 cc.
1/22 | Bled 270 ce.
1/23 | Bled 105 ce.

1/24 | 588| 900| 603 | 306 | 34.0| 61 | 0.80| 3,8 | 7,8 l10 .40] 87 |

1/24 | Diet: 50 grams cane sugar, 25 grams glucose, 400 cc. water

2 /2 717| 1121) 684 | 437 | 39.0) 64 | 0.62) 5,2 | 7,2] 9.40) 120 | * Anis.
2/9 636] 1027] 637 | 390 | 38.0} 62 | 0.65) 4,8 | 6,2] 8.90) 115

2/16 | 634] 961] 586 | 375 | 39.0) 66 | 0.59) 5,6 | 10,0| 8.50) 113 | Diarrhea +

2/23 | 541) 933] 562 | 373 | 40.0) 58 | 0.56] 5,2 | 9,0| 8.00) 117

* Anisocytosis of red cells.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
Experimental history, see table 6-b.

gliadin diet which holds the pigment volume curve about on a level—
no gain nor loss.

Table 19 is unsatisfactory from the standpoint of the blood volume
and pigment volume figures but in view of the other experiments we
venture to include it because this dog illustrates a reaction which is not
uncommon in dogs bled for the first time. In dogs used for the first
time in anemia experiments we often note a remarkable regeneration of

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA ~~ 185

TABLE 16-a
Total urinary nitrogen—sugar. Dog 17-28
DATE, 1917 sce 1 Wat labia hi jini WEIGHT REMARKS
q grams ~ ce. pounds
_ January 17 2.46 482 25.9 Fasting
‘Sea 18 2.10 340 25.8
19 2.46 425 25.6 Diarrhea
20 +2:21 519 24.8 Bled 373 ce.
21 | 2.80 375 23 .6
22 3.08 320 23 .6 ‘Bled 270 ce. Dyspnoea
23 2.49 493 23 .6 Bled 105 cc.
January 24 | Anemia period begun. Diet: 50 grams sugar, 25 grams glucose
24 2.46 495 22.8
25 1.96 470 22.4 0 feces
26 1.99 432 22-3 0 feces
27 2.86 427 22.1 0 feces
28 2.69 440 21.8 0 feces
-29 | . 2.07 446 21.5 Diarrhea +
30 2 .04 406 21.3 ~-|.0 feces
31 Evi 398 212 0 feces
February 1 1.62 391 20.9
2 1.79 396 20.6 Trace feces
3 1.60 452 20.4 0 feces
4 1.74 407 20.3 0 feces
5 1.68 395 20.1 0 feces
6 1.23 383 20.0 0 feces
7 . . 19.9 0 feces
8 1.74 425, 19.7 Feces +
9 1.48 398 19.6
10 1.51 415 19.4 0 feces
11 1.43 356 19.3 0 feces
12 1.40 387 19.2 0 feces
13 1.48 394. 19.1 0 feces
14 1.51 388 18.9 0 feces
15 1.40 391 18.8 — | 0 feces
16 1.26 390 18.6 Diarrhea +
17 1.51 378 18.4 0 feces
18 1.15 390 18.3 0 feces
19 123 399 18.1 0 feces
20 1.405 385 18.1 0 feces
21 Le 409 17.9 0 feces
22 1.20 384 17.8 0 feces
23 1712 346 17.6 0 feces
24 1.82 374 17.5

Dog given 400 cc. water daily by stomach tube.

186 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

red cells and hemoglobin on unfavorable diets. This was not under-

stood at first but it may be stated as a fact to be explained or not. It
is possible that this type of dog has a greater reserve stored in its body
from which it can construct red cells and hemoglobin on demand but a
dog which has been bled at various times has not this large reserve.
At any rate, this dog was able to give a remarkable exhibition of regen-
eration of red cells and hemoglobin during a short period of sugar feed-
ing. The red cell hematocrit rose from 26 per cent to 47 per cent and

TABLE 17

Blood regeneration—sugar feeding—metabolism—splenectomy. Dog 17-34. White
bull mongrel, female, adult

ug P|
Se <>] al c
DES a | 3 ; Zz °
3 Z 5} § S si & 9 =

Pung ie) a I REMARKS
ee a>) 8 iS) 5 a 4 4 8
S oF ER a] > J > fA Z a : a as
- {Be8| @ $ 3S 3 2 3 . Ge a

pf nm . . ° ; Q =
: om 8 < A a ze a ¥ ; a 8
a |® Fs a Fe ed ss] 5 Ps op eae
per-| per
cc ce. cc seit | aeni kgm. | ce. ;
1/19 | 886 | 883 | 459 | 424 | 48.0) 103 | 0.69] 7,3 | 14,8] 10.9} 81 | Fastin
b] ’

1/20 | Bled 221 ee.
1/22 | Bled 221 ce.

1/24 | 414 | 780 | 546 | BA | 30.0 53 | 0.80) 3,3 | 11,4| 10.1| 77 |

1/24 | Diet: 50 grams cane sugar, 25 grams glucose, 400 cc. water

2/2 | 528 | 979 | 676 | 303 | 31.0) 54 | 0.60) 4,5 | 6,4) 9.3) 105
2/9 | 536 | 893 | 607 | 286 | 32.0} 60 | 0.62) 4,8 | 6,2) 8.8} 101 | * Anis.
2/16 | 497 | 888 | 604 | 284 | 32.0) 56 | 0.57] 4,9 | 7,8] 8.3] 107
2 /23 | 556 | 868 | 567 | 304 | 35.0} 64 | 0.60} 5,3 | 6,4] 7.8} 113

* Anisocytosis of red cells.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

hemoglobin from 62 to 121 per cent. This regeneration was almost

completed during 4 weeks on a diet which is least favorable to blood

regeneration.

Tables 20, 20-a and 20-b present data on sugar feeding which support
the other experiments given above. This dog was observed during a
fasting period (table 9) as well as during this sugar period and subse-
quently during other diet periods of hemoglobin regeneration. This
experiment shows only a 2-week period of pure sugar feeding during

es eee ee oe «

|
/
|

BLOOD REGENERATION FOLLOWING SIMPLE: ANEMIA

187

TABLE 17-a
. Total urinary nitrogen—sugar. Dog 17-34
DATE, 1917 — Gex 24 nouns gg ger WEIGHT REMARKS
grams cc. pounds
January 17 3.42 303 25 .0 Fasting
vege F 3.30 424 24.4 Diarrhea +
19 2.63 406 24.0
20 2.52 412 23 .6 0 feces. Bled 221 cc.
21 2.58 326 22.9
22 2.86 316 22.8 Bled 221 cc.
January 23 | Anemia period begun. Diet: 50 grams sugar, 25 grams glucose
23 2 .66 526 22.8 Trace soft feces
24 2.35 436 23.2
25 1.99 371 22.0 Trace feces
26° 21.8 0 feces
27 2.46 351 21.6 Slight diarrhea
28 2.69 431 21:3" 0. feces ;
29 2.60 31 21.2 0 feces
30 2.77 397 21.0 Trace of feces
; 31 1.68 382. 20.8 0 feces
February 1 1.54 396 20.6 0 feces
2 1.46 396 20 .4
3 1.74 381 20.2 0 feces
4 1.63 426 20.0 ~ | 0 feces
5 1.54 341 20.4 0 feces
6 1.51 406 19.8 0 feces
7 19.6 Diarrhea +
8 1.51 420 19.6 0 feces
9 1.48 406 19.3
10 1.34 386 19.0 0 feces
11 1.51 380 18.8 0 feces
12 1.34 361 18.9 0 feces
13 1.60 412 18.7 0 feces
14 1.40 401 18.4 Trace of feces
15 1.40 381 18.4 0 feces
16 1.40 386 18.3 0 feces
17 Loe... 396 18.0 Trace of feces
18 1.46 376 . 17.8 Trace of feces
19 1.46 432 17.7 0 feces
20 1.48 367 Yi .7 Trace of feces
21 1.34 411 17.6 0 feces
22 1.48 391 17.4 0 feces
23 1.62 431 17.1 0 feces ‘
| 24 1.74 384 17.13 Diarrhea. Boiled milk diet

Dog given 400 cc. water daily by stomach tube.

TABLE 17-3
Experimental history. Dog 17-34. Splenectomy
BLOOD REGENERATION
EXPERIMENT
NUMBER DIET Pieant Blood BS WEIGHT REMARKS
volume | kilogram
kgm "i
Begin 12/15/16 | Meat and 10.9 | No blood volume
Bled 390 ce. bread only data
End 1 /11 /17 972 85 11.3 | Complete regenera-
12.5 tion of Hb. and
R. BYONe
Begin 1/20/17 | Sugar. 910 81 10.9 | Table 17
Bled 442 ce. Metabolism 413 77 10.1
End 2 /23 /17 556 113 7.8 | Bread and milk diet
5 months
Begin 6 /3 /18 Powdered 1331 63 16.6
Bled 807 ce. liver, bread 664 62 15.7 | (3 bleedings)
End 6 /28 /18 and milk 960 70 15.3
Splenectomy 10/3 /16.
TABLE 18 |
Blood regeneration—sugar feeding. Dog 16-160. Bull mongrel, female, age 2
years +
Aw pe &
a. g o
eel aa | 2] 2] § :
sos] B 3 5 = g = . REMARKS
2 ap] & Q 6 a a =I a
=~ Mel, & f > tl Z 2 i
eel b 5 5 3 2 cS 8 eI a
Sees) Sot So gs | ela £8 lees bee ee
< o ma 4 Wa : : 2 3 : f ed =)
a | & #2 cy ro rf a) 5 ns z = ce)
ce. ce. ce. pe ee kgm. | ce.
8/28 | 1835} 1103} 456 | 654 | 58.2} 166 | 1.1 | 7,6 | 9,6 |10.15| 109 | * Slight
poik.
8/28 | Diet: Crackermeal and milk
8/28 | Bled 276 cc.
8 /29 | Bled 276 ce. :
g/31| 64s] 771| 524 | 243 | 31.5] 84| | | 9.55] 81 |
9/1 | Bled 198 ce.
9/3 | 448| 696] 513 | 177 | 25.5| 64 | 0.8 | 4,0 }15,4| 9.5] 73 | * Poik.+
9/3 | Diet: 75 grams sugar, 25 grams dextrose by stomach tube
9/10 | 561) 792) 577 | 211 | 26.7) 71 | 0.96) 3,7 | 9,4] 8.65) 92 | * Poik.+-+-
9/16 | 488} 703) 500 | 192 | 27.3} 69 | 0.78} 4,4 | 7,8} 8.0} 88] * Poik.++
9/25 | 429) 664) 474 | 182 | 27.4) 64 | 0.61) 5,2 | 9,6) 7.35) 90 | * Poik.+-+

* Poikilocytosis of red cells.
For continuation of experiment see table 80.

188

Ne ee ee ee

EE ee ee ee Re ee ty

TABLE 18-3

Experimental history. Dog 16-160

BLOOD REGENERATION
coe pong DIET Ugaat | wlodh ae WEIGHT REMARKS
volume | kilogram
kgm.
Begin 9 /26 /16 Mixed 5.70 | No blood volume
data .
Bled 440 cc. 6.10 | (4 bleedings)
End 11 /29 /16 7.30 | Complete regen-
eration of Hb.
and R. B. C.
Begin 2/12/17 | Bread, milk and 938 127 7.70 | Table 68
Bled 488 ce. Blaud’s pills 423 80 7.40
End 7 /16 /17 456 97 5.90 | Maximum regen-
: eration 17 weeks
Begin 10/17/17 | Sugar and glia- | 1278 122 8.90 | Metabolism
Bled 542 ec. din 599 95 8.90 | Table 21
End 12 /3 /17 755 106 6.30 | Crackermeal, gel-
te4 atin, lard, but-
ter, 2 weeks.
Mixed diet 5
weeks. Com-
plete regenera-
tion
‘Begin 5/20/18 Fasting 1316 106 10.60 | Metabolism
Bled 562 cc. 482 74 10.20 | Table 10
End 6 /12 /18 636 94 7.55 | Bread and milk 3
weeks
Begin 8/28/19 | Sugar 3 weeks. | 1835 109 10.15 | Table 18
Bled 745 ce. Sugar and Hb. 448 73 9.50 | (8 bleedings)
End 10/16/19 intravenously 586 95 7.80 | Dried yeast and
1 week crackermeal, 2
weeks. Slight
regeneration.
Table 80
Begin 2/20/19 | Sugarand carrot | 1875 99 11.30 | Table 22
Bled 781 ce. juice 548 81 | 10.10 | (3 bleedings)
End 3 /18 /19 Dried yeast, 474 86 8.75 |.
bread and milk
Beef liver, bread
and milk
Begin 8 /8 /19 Beet tops, bread | 1220 97 10.80
Bled 526 cc. and milk 600 66 13.15
End 12/5/19 Spinach, bread 799 95 8.60
and milk ‘
Compressed
yeast, spin-.
ach, bread and
milk

189

190 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

which time is recorded a moderate increase in red cells and pigment
volume. This increase is somewhat above the average reaction under
similar conditions and if sugar feeding had been continued for 2 more
weeks a drop in hemoglobin, pigment volume and red cell hematocrit
was to be expected.

Histidine (1.5 gram per day) added to this diet actually prevented
_ the expected fall and is responsible for a definite gain in the first week of

TABLE 19 <i:
Blood regeneration—sugar feeding. Dog 19-28. Fox terrier, female, adult
la ss z
Beal e | 3] a | 8 | g Sag
ofa! 6 2 p 3 x 5 i
2 [rahi s | 8] 8 | } we + ne
= ° > 3 =
& |Epal > _ > Z a ‘ fy
af As6 = s oO ie) ion] 3 ” = =
a |g o | 3 | a | a ck Sey om fe a ae
< = a ~ 4 . . fon rs ° ig = pe
& uy a a & = en) is) = e e ios)
cc cc. cc joe adel ion ce.
9/18} 1193} 790 | 341 | 441 | 55.8} 151 | 0.94] 8,0 | 8,4 | 8.35} 94
9/18} Diet: Crackermeal and milk
9 /20| Bled 198 ce.
9/21} Bled 178 ec. Dyspnoea
9/23 316| 509 | 370 | 137 | 26.9| 62 | 0.80| 3,9 | 22,6 | 7.85] 65 |
9/23) Diet: 50 grams sugar daily with 150 cc. water
10/2 | 637| 637 | 349 | 284 | 44.5] 100 | 0.81) 6,2 | 8,6] 6.90) 92
10/11 47 .2| 107 6.10
10/18} 562| 464 | 242 | 219 | 47.3] 121 | 0.89| 6,8 | 7,8| 5.75) 81] *

* Hemolysis in tubes containing blood and dye. Reading of color unsatisfac-
tory.
No previous anemia experiments on this dog.

histidine feeding. In view of the constancy of the sugar feeding reac-
tion in the third week of blood regeneration we attach considerable
importance to this reaction. We feel that histidine may be in part
concerned in the complicated endogenous reaction which is responsible
for the final elaboration of the complex protein hemoglobin.
The last 2 weeks of histidine feeding show merely a level curve of |
hemoglobin and pigment volume indicating that the maintenance fac- —
tor alone is being supplied. Under sugar feeding alone a slowly fall-

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 191

_ ing curve would be expected, so that the reaction as a whole is strongly
- in favor of the histidine feeding as being a contributory factor in the
_ hemoglobin regeneration under these experimental conditions. The
' small amount of histidine given is significant when we consider the

percentage content of histidine in casein (2.5 per cent). It will be noted
in a subsequent paper that casein is not an efficient food for hemoglobin
regeneration.

TABLE 20

; Blood regeneration—sugar and histidine—metabolism. Dog 17-38. White bull
- mongrel, female, young adult

: i ‘ -
} BE g Fe
mee eae S :
iy 5 oO = ia Ss a) he

al os ar 3 2 ° (<3) a = S
a ba D4 5 a 4 < P a a a ° Ay
eee | Bas | 8 pe ape aru et rae 8
ee pee} © | 2} ej) et] ea] g-] a} §] a] sg
qi a y a ®y 8 ea ee) is) a E E Q
4 ce. ce. ce. |per cent|per cent kgm. ce.

wa : 5

4 9/11 | 1342 | 1231 505 726 | 59.0 109 | 0.59 9,3 | 14,0 |; 11.60} 106

9/11 | Diet: Bread and milk

9/12 | Bled 308 ce.
9/13 | Bled 308 ce.

9/15 | 468| s34| 619 | 265|30.0| 53|0.68| 3,9 | 12,8 | 11.30| 78

9/15 | Diet: 50 grams cane sugar, 25 grams dextrose; 300 cc. water

78
79

0.71
0.58

10.30
9 .60

71
76

550
468

290
287

36 .0
38 .0

805
755

9/21 | 572
9/28 | 574

5,0
6,5

11,3)
6,6

10/1 Diet: 50 grams cane sugar, 25 grams dextrose, 14 gram histidine, 300 cc.
water

a 10/5 668 | 795 | 468 | 326 | 41.0 84 | 0.68 | 6,2 | 8,4 | 8.90 76
j 10 /12 735 | 826 | 471 | 355 | 43.0 89 | 0.58 | 7,7 | 7,2 | 8.00 | 103
' * 10/19 | 684} 786} 456 | 330 | 42.0 87 | 0.60 | 7,3 | 9,2 | 7.30] 107

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

pected low level except during the last week of the experiment when
a decided increase is noted. This type of intoxication is not infrequent
after long sugar diet periods and we believe. is of gastro-intestinal ori-
gin. It is a fact that the hemoglobin and pigment volume show no

The total nitrogen elimination is uniform and sustained at the ex-
;
7
|
| increase during this period of increased protein katabolism but rather

192 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

Total urinary nitrogen—sugar and histidine. Dog 17-38

TABLE 20-a

TOTAL NITRO-

=

DATE, 1917 GEN 24 nouns ba ry reece WEIGHT REMARKS -
September 15 | Diet: 50 grams cane sugar, 25 grams dextrose, 300 cc. water
grams ce. pounds
16 2.52 415 23.9 Diarrhea. Vomited 55 ce.
17 1.93 370 23.7 Diarrhea. Vomited
18 1.46 401 23.4 Soft feces. Vomited 100 ce.
19 1.40 315 25.2 Vomited 50 ce.
20 1.34 240 23.1 0 feces
21 1.65 290 22.5 Diarrhea
22 1.23 285 22:5 0 feces
23 1.34 346 22.1 Slight diarrhea
24 1.34 247 21.8 0 feces
25 1.28 271 21.8 Slight diarrhea. Vomited
26 1.40 275 21.4 0 feces. Vomited 45 ce.
27 1.29 225 21.4 Soft feces. Vomited 25 cc.
28 1.20 245 21:2 Slight diarrhea
29 1.29 336 20.8 Soft feces
30 1.29 251 20.7 0 feces
October 1 | Diet: 50 grams cane sugar, 25 grams dextrose, 1.5 gram histidine,
300 cc. water :
October 1 1.15 317 20.5 Feces +
2 1:23 290 20.5 0 feces \
3 1.57 303 20.2 Slight diarrhea
4 1.34 264 19.9 0 feces. Vomited
5 1.65 19.6
6 1.48 152 19.5 0 feces. Vomited 55 ce.
7 1.62 620 18.6 0 feces. Vomited
8 1.85 416 18.3 0 feces. Vomited
9. 1.68 111 18.6
; 10 2.63 401 18.1 0 feces
11 2.35 261 18.3 Vomited
12 3.25 453 17.6 0 feces: Vomited
13 3.08 341 17.6 0 feces
14 3.14 300 17.4 0 feces
15 2.86 415 17.1 + 0 feces
16 2.88 266 16.9 0 feces
17 2.58 329 16.8 0 feces
18 2.24 308 16.3 Moderate diarrhea
19 1.68 281 16.1 Diarrhea. Fair condition

ee

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA

TABLE 20-38

Experimental history. Dog 17-38

193

BLOOD
REGENERATION
pend eg DIET WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
kgm.
Begin 12/18/16 | Sugar and gela- 8.60 | No blood volume
tin data
Bled 600 cc. 8.20 | (4 bleedings)
End 1/11/17 532 93 7.00 | Slight. regenera-
tion of Hb. and
hh. B.C,
Begin 3/26/17 | Fasting, metab- | 1395 111 9.80 | Table 9
Bled 544 ce. olism 443 79 9.30
End 4/20/17 585 119 6.30 | Bread and milk 3
months
Begin 9/11/17 | Sugar 1341 106 11.60 | Table 20
Bled 616 ce. Sugar and histi- | 468 78 11.30
End 10/19/17 dine 684 107 7.30 | Beef heart fol-
Metabolism lowed by mixed
diet
Begin 6/3/18 Gelatin, bread | 1621. 86 12.15
Bled 753 cc. and milk 723 76 11.25 | (3 bleedings)
End 6/28/18 1071 85 10.70°| Mixed diet
Begin 8/8/18 |Cooked brain, 2060 109 11.65
Bled 825 ce. crackermeal 526 73 11.20 | (3 bleedings)
and milk Pregnant
End 9/27/18 1100 84 11.95 | Maximum regen-
eration 3 weeks
Begin 2/6/19 Bread (348 | 1873 117 12.15 | Table 26
Bled 710 cc. grams) milk 520 77 11.10
End 3/19/19 (200 ec.) 1092 95 11.35 | Maximum regen-
eration 3 weeks
- Begin 3/31/19 | Bread (100 | 1275 96 | 12.00 | Table 26
Bled 580 ce. grams) milk 499 78 11.40
End 4/9/19 (500 cc.) 691 91 10.00 | Maximum regen-
eration 4 weeks

194 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

a slight loss in red cell hematocrit and pigment volume. This will be
shown to be true in other abnormal conditions in which increased protein
katabolism and urinary nitrogen excretion are observed.

TABLE 21

Blood regeneration—sugar and gliadin—metabolism. Dog 16-160. White bull
mongrel, female, young adult .

B =
; 5 & a <
ee 2 3
A a 5 x °
BEB 8 = = & ° =|
of 5 Pp p < va 5 td F
roo! 5 I re] a a q 8 REMARKS
> eel 5 9 fe) al A = sf ;
S BA al) 5 > ad ie) a a my
= |}Zam6 < x H : 3 5
: BS a s o o re 1) : a
ay sO 5 ° D o By . } ° ae a S °
o otal 8 < m m He! 3 a : a 3
A ry a oy 8 rs ja) =) ro z 3 =)
per per
cc ce. cc cont 1 eens kgm. | ce
10 /17| 1397} 1083) 509 | 627 | 53.0] 118 | 0.74) 8,0 | 8,2} 8.90) 122°

10/17} Diet: Bread and milk

10/19} Bled 271 ce.
10 /20} Bled 271 ce.

10 /22| 600 | 844] 574 | 270 | 32.0| 71 | 1.04 3,4 | 11,4 | 8.90 95 |

10 /22| Diet: 75 grams cane sugar, 25 grams glucose, 300 cc. water

812} 552
794) 548

260 | 32.0] 76
246 | 31.0) 97

0.88
0.87

8.0
7.4

101
105

10/29} 617
11/5 | 770

43 | 82
5,6 | 6,0

*

11/5 | Diet: 75 grams cane sugar, 25 grams glucose, 20 grams gliadin, 300 ce.water

11/12} 762} 838) 536 | 302 | 36.0) 91 | 0.78) 5,8 | 3,6} 7.10) 118
11/18} 764) 813) 496 | 317 | 39.0) 94 | 0.82) 5,7 | 7,2] 6.70) 121
11/26} 780) 777) 466 | 311 | 40.0) 103 | 0.76) 6,6 | 4,4] 6.40) 106
12/3 | 755) 770) 485 | 285 | 37.0) 98 | 0.71) 6,9 | 3,6| 6.30) 106

+ 4%

* Poikilocytosis and anisocytosis of red cells.

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

Gliadin prepared in the usual way by dilute alcohol extraction of wheat flour.
Experimental history, see table 18-b.

Tables 21 and 21-a give more data on sugar feeding periods. The
hemoglobin regeneration is rather more marked than in the average
experiment during a 2-week period. We note again a remarkable in-
crease in red cells (2,000,000) and hemoglobin (26 per cent) with a
stationary red cell hematocrit (32 per cent). If this observation had
not been recorded in other experiments we might suspect an error.

TABLE 21-a
Total urinary nitrogen—sugar and gliadin. Dog 16-160

DATE, aed GE 24 HOURS jie ate a WEIGHT REMARKS
4 October 22 | Diet: 75 grams cane sugar, 25 grams glucose, 300 cc. water daily
: grams cc. pounds
23 2.77 413 18.6 0 feces
24 2.02 267 18.3 Solid feces
25 1.74 232. 18.2 0 feces
26 1.46 178 17.8 Trace of feces
27 1.48 101 17.8 0 feces
28 1.62 251 17.8 0 feces
29 1.74 261 17.6 0 feces
30 2.10 256 17.3 Diarrhea +
EQ] 1.48 217 17.5 0 feces. Vomited
November 1 1.51 131 16.8 0 feces
2 1.57 241 16.8 0 feces
3 1.46 471 16.0 0 feces
4 1.79 151 16.3 0 feces
November 5 | Diet: 75 grams cane sugar, 25 grams glucose, 20 grams gliadin,
300 cc. water daily
5 1.71 301 16.2 Trace feces
6 3.16 241 16.1 0 feces
7 3.53 192 15.9 Feces +
8 3.47 186 15.9 Diarrhea +
9 3.20 173 15.8 Diarrhea +
10 3.58 311 15:5 Diarrhea ++
11 3.25 277 15.4 0 feces
12 3.33 322 15.6 0 feces
13 3.47 301 16.3 Solid feces
14 3.53 319 15.1 Diarrhea +
15 3.50 156 15.1 Trace of feces
16 3.30 132 15.0 0 feces
17 3.25 176 14.9 Soft feces
18° 3.30 151 14.7 Trace of feces
19 3.36 192 14.8 Diarrhea +
20 3.25 161 14.6 0 feces
21 3.36 156 14.5 Soft feces
22 3.22 151 14.4 Solid feces
23 3.58 156 14.4 Soft feces
24 3.47 182 14.3 Soft feces
25 3.36 . 181 14.2 Soft feces
26 3.33 173 14.1 Diarrhea +
27 3.47 222 13.9 Soft feces
28 3.22 152 14.0 Trace of feces
' 29 3.36 181 13.9° | 0 feces
30 3.08 168 13.9 Soft feces
December 1 3.70 166 13.9 0 feces
2 3.28 181 13.8 Trace of feces
3 3.42 163 13.8 0 feces

195

196 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

TABLE 22
Blood regeneration—sugar plus carrot extract—yeast, bread and milk. Dog 16-160.
White mongrel, female, adult

NM
"g a E
= & i] 34 o
SHS] «a s a ) Zz ii
a Zap s a Ss a ° =
O83 5 B p < a 5 iw
Pee eae 0 4 ee 3 3 a B 3 os REMARKS
= baa) & < 3 zs & . ; & r
- 1828] 8 = ° ° e 3 © fd Q
& s2y ° mn . . ,: fo) . re] 5 °
& | otal 8g < a i re) a . ; a 8
Ale a oy 3 Pe ss 5 Pe Es es Fs
per per
cc cc. ce als Vleent kgm. | ce
2/20 | 1375) 1114} 508 | 601 | 53.9} 123 | 0.70) 8,8! 6,0 |11.30} 99

2/20 | Diet: Bread and milk

2/21 | Bled 280 ce.
2/22 | Bled 280 ce.

2 /24 690| 884| 473 | 307 | 34.7| 78 | 0.85| 4,6 | 19,2 [10.45] 85 |

2/24 |Diet: 100 grams sugar, 100 cc. carrot juice, f 150 cc. water

2/27 | 548] 816] 569 | 242 | 29.7/ 67 | 0.90} 3,7]|12,8|10.10) 81
3/5 500} 818} 568 | 238 | 29.1) 61 | 0.71} 4,3 | 11,6 | 9.65) 85
3/12 | 444) 776) 555 | 206 | 26.5| 57 | 0.73) 3,9| 5,2| 9.15} 85.| * Poik.++
3/18 | 474] 750} 530 | 211 | 28.2; 63 | 0.79] 4,0| 6,8| 8.75} 86 | * Poik.++

Shadow

cells
3/18 | Diet: 200 grams bread, 500 cc. milk
3/26 | 520 812| 575 | 232 | 28.6| 64 | 0.71| 4,5| 7,4| 9.20| 88 | * Poik.++
3/26 | Diet: 200 grams bread, 3 grams yeast, 300 cc. milk
4 /2 516} 816) 556 | 251 | 30.8} 63 | 0.63) 5,0} 6,2] 9.45} 86 | * Poik.++
4/8° | 433} 761) 542 | 211 | 27.7) 57 | 0.54) 5,3] 9,4] 9.35) 81 | * Poik.++
4/14 | 416) 726) 517 | 197 | 27.5) 57 | 0.57) 5,0} 7,2| 9.30) 78 | * Poik.
4/21 | 535} 828) 553 | 275 | 31.2) 65 | 0.59) 5,5] 9,4) 9.40) 88

4/21 | Diet: 200 grams cooked beef liver, 200 grams bread, 300 cc. milk

4/28 | 631} 851) 542 | 305 | 35.8] 74 | 0.70] 5,3] 20,4 |10.30} 83) R. B. C.
fairly
normal
5/7 | 1087} 1055) 548 | 496 | 47.0} 103 | 0.71) 7,3} 10,4 {10.70} 99
5/12 | 1153} 1082} 556 | 517 | 47.6} 107 | 0.69} 7,8 | 16,2 |10.85) 100

* Poikilocytosis of red cells.

+ Carrot juice = water extract of cooked carrots filtered and concentrated to
one-third of its original volume.

Experimental history, see table 18-b.

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 197

This shows the possible fluctuation in hemoglobin content and size of

q red corpuscles which may be observed with a constant hematocrit.

Probably many factors enter into this reaction which will be taken up
again. ‘The clinical summary of.this dog shows a variety of diet peners

including a fasting period (table 18-a).

Gliadin (20 grams per day) added to the sugar diet causes no increase
in hemoglobin but merely a uniform maintenance factor. There is
still further increase in red cells with poikilocytosis and we believe the
evidence favors some red cell fragmentation under these conditions.
The urinary nitrogen shows a uniformly low level during the sugar
period and the expected level during gliadin feeding (table 21-a).

Table 22 gives the results of a second sugar regeneration period on
the same dog (16-160) used in the preceding experiment (table 21). It
will be observed that this sugar regeneration period is not as favorable
and there is actually a loss in pigment volume amounting to about 30
per cent during a period of 4 weeks. This experiment includes another
factor (carrot juice) which is obviously inert under these experimental
conditions. This point will come up again in subsequent papers dealing
with pigment derivatives. We wish to point out an unusual condition
on February 24 after the 2 bleeding days when a low plasma volume
(473 cc.) is recorded against the norma! during the entire experiment of
500 to 550 cc. When the plasma volume returns to normal we note a
fall in hemoglobin and red cell hematocrit. It is probable that the
reaction is the result of the shock of the hemorrhage which is usually
adjusted during the resting day intervening between the second bleeding
and the second blood volume determination.

The second period of bread and milk feeding is included to substanti-
ate the plasma volume figures. This reaction will be discussed in detail
in the next paper.

Table 23 is given at the end of the series because one important factor
separates it from all the other experiments,—a bile fistula. This dog
(15-22) has been under observation in this laboratory for several years
and many reports on bile excretion include experiments on this animal
(4). It is known that traces of bile can gain entrance into this dog’s
intestine but the general condition of the dog is perfect. The reaction
to anemia under fasting conditions in this dog is of particular interest
as the absorption of pigments from the intestine may be excluded. It
has been suggested by Addis (5) that absorption of some pigment com-
plex from the intestine is a part of the body conservation of pigment
materials. This and other experiments give no support to this inter-
esting suggestion.

198 G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

Bile pigment figures in. a fasting period are given for this same dog
in another publication (6). The constant presence of urobilin in the
fasting bile of this experiment is noted in table 23 and discussed below.
Total urinary nitrogen figures are not given here but were obtained in
this experiment and average 3.08 grams per 24 hours. This is an aver-
age figure for a normal dog of similar weight and activity. Evidently

TABLE 23

Blood regeneration—fasting—metabolism—bile fistula. Dog 15-22. Brindle bull,
male, age 4 years +

ee: ; 3
AH es

SH 8 g
io] rca) Zz °
REB| & 2 5 e o =
ofa d = S) < re 5 4

Fe oP ro) ° Q a _
= a > > i Z = a
> 2 na > < : ' 4 ‘ . & 4
3} os a) s is) iS) 2 i) br = 2)
i=} sO 5 ° D . " 2 re) a Pe io} °
Bi RRR) Su pod: |g dy Maal ade ho gol) aa hia Se
A Ey Ey y 3 a ss] 3 3 E z a
ce. | ce. ce. |per cent|per cent kgm. ce.

3/26 | 2200 | 1833 770 | 1063 | 58.0 120 | 0.77 7,8 | 12,6 | 15.40) 119

3/26 | Diet: Bread and milk

3/28 | Bled 460 cc.
3/29 | Bled 460 ce.

3/30 | 673 | 962| 654| 308 | 32.0| 70|0.83| 4,2 | 18,6 | 15.10| 63

3/30 | Fasting begun

4 /2 890 | 1186 | 747 | 4389 | 37.0 75 | 0.66 | 5,7 | 28,2 | 14.00) 85
4/9 874 | 1136 | 625 | 511 | 45.0 77 | 0.70} 5,5 | 14,6 | 12.50} 91
4/16 | 1122 | 1069 | 556 | 513 | 48.0| 105 | 0.71 | 6,4 | 6,2 | 11.40} 94
4/23 | 1133 | 1059 | 540 | 519 | 49.0 | 107 | 0.66] 8,1 | 7,2 | 10.40) 116

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
No previous anemia experiments on this dog.

Urobilin constantly present in bile after 1st day of fasting period.
Total nitrogen in urine—average 3.08 grams per 24 hours.

the bile fistula does not modify the total nitrogen figures in this experi-
ment nor in many others.

The curve of hemoglobin and pigment volume is quite remarkable.
There is even more marked a new formation of red cells and hemoglobin
than is noted in an average normal dog. We must not forget the occa-
sional abnormal regeneration which is noted in a normal dog which
has never been bled—a reserve being forthcoming which returns the

lee Ye a ee ee

4
BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 199

hemoglobin almost to normal in 3 weeks of fasting. This bile fistula
dog had never been bled in any large amounts before. But the reserve
reaction in this dog is truly remarkable and at the end of the fasting
period we note a normal red count, a hemoglobin and red cell hemato-
erit figure close to normal and a considerable increase in pigment vol-
ume. It is noted that the plasma volume shows a decrease during the
fasting period which gives the reason for the relatively low pigment
volume. At any rate the capacity of this bile fistula dog for hemoglobin
regeneration is in no way impaired and may be greater than normal.

TABLE 24

Fasting summary

iesimer Troan | els wee) Seve
DOG NUMBER E €y 3 5. Eo 5 E Eu 3 E 5 3
So| S45 | | sel aa| 2] Se] so | Bl Se] aa |
B5| Gi | & | S5| sai) 8 G8) ex | & | G5) se | &
Ay an) tie |e 1a x ta a) xq
15-22 673/32.0 | 70 | 874/45.0| 77|1122/48.0 | 105/1133/49.0 | 107
18-103 590/25.3 | 62 | 852/86.4| 97) 745/35.5 | 96} 700/37.7 | 89
18-114 620/30.1 | 62 | 899/38.7| 93/1103/44.4 | 102
18-116 778|28.5 | 62 |1025/36.0| 85/1176|40.7 | 95
17-38 443/28 .0 | 60 | 491/39.0| 68] 585/43.0 | 76) 585/44.0 | 78
17-37 313/28 .0 | 50 | 492/35.0| 64] 532/385.0 | 73) 540/35.0 | 70
16-160 482/33.2 | 64 | 591/83.5| 88] 636/34.3 | 90
17-28 631/30.8 | 66 |1007/43.8} 102
17-27 1005/31.0 | 90 |1086|/39.0} 101)1213/41.0 | 115)1240|38.0 | 124
Fasting average...... 615/29 .65} 65 | 813/38.5} 86) 889/40.23} 94) 840/40.74; 94
Sugar average, table | _
Re welll cea «ak 515/30.87| 63 | 648|35.5) 75) 593/36.4 | 76] 614/37.04| 74

For example, we know that the bile fistula liver contains more pigment

as demonstrated by the microscope. ‘There may be stored away in the
liver or other tissues more of the pigment complex from which the pig-
ment reserve is derived—to be turned into finished hemoglobin on emer-
gency demand.

The last three tables (tables 24, 25 and 25-a) give the summary fig-
ures of all the experiments in'this paper and one fact stands out clearly
in table 25-a. The average blood regeneration is distinctly greater during
a fasting period of 2 weeks than during a similar sugar period of 2:

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 2

200. G. H. WHIPPLE, C. W. HOOPER AND F. S. ROBSCHEIT

weeks. The difference is even more striking at the end of 3 weeks when
the average gain in pigment volume during fasting is 274, contrasted
with 78 on sugar feeding. Hemoglobin figures show an average gain
of 29 per cent on fasting contrasted with 13 per cent on sugar diet.

TABLE 25.
Sugar diet summary
AFTER BLEEDING Bh ib aye eas eee ha ahaa ett:
a Pe a jet ve - as a oo
DOG NUMBER |= £5 | 2 |3 £5 3 E £55 3 8 ES 8
So| Sc | # | Selan| 2 | Se] #4 | Bl e| sa] ®
fel es] 2/88/85] 2 | 88] gs | 2 | Be] ga | @
00 3 oR o | &5| of o | 85] of o | ws Of o
a ns ch | m 1a a oie | & q
17-28 588/34.0 | 61 | 636/38.0} 62) 634/389.0 | 66) 541/40.0 | 58
17-34 414/30.0 | 53 | 536/32.0| 60] 497/32.0 | 56) 556/385.0 | 64
17-38 468|30.0 | 53 | 574/38.0| 76] 668/41.0 | 84) 735/43.0 | 89
16-160 600|32.0 | 71 | 770|31.0| 97| 762/36.0 | 91] 764/39.0 | 94
16-160 690|34.7 | 78 | 500/29.1|} 61| 444/26.5 | 57| 474/28.2 | 63
19-28 316/26.9 | 62 | 916/47.2| 107] 562/47.3 | 121
17-27 598/34.0 | 63 | 763/41.0| 70] 750/42.0 | 73
16-160 448|25.5 | 64 | 488]27.3) 69] 429|27.4 | 64
Sugar average........ 515|30.87) 63 | 648/385.5) 75) 593 36.4 | 76 614|37 .04| 74
Fasting average, 7
thle FA). Sy sign es 615/29 .65} 65 | 813/38.5| 86) 889/40.23) 94] 840/40.74) 94
TABLE 25-A | .
Gains made above minimum level after bleeding 7
DURING TWO WEEKS DURING THREE WEEKS
(TOTAL) (voTaL)
Pig- | Hema- H Pig- | Hema- H
ent, eG, | elobin | gheme [Be,| elobin
Nine experiments, fasting average...... 198 | 8.85} 21 274 | 10.58] 29 ;
Hight experiments, sugar average.......| 133 | 4.63 | 12 78 | 5.53) 13 )
DISCUSSION

The term “sparing action of carbohydrates” is familiar to all work-
ers in metabolism and means that carbohydrate feeding will decrease —
the excretion of total urinary nitrogen by man or animal as compared
with the fasting excretion of nitrogen. It is therefore assumed that

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 201

( _ the administration of carbohydrate actually spares the body protein.

There are two theories for this sparing action of the carbohydrates:

a, That the sparing action is at the source—that is, the sugar prevents

tissue katabolism or spares protein in tissue cells; 6, That the sparing |
action is a conservation of split products which, with the aid of carbo-—
hydrate radicles, are reconstructed into a variety of protein complexes
and used in various parts of the body. Both theories have able advo-
cates, but it is fairto say thatmost of the experiments can be used hy a
skilful proponent to support either hypothesis. An admirable review
of the work in this field has been published recently by Janney (7).

Some experiments by Davis and Whipple (8) concerning the regen-
eration of liver cells following a unit injury can be said to support con-
vinecingly the theory of conservation of protein end products. In these
experiments there is little liver cell repair during fasting periods but a
rapid repair during sugar diet periods. This indicates that the body
can form many new liver cells when sugar is available but not during
a fasting period. These new liver cells must be formed from body pro-
tein split products, and this type of conservation of nitrogen is due to
sugar feeding and cannot be explained by any amount of protein sparing
at the source. : |

The experiments, summarized*in tables 24, 25 and 25-a, follow the

' normal regeneration of another type of body cell: the normal red blood

cell which is constantly being used up and reformed or reconstructed
day by day. This is an admirable cell for a study of cell regeneration
as its main constituent (hemoglobin) is very complex and easily and
accurately measured. The amount of hemoglobin circulating in the
body can be measured with considerable accuracy and therefore its
curve of regeneration can be established with reasonable precision.
When we compare periods of regeneration of hemoglobin during fasting
and during sugar diet periods we find a constant difference which comes
out clearly in an average figure of many experiments (table 25-a).
This table shows that the fasting dog can regenerate more red cells and
hemoglobin than a dog on a sugar diet. This figure is over and above
the maintenance supply of hemoglobin which is needed to keep the level
uniform and furnish the hemoglobin used up by the daily wear and tear
of the circulation in the body.

This actual reconstruction of new red cells and hemoglobin must
come from the body protein or its split products as no nitrogen is being
supplied to the body. Evidently the body conserves very carefully the
substances which are suitable for the elaboration of hemoglobin. The

202 G. H. WHIPPLE, C. W. HOOPER AND F. 8S. ROBSCHEIT

pyrrol complex is a peculiar feature of the hemoglobin molecule which -

can scarcely be formed in the body and may be an important determin-
ing factor in its reconstruction. How may we explain the increase in
hemoglobin during fasting periods in excess of the reaction on sugar
feeding? This surely cannot be explained by increased synthetic
capacity due to the presence of sugar or the conditions should be re-

versed. If we assume that sugar may have a certain sparing action at

the source we are able to suggest a plausible explanation. Suppose the
sugar feeding does protect body protein from katabolism and therefore
lessens the amount of available protein split products, we are then able
to explain the smaller amount of hemoglobin produced, provided we
assume that under all circumstances of need or limited diet the body
conserves all the available protein building stones which go to make up
the hemoglobin molecule. This seems to us to be the best explanation
of the observed facts, but this opinion may be modified by further work.
It may be objected that the “maintenance factor” of red cells may
vary in fasting as compared with sugar periods. This factor is not to
be determined at this time, but we have no reason to suppose that the
daily wastage of red cells should be greater on sugar feeding than during
fasting periods. This question must be left open for the present. |
Granting the facts as outlined above we may say that we have good

proof to explain the “sparing action of carbohydrates” as due to a con-

servation of protein split products which aid in new protein con-
struction as observed in liver repair (8). But these anemia experiments
may be best explained by a “sparing action of carbohydrate” which
protects at the source the body protein from katabolism. If this work
is correct we may assume that the carbohydrate in the diet may have
a double “sparing action’’—to protect the body protein at its source and to
aid materially in the conservation of protein split products, which are recast

into new body protein. That one or the other of these two reactions —

may be dominant under varying conditions may be granted as probable.

No discussion of any phase of pigment metabolism is complete with-
out proper consideration of the pigment output in the bile. The bile
pigment represents in part at least under certain conditions the end
product of hemoglobin degradation in the body, but in addition under
certain conditions this bile pigment may represent certain constructive
activities of the liver (9). It is possible that a part of this bile pigment
produced by the liver and not derived from the degradation of hemoglobin
may be an excess of pigment substance available for hemoglobin pro-
duction but not so used and later discarded by way of the bile. This

te ee Lec eee Se ie, oe oe hae Sa re be Sie Pen eS seco eb ee a se | a a al nl ec ila Y U — ae a a ay < > a

TSE hear amon ca ge eG ee TR SL Rane Se Gene bad raed Min NS get oe A oes ee oie ce aaa = , aim sae? Pecan hd Simin. EN LE Parr
. ms = lh iN td ge ee ee 5 <A Ss si Sak Ren ie ee = as z+ “ =e ,, 7 . we

r _ 7 “+ Fg . 7 ie Cu Sey = oo ia ins ho) = =>, — were ee ee hy ee eS ee

: ; oe gah Ht in ci i IE Ie eee nee

a A Sit eh aa a at ie | i Bae

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 203

‘ _ reaction might only take place in the liver when there was a consider-

able surplus of pigment elements in the food or elsewhere. Such con-
ditions might not obtain in the body when it was deprived of all protein
intake (fasting or sugar diet).

_ The bile pigment output, therefore, must be reviewed briefly in this

3 place. We are able to refer to observations of this nature (6) and state

that there may or may not be differences in the bile pigment excretion
during fasting periods as compared with sugar feeding periods in bile
fistula dogs. Certain experiments appear to show greater bile pigment
elimination during sugar periods than during fasting periods but other
experiments show little or no difference in the bile pigment figures. These
differences may represent individual variations in these bile fistula dogs
and the available data are not sufficient to establish any difference in
bile pigment excretion under these conditions. One thing is quite
clear—these bile fistula dogs during fasting or sugar periods do excrete
a measurable amount of bile pigments (average of 15 to 30 mgm. bile
pigment per 6 hour daily collection). This pigment results from the
degradation of hemoglobin in the body or the production of pigment
complex from other substances in the liver—in other words, a distinct
loss of pigment material from the body.

One other point must be mentioned in this connection. During
fasting periods in bile fistula dogs we have noted the invariable appear-
ance of urobilin in the bile. In our experience this is the only condition
which is constantly associated with urobilin production in the dog’s
liver. At times the bile pigments may be almost completely replaced
by the urobilin pigment and this introduces a serious error in our analy-
sis of bile pigment. It is probable (if not certain) that this urobilin is

derived from the bile pigment and its increase therefore will be asso-

ciated with a corresponding decrease in bile pigments. But we have

no accurate quantitative analytical method for urobilin, although we

can estimate bile pigments there present by precipitation of the cal-
cium pigment compound, filtration and analysis of the acid alcohol
derivative. It seems probable to us that the urobilin appearing in the
bile fistula dogs is derived at least in part from the bile pigments in the
bile ducts, due to the activity of bacteria which we know are responsible
in part at least for thisreactionin the intestine. During fasting periods
the flow of bile is very sluggish and this inferior drainage of bile gives a
favorable opportunity for the bacteria to multiply. It can be stated
that bacteria are numerous in all bile fistula tracts and may at times

‘set up an inflammatory reaction in the bile passages which will cause

204. G. H. WHIPPLE, C. W. HOOPER AND F. 8. ROBSCHEIT

trouble. Flushing out the bile passages by cholagogue pata as a rales
gives relief to this condition.

It has occurred to us that this observation may have some signifi-
cance as regards urobilin in the urine in a variety of conditions. ‘There
is no conclusive proof that urobilin is ever absorbed from the intestine.
We have here proof that urobilin may be formed in the liver. It would
seem safe to assume that the hepatic origin of urobilin should be consid-
ered in any analysis of this complex question. When the possibility is —
suggested that urobilin may be formed at times in the liver, it is obvious
how difficult it is to exclude this possibility in the clinical conditions
associated with which we note urobilin in the urine.

SUMMARY

During fasting periods after unit hemorrhages the normal dog can
regenerate measurable amounts of red cells and hemoglobin.

This regeneration of red cells and hemoglobin includes the daily wast-
age of these elements, or the maintenance factor of the blood. The
curve of hemoglobin regeneration represents the production of hemo-
globin in excess of this unknown maintenance factor.

Bile pigment excretion under fasting or sugar diet conditions may be
considered as uniform. A bile fistula dog may regenerate hemoglobin
and red cells with at least equal and perhaps greater speed than a nor-
mal dog. The constant presence of wrobilin in the bile of the fasting
bile fistula dog is recorded and discussed from the standpoint of uro-

bilinuria.. The hepatic origin of urobilin is suggested.
During sugar diet periods the regeneration of hemoglobin and red cells
is distinctly less than during fasting periods (table 25-a).

We believe that this observation may be explained by a double
“sparing action of carbohydrates’”—both sparing at the source or pro-
‘tecting body protein from katabolism as well as effecting synthetically —
a distinct conservation of protein split products. This postulates a
strict conservation by the body of certain protein fractions which may
be recast into hemoglobin. The presence of carbohydrate may facili-
tate this reaction but the actual new formation of hemoglobin may de-
pend in part upon the type and amount of amino acid groups available
from normal protein katabolism. }

, Histidine given with sugar appears to cause a production of hemo-
globin over the control level. This amino acid may be one of the im-
portant elements in this hemoglobin regeneration complex.

Gliadin in the amounts used does not modify the hemoglobin reaction. 4

ee ee ee ee mn

i a lh

sotto

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 205

%
BIBLIOGRAPHY

L “AsuBy: Journ. Exper. Med., 1919, xxix, 267.
fe ere Vublio Health Repts., U. 8. Public Health Service, no. 325, 1916,

ek: soe Biol. Chem., 1919, xl, 62.
) Wurppie anp Hoopzr: This Journal, 1917, xlii, 256.
| AppIs: Arch. Int. Med., 1915, xv, 413.
) ‘Fosmm, peores anp Wurppte: Jour. Biol. Chem., 1919, xxviii, 393.

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA

III. InFLuENcE OF BREAD AND MILK, CRACKERMEAL, RICE AND
Potato, CASEIN AND GLIADIN IN VARYING AMOUNTS
AND COMBINATIONS

C. W. HOOPER, F. 8S. ROBSCHEIT ann G. H. WHIPPLE

From the George Williams Hooper Foundation for Medical Research, University of
California Medical School, San Francisco

Received for publication April 3, 1920

Early in our work it became evident that bread and milk did not

constitute a favorable diet for the rapid regeneration of blood. This —
diet is palatable to dogs and maintains them in good nutritional condi-

tion for many weeks. As a result we began to use this diet as a main-
tenance diet to which other factors could be added which did or did
not modify the curve of blood regeneration to be expected from the
bread and milk factors alone. Because in many experiments we use
bread and milk as a part of the diet it is essential that we understand
clearly the effect of this diet under a variety of conditions. We there-
fore submit many experiments tabulated below to establish the normal
blood regeneration of the dog which is limited to varying amounts of
dried white bread and skim milk.

We must mention in passing the dietary deficiency disease which
develops in dogs kept for long periods on a strict bread and milk diet.
This disease condition resembles scurvy in human beings and is
rapidly fatal if not energetically treated by antiscorbutic measures.
This condition will be reviewed in a subsequent publication.

It will be noted from the experiments here outlined that bread and
milk alone when given in large amounts may return the blood picture
to normal in six weeks or longer. But when given in moderate amounts
(100 grams dried bread and 500 cc. skim milk) this diet will rarely per-
mit of complete blood regeneration. On this diet the hemoglobin,
pigment volume and red cell hematocrit may be kept at a permanently
subnormal level following the unit hemorrhages used in our experi-
ments to produce uncomplicated secondary anemia. ‘The value of

206

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 207

establishing this fact is obvious when it is found that some food mate-
rials added to this diet will profoundly modify this reaction expected
from bread and milk alone. These observations will be reported in
subsequent communications.

A few experiments with two of the important constituents of the
bread and milk diet are included. Casein and gliadin in sufficient
amounts are able to modify somewhat the reaction expected from >
sugar feeding alone. Some work with incomplete proteins and mix-
tures of amino acids has been completed but the evidence, so far, is not
conclusively in favor of any single amino acid as being responsible for
this peculiar reaction which depends in great measure upon the capac-
ity of the body to construct hemoglobin.

Crackermeal and milk were used at one period during the war when
white bread or in fact any kind of bread was not available for obvious
reasons. This crackermeal was purchased on the open market during
the war period and its constitution is not accurately known. We
were able to ascertain with reasonable certainty that this crackermeal
contained 70 to 80 per cent wheat flour, but a considerable percentage
of barley and rice flour. Other grains may possibly have been con-
cerned. The fact remains that this mixture of wheat flour and other
grain flours did not modify in any manner the reaction established
recently for commercial white bread which at present is made almost
wholly from wheat flour.

Rice, potatoes and milk are used in one large series of experiments.
The amount of blood regeneration on this diet closely parallels that
observed on a bread and milk diet. This diet also includes one other
substance sometimes used in bread,—potato or potato flour. Any one
of these three diets is a favorable maintenance diet to which other
factors may be added to determine the value of the unknown substance
in its relation to blood regeneration.

EXPERIMENTAL OBSERVATIONS

Unless otherwise noted, the same technique is used in these experi-
ments which has been described above (paper I). The food mixtures
were all palatable and readily eaten unless note is made to the con-
trary. With few exceptions the dogs maintained their weight, general
activity and health throughout the experiments.

Bread and milk diet. The first experiment (table 26) in this series
illustrates many points which are established by the succeeding experi-

208 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

ments given below. The dog presented a very high hemoglobin (130

per cent), high red count (10 million) and blood per kilo (127 to 117
ce.). Following the unit hemorrhages there is recorded as usual a
volume of red cells (239 cc.) which is much below the calculated ex-
pected red cell volume (438 cc.). The low level in this experiment is
more noticeable than in the average experiment. The plasma volume
is promptly made up to normal after the bleedings and remains as usual
relatively constant. The curve of regeneration is quite steep during
the first 2 weeks of the bread and milk diet but thereafter remains at a
uniform level. This statement applies to the pigment volume, red cell
hematocrit, and hemoglobin, but the red cell count shows a slow in-
crease toward normal in the last 3 weeks of the experiment. __

In this first experiment the diet was abundant and sufficient to
maintain the body weight and even to allow of slight increase. This
point is of much importance, as will appear later. This and other
similar experiments show that a liberal bread and milk diet sufficient to
maintain or increase the body weight will cause a certain degree of
hemoglobin regeneration over and above the daily maintenance hemo-
globin factor. Blood regeneration may be rapid for a week or two
and may even return the blood picture almost to normal in certain
experiments.

The repeat experiment on this same dog (table 26) shows a lower
initial hemoglobin and red count. The hemorrhages are less in amount
but the anemia level for pigment volume is much the same as in the
preceding anemia period. The diet now is not abundant and contains
only 100 grams dried bread as compared with 343 grams in the first
period of regeneration. This 100 grams bread diet is not sufficient to
maintain the body weight at its normal level and there is a loss in
weight of 1.4 kilos during the 5 weeks of blood regeneration. There is
a striking difference in the amount of hemoglobin regeneration -which
shows only a trivial increase from week to week over the maintenance
factor. In using the term maintenance factor we wish to indicate that
unknown replacement fraction which represents the daily wastage of

red cells used up in the body metabolism. There are experiments to

indicate that this fraction may be 3 per cent per day in human beings,

but there are no data to establish this important point for the dog.
It is obvious from these two anemia periods (table 26) that a dog

will regenerate more hemoglobin on an abundant bread and milk diet

than on a limited bread and milk diet. This applies particularly .

to the bread portion of the diet. The term ‘bread’ as used in this

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 209

x TABLE 26 .
Blood regeneration—bread and milk—repeat experiment. Dog 17-38. Bull mon-
grel, female, young adult

la |
e u z
: 5 a ‘a Q a a pers S
a 5 a B 5 is s e m1 g .
oe eats 3 6 a a a fc ae REMARKS
oS 5B a al ip : > | Z = ; : .
} = ee 3 = rf 3 8 iS) ° 8 2
ee eee 8) fi) ala |g | 8 | «| el gg] 8
Re Rept) et) ae) ec] Se] os) Re Re Ue
e ce. ce. ce. pwd on kgm. | cc.
1/30 | 1910} 1467] 585 | 877 | 59.8} 130 11.5 | 127
q 2/6 1873} 1418} 600 | 812 | 57.2} 132 | 0.66] 10,0) 9,0/12.15} 117

4 1/30 Diet: White bread and milk

_ 2/7 | Bled 355 ce.
2/8 Bled 355 ce.

2/10 | 520| 852| 608 | 239 | 28.1] 61 1.0| 3,1] 284{i1.1.| 77 |

2/10 Diet: Dried, ground white bread, 343 grams, skim milk, 500 ce.
2/17 | 852| 1074) 654 | 404 | 37.6| 79 | 0.88| 4,5| 12,0(11.6 | 93 |

2/17 | Diet: Dried, ground white bread, 343 grams, skim milk, 200 ce.

2/26 | 1120) 1133] 588 | 534 | 47.1| 99 | 0.72) 6,9} 11,8]11.6 | 98
3/3 | 1137} 1110) 560 | 544 | 49.0) 102 | 0.82) 6,2) 7,411.35! 98 | * Poik.+
3/10 | 1138} 1083) 575 | 497 | 45.9} 105 | 0.72) 7,3) 7,8/11.5 | 94] * Poik.+
3/19 | 1092) 1072) 561 | 493 | 46.0) 102 | 0.66) 7,7} 6,8)11.35) 95 | * Poik.+

3/21 | Mixed diet

4g 3/31 1275 1155 558 | 586 | 50.7| 110 | 0.67| 8,2| 7,2] 12.0| 96 | * Poik. ++
3/31 | Diet: White bread and milk
4/1 | Bled 290 ce.
4/2 | Bled 290 cc. No distress |
4/3 | 499| 887| 633 | 249 | 28.1) 56 | 0.67| 4,2 9,6|11.4 78 | * Poik. +

4/3 | Diet: Dried, ground white bread, 100 grams, skim milk, 500 ce.

4/11 | 566) 989) 662 | 307 | 31.0) 64 | 0.68) 4,7) 11,0)11.25) 77 "
4/18 | 662) 988) 638 | 335 | 33.9) 67 | 0.56) 6,0) 9,2)11.0 | 90 3
4/25 | 652) 920) 578 | 304 | 35.4) 71 | 0.55) 6,5) 6,6/10.7| 86 7

5 /2 734| 955) 585 | 367 | 38.4) 77] 0.57] 6,7| 7,2|10.35} 92] * Poik.++
5/9 691; 909) 564 | 341 | 37.5) 76| 0.58} 6,5) 5,610.0} 91 | * Poik.++

* Poikilocytosis of red cells.
Experimental history, see table 20-b.

210 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE |
TABLE 27 i
Blood regeneration—bread and milk—repeat experiment. Dog 19-94. Bull mon- i
grel, male, age § months :
P é
oe ; : t
saa g a s 3
eee] B@ | 2 | gs | & B = 3
SRST b 5 » 3 al | | -
pelo fe" 5 4 a - mi 2 REMARKS
2 a>) 3 2 iS) SI a = =
S |baa| > < . . q a ( & ey
es ee Bo discl (Sel Malte |
a s23/ © a : : y ° ; Fs] S ° <
& | om < . " re) 2 a ; 8 g ;
ie a & a Pe x 5 a B B a j
ce. ce. re. pad | ns kgm. | cc. :
1/16 | 1250) 1343) 790 | 540 | 40.2) 93 | 0.57) 8.1 | 21,6)10.65) 126
1/16 | Diet: Bread and milk
1/17 | Bled 335 ce. |
1/18 | Bled 285 ce. No distress .
1/20 | 462! 830| 645 | 190 | 22.6 55 | 0.95] 2,9 | 14,6|10 09] i ie |
1/20 | Diet: 250 grams dried, ground white bread, 500 cc. skim milk
1/27 | 576} 1020} 683 | 327 | 32.0) 56 0.55 5,1 | 11,0/10.25} 100 .

2/3 | 900} 1088] 651 | 485 | 39.7) 83 | 0.57] 7,3 | 13,4/10.25| 106 | * Poik. + )
2/12 | 750) 1000] 626 | 362 | 36.2) 75 | 0.72|.5,2 | 10,2/10.80) 93 | * Poik.+ |
2/19 | 907| 1085] 647 | 439 | 40.4) 84 | 0.69] 6,1 | 9,8/11.15| 97 *

2/28 | 994] 1126] 667 | 448 | 39.8] 88 | 0.76) 5,8 | 6,2/11.50| 98 *
3/7 | 899] 1098} 650 | 437 | 39.8] 82 | 0.73] 5,6 | 8,4]11.70| 94 *

———

3/10 | Diet: Mixed diet

x

3/17 1146) 1216| 687 | 523 | 43.0| 94 | 0.70| 6,7 | 10,6|13.05| 93 | * Slight
3/17 | Diet: White bread and milk

3/18 | Bled 304 ec.
3/19 | Bled 304 cc. No distress

3/21 | 542| 968] 709 | 255 | 26.3) 56 | 0.82] 3,4 | 14,4|12.40| 78

3/21 | Diet: 300 grams dried, ground white bread, 500 ec. skim milk

3/28 | 736] 979] 640 | 324 | 33.1) 75 | 0.89} 4,2 | 12,6/12.50| 94 | * Slight
4/2 811) 1112) 712 | 395 | 35.5) 73 | 0.78] 4,7 8,4'12.45, 89} *

4/9 864 1110) 680 | 420 | 37.8} 78 | 0.65) 5,9 | 11,4/12.40) 89 | * Slight
4/16 | 1005) 1200) 718 | 469 | 39.1) 84 | 0.70) 6,0 | 8,0)12.75) 94 | * Slight
4/23 | 1094) 1236) 740 | 485 | 39.2} 88 | 0.71) 6,9 6,0)12.75| 97 | *Slght |
4/30 | 1031} 1311} 800 | 498 | 38.0; 79 | 0.55) 7,2 | 10,0/13.20| 99 | * Slight

* Poikilocytosis of red cells.
No previous anemia experiments with this dog.

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 21F

_ report indicates white bread of the first quality, obtained from the
_ University Hospital, sorted, dried in an oven and pulverized.

Table 27 gives a repeat experiment which shows that the curve of
blood regeneration following a very short resting period is practically
identical under uniform diet conditions. It is of considerable impor-
tance to know beyond question whether we may expect a uniform
_ reaction under uniform conditions when a dog is used for different
_ experiments at different intervals of time. We believe this communi-
cation includes sufficient data to establish this point beyond question.

_ Therefore we may feel secure in using for anemia work the same set of

_ dogs, provided the blood picture has returned to normal and the weight
and general health is also normal. With repeated anemia experiments
_ the dog does not increase in its capacity to regenerate hemoglobin nor
does this reparative mechanism fail under the conditions of these
repeat experiments. We may then attach considerable significance to,
deviations from the standard reaction in any given animal.

The repeat experiment (table 27) gives a reaction curve of pigment
volume, red cell hematocrit and hemoglobin which is practically iden-
tical with the first anemia period. There is slightly more gain in the
repeat experiment than in the first anemia observation. In both
anemia periods the diet was liberal and permitted a gain in body weight
of approximately 1 kilo per 6-week period. The repeat experiment
brought the hemoglobin back more nearly to normal but the initial
anemia level was not as low nor was the loss by hemorrhage as great.
In the repeat experiment the dog received 300 grams dried white bread
in contrast to 250 grams bread in the first period, but this was only a
proper proportion per kilo body weight. These dogs, moreover, were
in a period of rapid body growth (5 to 8 months).

The anemia experiments ‘given in table 28 are very similar to those
just described. In this instance, too, the bread and milk diet was
sufficient for maintenance plus a definite growth factor with a gain of
2 kilos in body weight during the 5-week periods. In both periods the
regeneration brings the pigment volume and blood picture back to the
normal level. The steady gain in weight and body growth must not
be lost sight of in reviewing the same gain in plasma volume. In the

q adult normal dog the plasma volume is now known to be quite con-

stant under these experimental conditions.

The experiment given in table 29 is slightly different from those
preceding. After the anemia period we have 1 week’s fast which shows
as usual a definite gain in pigment volume. The subsequent 3 weeks

212 ' C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE
TABLE 28
Blood regeneration—bread and milk—repeat experiment. Dog 19-102. Bull mon-
grel, male, age & to 6 months
ll B = 4
Bes] 2 | 8 | a | 8 z 3
See aede B® dake lows wm | 8. a
« > Pe 9 a 3 3 a 7 3 2. REMARKS
S |eBal & > Be by Z s a ‘
eee] ag = 3 ; ; 3 I "
Paes] Bip Bodog Sg hale Paegehie i>! 11 gedaan ae
a | z * Fe ¢ | a Ssh. obs: doa Riedion
| ce ce. cc peta’ port kgm. | ce.
1/30 | 972) 944) 482 | 453 | 48.0] 103 9.75) 97
2/6 | 1026) 1068) 606 | 452 | 42.3) 96 | 0.63) 7,6 | 16,0/10.90) 98 ,
2/6 | Diet: Bread and milk
2/7 | Bled 267 ce.
2/8 | Bled 267 ce. No distress
2/10 | 418| 803| 594°| 205 | 25.5| 52| 0.79| 3,3 | 11,410.25] 78 |
2/10 | Diet: 283 grams dried, ground white bread, 500 cc. skim milk |
2/17 | 720) 951) 608 | 337 | 35.5) 76 | 0.93) 4,1 8,6|10.65) 89

2/26 | 888] 1014] 592 | 417 | 41.1] 87 | 0.65] 6,7 | 11,6/11.25] 90| *Poik.+ —
3/3%| 1045] 1068| 575 | 488 | 45.7| 98 | 0.76] 6,4 | 12,8/11.35| 94 | * Poik.+
3/10 | 1020) 1045] 578 | 462 | 44.2| 97 | 0.73] 6,6 | 11,2/11.55| 91| *Poik.t+
3/19 | 1026] 1056] 576 | 467 | 44.2] 97 | 0.70] 6,9 | 12,0/12.55| 86] *Poik.++ —

3/22 | Diet: Mixed diet

3/31 | 1220] 1220| 644 | 564 | 46.2| 100 | 0.67] 7,5 | 12,6|13.20| 93 | * Poik. ++
3/31 | Diet: Bread and milk

4/1 | Bled 305 ce.
4/2 | Bled 305 cc. No distress

4/3 | 470| 874] 634 | 232 | 26.5| 54 | 0.68| 4,0 | 16,|12.50| 70 | * Poik. ++

4/3 | Diet: 283 grams dried, ground white. bread, 500 cc. skim milk

4/11 | 758) 1085) 702 | 361 | 33.3) 70 | 0.69) 5,1 7,4/13.30) 82 =
4/18 | ~ 884) 1083) 650 | 421 | 38.9) 82 | 0.64) 6,4) 12,8)13.50) 80
4/25 | 1099) 1163) 660 | 498 | 42.8) 94 | 0.64) 7,3 9,6]13.85| 84.
5/2 | 1648) 1485) 701 | 769 | 51.8) 111 | 0.74) 7,5 | 11,2)14.55) 102 Sele
5/9 | 1280) 1268) 667 | 584 | 46.0) 101 | 0.64) 7,9 | 19,814.50) 87 *

* Poikilocytosis of red cells.
No previous anemia experiments with this dog.

=a STS ey

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 213

_ show a stationary pigment volume up to the last week, when there is a
_ definite gain. This diet was sufficient for maintenance of body weight
__ and the general condition was uniformly excellent.

ae. : TABLE 29 :
g Blood regeneration—bread and milk. Dog 18-123. Brindle bull mongrel, male,
young adult
a
ao. 0 Q S 8
i=) a ° Z
se] |e] 2 | § |S
Re Sot 4 2 5 3 | a | 8 REMARKS
& |eRal > a > = q 2 2 Fy
~imces! @ | os |} sic a | fe See
Sa e | Sia fh og | 8] a | a 8
ae Rest | we) 8 oe hee
= ce. cc ce. pene Fate kgm. | ce
5/1 1692) 1270) 546 | 716 | 56.9} 133 | 0.68) 9,8 | 12,2/12.55) 101

5/1 | Diet: Bread and milk

5/2 | Bled 318 ce.
5/3 | Bled 318 cc. No distress

515 | 592| 826| 552 | 270 | 32.7/ 72 | 0.64] 5,6 | 18,8|11.60| 71 |
5/6 | Bled 200 ce.

5/8 440| 668| 450 | 203 | 30.3| 66 | 0.85] 3,9 | 12,0|10.90| 61 |
5/8 Fasting

5/14 | 620| 802] 515 | 283 | 35.3] 77 | 0.73| 5,3 | 12,2|10.30 78 |
5/14 | Diet: 100 grams dried, ground white bread, 500 ec. skim milk

5/21 | 638) 852) 560 | 270 | 31.7| 75 | 0.73) 5,1 | 17,0] 9.95) 96 | * Poik.+
5/28 | 633) 891) 582 | 299 | 33.6) 71 | 0.66) 5,4 | 14,2) 9.90} 90] * Poik.+
6 /4 798} 929) 574 | 345 | 37.2! 86 | 0.63] 6,8 | 10,6) 9.75) 95 | * Poik.+

* Poikilocytosis of red cells.
No previous anemia experiments on this dog.

The experiments given in tables 30 and 31 are very similar and may |
be discussed together. In both experiments the amount of bread and
milk was not measured, but it is significant that there was a slight
but definite loss of body weight during the bread and milk periods.
It is to be expected, therefore that the diet would not suffice to raise
the level of hemoglobin and pigment volume much above the anemia

214 C. W. HOOPER, F. 8S. ROBSCHEIT AND G. H. WHIPPLE

level. The larger dog (table 30) does show a slow regeneration toward

normal in 4 weeks, but the smaller dog (table 31) shows almost no net
gain during 3 weeks. ‘There is a little gain in the first week which is
lost subsequently.

The mixed diet reaction is very nicely shown in both experiments
(tables 30 and 31) and a week or two is sufficient to make up the deficit
in hemoglobin and establish the normal level.

TABLE 30
Blood regeneration—bread and milk. Dog 18-113. Bull mongrel, female, age 4 to
5 months
mM
=
oo f
sha . 8 3
ree} 2 | 2 | 2 | & 6 =
osx 5 5 p < ‘al 5 i
on > ab ag 5 3 a bs 3 - REMARKS
S Geel Sie fs ee are it
- |eoel 2 3 S S e S 3 B a
R 30 q {o) nm ° . hi (o) . Fes} S °
2 | oa § s - “ a a . : a 3
Aa |e a Ey ne re q 5 3 B a
cc cc. cc pac pea kgm. | ce
4/24 | 970 | 1000} 561 | 426 | 42.6} 97 | 0.63) 7,7 | 9,6] 9.75} 103

4/24 | Diet: Bread and milk—amount not measured ‘

4/25 | Bled 250 cc.
4/26 | Bled 170 ce. No distress

4/29 | 418 | 853] 559 | 277 | 32.5] 49 | 0.52] 4,7 | 12,6] 9.50] 90

5/8 | 597 | 776| 468 | 295 | 38.0/ 77 | 0.61/ 6,3 | 8,6] 9.13] 85

5/15 | 878'| 1140] 674 | 445 | 39.1) 77 | 0.49] 7,9 | 9,6] 9.10] 125 |*Fragm.
5/22 | 666 | 912] 555 | 341 | 37.3] 73 |-0.52| 7,0 | 10,2] 9.30] 98 |*Fragm.
5/29 | 701 | 788] 473 | 315 | 40.0] 89 | 0.55] 81 | 9,8} 9.10] 87 |*Fragm.++

5/29 | Diet: Mixed diet

6/10 | 940 | 940| 530 | 397 | 42..2| 100 | 0.60 8,4 | 10,2|10..45| 90 |*Fragm.

* Fragmentation of red cells.
No previous anemia experiments on this dog.

A bile fistula experiment is included (table 32) to show that these
dogs react like normal dogs as regards blood pigment production after
simple anemia. This dog was known to have complete exclusion of
bile from the intestinal tract (autopsy notes). His general condition
during the entire experiment was excellent and the bread and milk
diet was sufficient to maintain the body weight close to normal. Dur-
ing the entire period there was a loss of only 1.4 kilos, which is not

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA |. 215

great when we consider the size of the dog and the length of the experi-
- ment (7 weeks). There was a slight initial gain in hemoglobin and
- pigment volume which was subsequently lost. The general level of
' pigment volume, red cell hematocrit and hemoglobin is pretty nearly
- uniform with occasional temporary gains and losses. It is of interest
_ to note a steady gain in number of red cells from 3,400,000 to 6,600,000.
- The hemoglobin gain was much less, which gives a fall in color index

2 TABLE 31

Blood regeneration—bread and milk. Dog 18-115. Bull mongrel, female, young
adult
"g : z
gh a f e
= a fs) z ¢
lege} $ | 2/2 | & | 8 2
mn pe eel 3 3 3 a a 3 ‘as REMARKS
BS |eBal & c > = Z A " ray
% =) :8 a s 3 3 Io 3 4 r a
a | 399! o A Hi ° Fs 5 °
& |ohal < m = o 3 a ; 3 2
a |e a cy e fe an d ee e B a
cc ec. ec pat bi kgm. | cc
4/24 | 655 | 780 | 394 | 368 | 47.2) 84 | 0.59] 7,1 | 14,2) 7.00) 111
4/24 | Diet: Bread and milk—amount not measured
4/25 | Bled 195 ce.
4/26 | Bled 140 ce.
4/29 | 314 | 582 | 375 | 200 34.3| 54 | 0.63 4,3 | 26,2! 7.30) 80
5/8 | 572 | 752 | 467 | 274 |36.45| 76 | 0.59) 6,5 9,4} 6.77| 111
5/15 | 675 | 888 | 522 | 353 [389.8 | 76 | 0.59] 6,4 | 25,0) 6.40) 188
5/22 | 468 | 593 | 354 | 233 |389.3.| 79 | 0.70) 5,6 6,0} 6.20) 96
5/23 | Diet: Mixed diet :
5/29 | 516 | 607 | 373 | 228 |37.5 "85 | 0.64] 6,6 | 10,8) 6.90) 88 | * Poik.
6/10 | 769 | 761 | 488 | 318 |41.8 | 101 | 0.73) 6,9 | 14,2) 8.00) 95 | * Poik.

* Poikilocytosis of red cells.
No previous ahemia experiments on this dog.

from 0.87 to 0.54. This is not uncommon in nopraat dogs under similar
experimental conditions.

Crackermeal, milk, lard and butter. These crackermeal Fy inanie
were performed in part during the war period when white bread was
not available. This crackermeal consisted of an unknown mixture
including at least wheat, barley and rice flours, possibly others. The
experiments show reactions which resemble accurately those observed

THE AMERICAN JOURNAL OF PHYS{OLOGY, VOL. 53, NO. 2

216 _C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

with white bread. A mixture of grain flours, therefore, is no more
efficient in promoting a regeneration of hemoglobin and red cells than
white bread alone, consisting mainly of wheat flour.

Table 33 is to be compared with table 30—the first experiment done
with white bread and milk, and the second with crackermeal and milk.

TABLE 32

Blood regeneration—bread and milk diet—bile fistula. Dog 17-151. White bull
mongrel, male, young adult

nN
Il - e 2
ag re 8
a8 2) 5 °
= | ° a

nap | § a | 5 . 3 a
205 B 2 jc = fe fc| mn
~ ar 3 9 3 a a | %
— ° > ; =
1 5 a8 e < . .. q . ; 5 Ae
‘ a8 Q = 3 3 - 3 > a
| sh ° D : : e fo) : Pe S °
e |otal] ° < a 5 S 3 a : a g

A yi ry ry a ee a0) 8 8 E 3
Ce; ce. ce. per cent\per cent kgm. ce.

5/28 | 2675 | 2158 | 971 | 1085 | 55 124 | 0.72 | 8,6 5,2 | 18.20) 113

5/28 | Diet: Bread and milk

5/29 | Bled 540 cc.
5/30 | Bled 540 ce.

5/31 830 | 1408 | 1000 | 4os| 29 | 59 | 0.87 |

a
a
—

-
jor)
_
~I

a i

5/31 | Diet: Bread and milk—amount not measured

6/8 | 1058 | 1557 | 1059 | 498] 32 "68 | 0.77 4,4 9,8 | 17.20) 90
6/15 | 1575 | 1852 | 1222 | 630 | 34 85 | 0.65 | 6,5 6,6 | 16.80} 110
6/22 | 1274 | 1464 | 937 | 527 | 36 87 | 0.63 | 6,9 | 10,8 | 16.40) 89
6/29 | 990 | 1415; 920°} 495 | 35 70 | 0.49 | 7,2 7,4 | 16.10} 88
7/6 1150 | 15382 | 950 | 582 | 38 75 | 0.54] 6,9 | 5,8] 15.90) 96
7/13 | 1480 | 1741 | 1097 | 644 | 37 85 | 0.55 | 7,7 | 13,8 | 15.90} 109
7/18 | 1118 | 1574 | 1039 | 545 | 34 71 | 0.54 | 6,6 | 13,8} 16.10) 98

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
No previous anemia experiments on this dog.

These experiments were performed during a period of rapid growth
and the diets in each instance were sufficient to preserve body weight.
There was a slight gain in weight during the crackermeal experiment.
Both experiments show a slow steady gain in pigment volume, hemo-
globin and red cell hematocrit. We may say the curves are as nearly
identical as one can hope to observe in this typeof experiment.

‘a

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 217

__ After a period of 8 weeks of crackermeal and milk diet the dog sud-
_denly developed acute dietary deficiency disease which resulted in

TABLE 33

_ Blood ‘regeneration—crackermeal and milk. Dog 18-118. Bull mongrel, female,

age 8 to 9 months

1g E ;
ag [a=]
s Fa o
sees | 8 |g g 3 3
68a| 2 B 5 < 5 i
wo | asl 2 3 3 a ry a ‘2 REMARKS»
SB |BBal > 6 > ti Z a ee ee Fy
= |@eel a = S 3 aa cS) ° id a
elgee} S|) ele] aetal]s}ale}a] g
ereree em | ek jot) a | |) Ss] @ fe pe Pe
ce ce. ce a ja kgm. | cc
8/14 1556} 1136) 571 | 557 | 49.0) 187 | 0.81) 8,4 5,2)12.85] 88
8/15} Diet: Bread and milk
8/16) Bled 284 ce.
8/17| Bled 284 ce.
gjis| 714| 978 | 699 | 270| 27.6] 73| | | — |ia.as| 79|
8/19} Bled 220 cc. &
8/21) 804| 1200| 858 | 323 | 26.9| 67 | 0.84 4,0 | 26,212.35] 97|
8 /21| Diet: 200 grams crackermeal, 500 cc. milk
8 /27| 788| 984| 667 | 308 | 31.3| 80 | 0.80] 5,0 | 12,4/12.50| 79 | * Poik.
9/4 | 894) 1048) 670 | 367 | 35.0} 85 | 0.66) 6,4 8,612.55} 84] * Poik.++
9/11) 1078) 1135) 688 | 441 | 38.9) 95 | 0.65) 7,3 6,4412.55} 90] * Poik.++.
9/19} 1136} 1152) 700 | 442 | 38.4; 99 | 0.67| 7,4 | 18,0)12.60} 92] * Poik.+-+
9 /27| 1380) 1315) 737 | 558 | 42.5) 105 | 0.67} 7,8 | 14,8/12.90} 102 | * Poik.+
10/9 | 1085) 1119) 640 | 497 | 44.4; 97 | 0.63) 7,7 | 11,0)12.80) 93
10/17} 1290} 1277| 728 | 536 | 42.0) 101 12.75} 100
10 /18| Diet: Mixed diet. Extra meat. Dietary deficiency disease
10/25] 788| 1050| 690 | 355 | 33.8| 75 | 0.58] 6,5 | 10,8|10.35| 101 |
10 /26) Killed. Autopsy

eee '

sa eagle Se

* Poikilocytosis of red cells.
Refer to table 30, bread and milk experiment.

death 1 week later. Note the fall in blood volume, hemoglobin and
red cell hematocrit during this short period. The autopsy findings will
not be discussed at this time.

218 C..W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

Table 34 presents a long experiment in which the diet is cracker-

meal, lard and butter in sufficient amounts to preserve the body weight
and permit of a gain of 0.7 kilo during the period of 12 weeks. No
previous anemia experiments had been performed on this dog, which

TABLE 34

Blood regeneration—crackermeal, lard and butter. Dog 17-205. Bull mongrel,
male, young adult

1a rl
& [<>] = oS
5 BS 3 oa Q 5 x °
nap 5 2 a C) =|
ofA B S) < a " : 4

5S o ° p wt a Ss i<3} 4y
b ae] 8 2 3 iS a 5 ; a
Ss Baa > < .. . z a & 9
4 3 -2 ra) s is) is) i is) o q Aa
ico] SOs ° m . 4 4 fo) 4 ro} o fo)
e |oma| ° < 2 fs 3 3 Ee es a Q
A Fy E: Fy 0 tia 8 aj e z Ey
ce. ce. ce. per cent|per cent kgm. ce.
10/29 | 1450 | 1058 | 571 | 487| 46 | 137] 0.87] 7,9 | 86 | 10.70] 99

10 /29 | Diet: Bread and milk

10 /30 | Bled 265 ce.
10/31 | Bled 265 ce.

11/2 | 522 | 790 | 563 | 229 | 29 | 66 | 1.14 | 2,9 | 27,0 | 10.40] 76

11/2 | Diet: 200 grams crackermeal, 10 grams lard, 10 grams butter

11/9 | 559| 860| 585| 275] 32 65 6,2 | 10.30] 83
11/16 | 948 | 1088 | 664| 425 | 39 87 | 1.01 | 4,3 | 18,8 | 10.30] 105
11/23] 969 | 1052] 589| 463] 44 92| 0.90 | 5,1 | 9,2 | 10.30] 102
11/28] 998] 998| 549] 449] 45 | 100] 0.78! 6,4 | 9,2] 10.30} 97
12/5 | 1111 | 1028] 555.) 473] 46 | 108|0.86| 63 | 88| 10.40] 99
12/10 | 1070 | 1008 | 544| 464] 46 | 106|0.82| 6,5 | 13,6 | 10.20| 99
12/19| 904] 913] 511| 404] 44 99 | 0.77 | 6,4 | 13,6 | 10.60] 86.
12/26 44 | 119|0.79| 7,5 | 8,0 | 10.60
1/2/18] . 48 | 12410.78| 7,9 | 10,2 | 10.50
1/9 46 | 1271 0.79] 8,0 | 12,8 | 10.70
1/17 46 | 1271 0.85] 7,5 |. 14,2 | 10.70
1/23 | 1480 | 1139 | 592] 546] 48 | 1301 0.69] 9,4.| 13,4 | 11.10} 108

No previous anemia experiments on this dog.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

may account for the fact that the blood regeneration finally carried the
level to normal. This reserve has been mentioned in the preceding
communication.: The gain is very slow and at times there appears to
be a slight loss in red cell hematocrit or hemoglobin or pigment volume.

= ee

Fors 2

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 219

. ‘The end result after 12 weeks of this diet may be accepted as normal.
_ It is unusual that this dog tolerated this diet for such a prolonged
_ period without any signs of dietary deficiency disease. A subsequent

@ experiment (paper V) shows dietary deficiency symptoms in this same

yg ips after a shorter period of a similar dict.
i 4 TABLE 35

a Blood regeneration—crackermeal, lard and butter. Dog 16-160. White bull mon-
grel, female, age 12 months

I a s
3 S
a a
a o
5 2| 8 3 S 3 g 3
3 = rs = e | |
Sos| 6 a B S g 4 _
“3 p55] & 2 2 Fe A c 0 REMARKS
S gaa = t > q a a pS a Ey
- [a.8| a ~ 3 3 . 3 ° tH a
dg |S) 9 a : f ; 3 ry = g
=< |ota) 8 3 : 3 i 8 ‘ : > :
“0 - ee ) iy a a q 5 r E B =)
per per
a cc ce. cc fond 1 Gand kgm cc
3/4 | 1218} 1006] 533 | 473 | 47 | 121 | 0.67] 9,1. | 7,2/10.00] 100

3/4 | Diet: Crackermeal, lard and butter

4 + 3/6 | Bled 262 cc.
8/7 | Bled 242 ce.

3/9 | 726 | 844] 501 | 256 | 30 | 86 | 0.86] 5,0 | 94| 9.60| 89 |

3/9 | Diet: 206 grams crackermeal, 10 grams lard, 10 grams butter

3/15 | 732) 842) 581 | 261 | 311] 87 | 0.72) 6,0 8,4; 9.30} 91
- 3/20} 762) 786) 517 | 267 | 34) 97 | 1.08) 4,5 9,8} 9.30} 84
_ 38/27 | 840) 785) 526 | 259 | 331] 107 | 0.89} 6,0 8,6} 9.20}. 85 | Slight diar-
i rhea
4/3 812} 805) 531 | 274 | 34] 101 | 0.83] 6,1 | 15,8) 8.90] 90

' 4/9 845} 836) 535 301 | 36 101 0.78} 6,5 | 12,6) 9.10) 92

Experimental history, see table 18-b.
_ Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

The experiment given in table 35 is very much like the preceding
one but for the fact that this dog had been observed previously in
’ anemia periods (see experimental history 18-b). The blood regenera-
tion is very slow on'the same amount of crackermeal, lard and butter.
There was a slight loss of weight on this diet which contains a sufficient
number of calories per kilo. This is to be explained by the presence
of diarrhea.

220 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

The addition of milk powder (table 36) to the lard, butter and crack-
ermeal diet does not modify the curve of blood regeneration nor does it

prevent the development of the dietary deficiency disease after a period

of 1 month. The peculiar reaction of the red cell hematocrit which
actually diminishes as the hemoglobin and red cell count increase may
be explained in part by the use of dry oxalate in varying amounts.

TABLE 36

Blood regeneration—crackermeal, lard, butter and milk powder. Dog 16-140. Bull
mongrel, male, young adult

nm
ug es -
Beg & 5
a

PRS eS | a | Be dg % 3
Seelce| Pal b ig w | & ri

». | heels a s 3 a “4 | e REMARKS
S |eeal & 2 > = z 2 . Fy
" ‘O] 4 m 3 3 3 8 F a
a | 253] 9 z : - : 6 . Ps o °
af pemeey8 dad. A. Rei wa] ae
Pay ry rs) ry a ro ea ) a E E Q

per | per
ce. ce. cc cent | cent kgm. | ce
3 /4 2212} 1616} 711 | 905 56 | 137 | 0.77) 8,9 6,8)15.10| 107

3/4 | Diet: Crackermeal, lard and butter

3/6 | Bled 414 ce.
3/7 | Bled 394 ce.

3/9 | 1027| 1229] sso | 465 | 38 | 84 0.87| 4,8 | 13,2/14.20| 85

3/9 | Diet: 163 grams crackermeal, 10 grams lard, 10 grams butter,
100 + grams milk powder ;

3/15 | 1215} 1322) 846 | 476 | 36] 92 | 0.88) 5,2 | 11,2)14.8| 89
3/20 | 1153} 1281) 883 | 448 | 35] 90 | 0.88) 5,1 9,6/15.0 | 85
3/27 | 1055) 1227) 834 | 392 | 32] 86 | 0.84) 5,1 8,0)14.80) 83
4/3 | 1350) 1324) 834 | 490 | 37 | 102 | 0.77| 6,6 | 19,8/14.60) 91
4/9 | 1408) 1257) 855 | 402 | 32 | 112 | 0.84) 6,7 | 11,6)14.10) 89 be

* Dietary deficiency disease. Death April 17.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

The pigment volume regeneration is about the expected amount in

view of the diet which was sufficient to maintain but not increase the |

_ body weight. :

The next three experiments may be discussed in a group (tables
37, 38 and 39). The influence of splenectomy is concerned in two
of these experiments and under these experimental conditions the blood
regeneration appears to progress in a normal fashion, at least for a time.

ws +
wane

TABLE 36-38
Experimental history. Dog 16-140

BLOOD REGENER-
ATION
DIET WEIGHT REMARKS

Pigment | Blood per
volume | kilogram

EXPERIMENT
NUMBER

P kgm
Begin 9/13/16 | Mixed 8.1

Bled 450 ce. | 8.4 | (3 bleedings)
_ End 10/13/16 10.1

Begin 11/6/16 | Fasting, metabolism 9.3
Bled 660 ce. Sugar, metabolism 8.2 | (4 bleedings)
_ End 12/16/16 | Gliadin, sugar 6.4
ae Gelatin, sugar

Begin 9/11/17 | Sugar, metabolism] 1570 109 13.1

Bled 712 ce. Mono-amino-acid 756 90 12.6

End 10/19/17 | fraction of gelatin| 1273 130 9.3

3 Begin 3/4/18 |Crackermeal, lard, | 2210 107 15.1 | Table 36

Bled 808 ce. butter, milk pow-}| 1025 85 14.3

End 4/9/18 der 1385 |° 89 14.1 | Dietary defi-
ciency dis-
ease

TABLE 37

¢ Blood regeneration—crackermeal, lard, butter, alfalfa meal. Dog 18-97. Bull
mongrel, female, young adult

Q

i : ;
s Be ie o
De peee | 8 lea) a] 8 g =
gos} 8 z 3 : i : .
~% ies 3 B. 3 = a S| S
& Baa > ia - a a a ; Ba
se a. a = 3 3 - 5 is) = a
re] S28 re} a : ; ; 3 ‘ : o °
& om a 8 < a a rr) a = a Ps S
A |e F Py Fs oi ry g 3 é E a
ce. cc. ce per cent|per cent kgm. ce.

3/11 | 2038 | 1358 584 774 Dz: 150 0.88 | 8,5 9,2 | 12.40) 109

| 3/11 | Diet: Bread and milk

3/13 | Bled 339 cc.
3/14 | Bled 340 ce.

3/16 | 908 | 966 | 570 | 306 | 41 | 94|1.04| 4,5 | 15,6| 11.40] 82

3/16 | Diet: Crackermeal, lard, butter, 40 grams alfalfa meal

3/22 | 1175 | 1118 | 548 | 570} 51 105 | 0.86 | 6,1 | 22,2 | 10.70) 103
3/29 | 1250 | 1058 | 561 | 498} 47 118 | 0.75 | 7,9 | 14,6 | 10.80) 98
4/4 | 13822 | 1076 | 581 | 495 | 46 123 | 0.74 | 8,3 | 12,4] 10.90) 99
4/10 | 1598 | 1102 | 595} 508} 46 145 | 0.75 | 9,7 | 11,4 | 11.00) 100

~ Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
No previous anemia experiments on this dog.

221

TABLE 38
Blood regeneration—crackermeal, lard and butter—splenectomy. Dog 17-163.
Bull mongrel, male, young adult

Te 5
ae : :
ee: “ :
Bea avr ee Bee nw | & e
mE IY eet 4 2 a -~ a “7. REMARKS
Sweet e low |e See ee a
Mag 21 he 8 eae = 3 3 3 = ra) ° a ®
BI ao 8 g < cs a s 3 rs a = g
a |e BOR a | ok RR Ge ee !
Ce. ce. ce. toe lh kgm. ce.
9/3 | 1110} 1069} 588 | 481 | 45 | 104 | 0.72! 7,2 6,2}10.0 | 106 | |
9/3 Diet: Bread and milk :
9/4 | Bled 279 cc.
9/5 | Bled 279 ce. © i ]
9/7 | 388| 719| 532 | 187 | 26 | 54 | 0.90| 3,0 | 22,4] 9.70| 74 |
9/7 | Diet: 200 grams crackermeal, 20 grams lard |
9/14) 610} 813) 512 | 301 | 37] 75 | 1.01) 3,7 | 10,2) 9.60) 85
9/21} 728) 867| 512 | 355 | 411] 84 5,8 5,6) 9.60} 91
9 /22) Diet: 200 grams crackermeal, 10 grams lard, 10 grams butter |
:
9 /28| 754; 820) 492 | 328} 40] 92] 0.72) 6,7 8,0} 9.60} 85 !
10/5 | 704) 800} 456 | 344 | 48] 88 | 0.73! 6,0 | 18,4) 9.80} 80 |
10/12} 806) 848) 517 | 331 | 39] 95 | 0.82) 5,8 5,6} 9.70) 87
10/17} 886} 914) 539 | 375 | 411] 97 | 0.88) 5,5 6,6| 9.70} 94
10/25} 868) 914! 585 | 329 | 36] 95 | 0.79) 6,0 8,0} 9.80} 93
10/31) 145) 596} 518 | 78 13 | 24 | 0.75) 1,6 | 20,4; 9.70) 61 ¥
* Death. (Internal hemorrhages, urobilin in urine ++.)
Blood volume by dry oxalate. Hemoglobin by Sahli tubes.
TABLE 38-3
Experimental history. Dog 17-163 (splenectomy) :
BLOOD REGENER-
EXPERIMENT ieee
NUMBER _. DIET WEIGHT . REMARKS
; Pigment | Blood per
volume | kilogram
kgm
Begin 4/24/17 | Meat, Blaud’s pills 918 102 9.0 | Table 75
Bled 458 cc. 340 76 8.9
End 6/6 /17 1063 117 8.6
Begin 9 /3 /17 Crackermeal, lard 1112 106 10.0 | Table 38 —
Bled 558 cc. Crackermeal, lard, 388 74 9.7 :
9 /21 /17 butter 728° 91 9.6
10 /25 /17 868— 93 9.8
End 10 /31 /17 2 143 61 9.7 | Death11/1/17

222

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 223

_ Table 38 illustrates a not infrequent condition which develops in sple-
- nectomized dogs made anemic and fed on a limited diet. We have
pointed out elsewhere (1) that there is a remarkable condition which
_ may develop in splenectomized bile fistula dogs. In these bile fistula
_ dogs if anemia is produced we may observe periods of spontaneous
blood destruction and enormous pigment overproduction. Under
4 such conditions it was suggested that the body was forming its maxi-

TABLE 39

i Blood regeneration—crackermeal, lard, butter, alfalfa meal—splenectomy. Dog
3 — 17-84. Bull mongrel, female, young adult

8/11 | Diet: Bread and milk

8/13 | Bled 336 ce.
—«- 3/14 | Bled 336 ce.

, n
F rg : g E
: 5 a a a 8 8
is Me) g Db E a s & 8 |
uf 3 ° a i R R =) s ba 3 i
4 cs) a> 5 2 o a a = Ps
Sue lere| § Ge bot Sabi goo poa ay sibpam
4 (igoeram Wb Bis Qa s 3 3 3 3 S cI A
B. = 2 ° ro) = A 4 " . Les)

<3] sO 4 n . ° Q 4 2
:: P ota) 8 5 " s = 3 “ d a 8
% a i ry my rs] r an) 5 ea -B e a
a : ce. ce. cc per cent|per cent kgm. cc.
i. 3/11. | 1776 | 1345 | 740 | 606); 45 132 | 0.94 | 7,0 11,8 | 16.10) 84
Ay :

fie | 828 | 1034| 765| 269| 26 | so|1.14| 3,5 | 22,0| 15.30| 68

3/16 | Diet: Crackermeal, lard, butter, 50 grams alfalfa meal

3/22 | 1128 | 1128 | 722| 407| 36 | 100| 1.00] 5,0 | 17,4| 14.801 76
3/29 | 1282 | 1187| 760| 427| 36 | 108] 0.93] 5,8 | 17,0 | 15.20) 78
4/4 | 1185 | 1162| 732| 430| 37 | 102/0.77| 6,6 | 16,2| 15.00) 77
Afio | 1371 | 1193 | 740| 453| 38 | 115] 1.08] 5,3 | 16,8 | 14.80| 81

: Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
- Experimental history, see table 17-b.

mum amounts of pigment material (hemoglobin as well as bile pig-
ment) but the red cell stroma was lacking in quantity or quality. It
_-was suggested that the spleen might be concerned in the development
q of red cell stroma. These present exeriments may point to the pro-
- duction of faulty red cell stroma under such: conditions (table 38) but
some readers may wish to postulate the development of some unknown
poison to explain the distintegration of the red cells in our splenectomy
experiments. ‘This condition develops very abruptly and may super-

224 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

vene in a dog with relatively normal red cell count and hemoglobin ~

values. Within 2 or 3 days the red cell count may fall to one-third
normal (table 38) and abnormal pigments appear in blood serum and
urine. Much more study must be given to this condition and we
expect to report further work in this line.

Alfalfa meal was added to the diets in two of these experiments.
These experiments give no evidence to indicate that alfalfa meal exerts
a definite influence upon the curve of hemoglobin regeneration in the
dog. The alfalfa meal used in our experiments was the usual grade
of finely ground alfalfa purchased on the open market. |

Rice, potatoes and milk. We may consider the next group of experi-
ments as a unit (tables 40, 41, 42, 43 and 44). In principle all these
experiments are similar and the results are remarkably uniform. In
the first four experiments the dogs were bled and placed upon a uni-
form diet of cooked rice, boiled potatoes and skim milk. The regen-
eration in most of the experiments was slow but uniform with the end
result after 5 to 6 weeks about normal or slightly below the normal
blood level. After this there followed a short period (7 to 10 days) of
mixed diet. ‘Then a second period of anemia and blood regeneration
upon the same rice, potato, milk diet was observed. These second
periods are replicas of the first regeneration periods on this same diet.

It is clear that a liberal diet of cooked rice and potato with skim
milk sufficient to maintain or slightly increase the body weight will
give a slow steady gain in blood pigment, red cell hematocrit, red cell
count, etc., which will often bring the regeneration curve back to nor-
mal or close to normal.

Two experiments are exceptions to the general reaction (tables 40
and 44). Table 40 shows a regeneration which is incomplete and not
back to normal in 5 weeks. In fact, during the last month the regen-
eration is not in evidence and the pigment volume, hemoglobin and
red cell hematocrit are stationary. There was a slight loss of weight
during this period but the dog was very active and normal in all re-
spects. The second anemia regeneration period shows an identical
reaction. Table 44 shows a still more striking difference from the
normal average regeneration. This dog refused to eat the amounts of
rice and potato and milk given at first. She ate the amounts recorded
in table 44, which amount to about 50 per cent that given to the other
dogs, or 50 calories per kilo body weight. During 6 weeks there was
a loss of 2.5 kilos and the blood regeneration was only slight during
this whole period.

ee ee a a ae

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 228

| TABLE 40
Blood regeneration—rice, potatoes and milk—repeat experiment. Dog 19-104.
Bull mongrel pup, male

ia 5 5
5 E = ic] a Q 8 Z g
gas, 2 | 2/2] § v | 8 :
& ann = 6 3 a a 4 : REMARKS
S E Bal p> 5 > mi ~ a &
epee 6 | 2) S| Sg a | ge eee
ies] ro) ) r) : F) a 4

Set el si el ele | el ae le) eee
cc. ec. cc. po ee kgm | ce.

1/30 | 1367| 1067) 455 | 602 | 56.4] 128 8.75} 122

2/6 1394) 1078) 468 | 594 | 55.1) 129 | 0.68) 9,5 | 14,8! 9.00} 120

2/6 | Diet: Bread and milk

2/7 | Bled 270 cc.
2/8 | Bled 270 cc. No distress

2/10 363| -630| 457 | 170 | 26.9] 58 | 0.76| 3,8 | 25,3 8.35| 75 |

2/10 | Diet: 363 grams rice, 417 grams potatoes, 500 cc. milk

2/17 | 692} 765| 451 | 311 | 40.6 90 | 0.82] 5,5 | 11,6| 8.00| 96
2/26 | 933] 859] 451 | 404 | 47.0] 109 | 0.75] 7,3 | 8,4] 8.00] 107
3/3 | 807} 816) 445 | 366 | 44.9 99 | 0.70] 7,1 | 10,2| 7.90] 103 | * Poik.++
3/10 | 910} 833] 419 | 410 | 49.2] 109 | 0.80] 6,8 | 10,0] 7.85] 106 | * Poik.

3/19 | 814 773 402 | 362 | 46.9] 105 | 0.67| 7,8 | 9,0] 7.85] 98 | * Poik.+t

3/21 | Diet: Mixed diet. Extra food
3/31 | 1036] 808] 433 | 456 | 50.8] 115 | 0.69| 8,3 | 12,2| 8.65 104 |
3/31 | Diet: Bread and milk 3

4/1 | Bled 225 ce.
4/2 | Bled 225 cc. No distress

4/3 | 462| 688| 462 | 223 | 32.4| 67 | 0.83] 3,8 | 24,8| 8.20 84 |

4/3 | Diet: 363 grams rice, 417 grams potatoes, 500 cc. milk

4/11 | 656) 780) 469 | 300 | 38.4) 84 | 0.70) 6,0 | 11,2) 8.20) 97 | * Poik.
4/18 | 701} 772) 435 | 330 | 42.7; 91 | 0.56) 8,1 8,8} 8.05) 96 |
4/25 | 760) 764) 421 | 339 | 44.4) 98 | 0.55] 8,9 8,0} 8.00} 95
5 /2 844) 816) 421 | 391 | 47.9) 103 | 0.64) 8,1 7,8] 7.90) 103
5/9 791} 804/ 431 | 369 | 45.9} 98] 0.58] 8,5 | 5,8) 7.65} 105

* Poikilocytosis of red cells.

tT Only 300 cc. of milk given.

t Gave 300 grams rice and 300 grams potatoes.
No previous anemia experiments on this dog.

226

C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE —

TABLE 41

Blood regeneration—rice, potatoes and milk—repeat experiment.
mongrel, male, age § months

Dog 19-95. Bull

a ee ee ee SPS ee ee ee

1g 3
: : ;
2 3 3 = a 5 Z 8
p
seal aa |e] a | Gg eee :
a |Pasl 2 ) 3 a a 3 pe REMARKS
— ° 2S =
S leks) F | EE | & Bu | ht gee
A a :8 A s i) .s) fo 5 - se] a
E (aoa) Steg fe] pea Bm |
a | c Fy ei a | 8 | E E a
ee. ce. ce. ie ital kgm. | cc.
1/16 | 1232} 1130) 595 | 528 | 46.4) 109 | 0.67| 8,2 | 18,0/11.35} 100
1/16 | Diet: Crackermeal and milk |
1/17 | Bled 282 ce.
1/18 | Bled 282 ce. No distress
1/20 | 560] 824] 602 | 218 | 26.4! 68 | 0.7| 4,4 | 30,4111 .00) 75 |
1/20 | Diet: 418 grams boiled rice, 490 grams potatoes, 500 cc. milk
1/27 | 742) 974! 574 | 395 | 40.6) 76 | 0.72) 593 | 16,2)12.75| 76 |
2/3 954, 976] 531 | 485 | 44.6} 98 | 0.65) 7,5 | 22,8)11.15) 87
2/12 | 982} 1013) 538 | 464 | 45.8) 97 | 0.75) 6,5 | 14,21/10.90) 93
2/19 | 1040} 1000] 511 | 480 | 48.0] 104 | 0.70] 7,4 | 10,6/10.60| 95
2/28 | 1242) 1058) 514 | 538 | 50.9! 118 | 0.88) 6,7 9,810.80} 98 | * Poik.+
3/7 983} 1006) 521 | 475 | 47.2! 98 | 0.87| 7,2 | 10,0)11.20} 90 | * Poik.+
3/10 | Diet: Mixed diet _ ,
3/17 | 1092| 1130| 640 | 484 | 42.8| 97 | 0.84! 5,8 | 13,0[12.80| 98 | * Poik.
3/17 | Diet: Crackermeal and milk
3/18 | Bled 283 cc.
3/19 | Bled 283 cc. No distress
3/21 | 570| 934| 664 | 251 | 26.9] 61 | 0.92] 3,3 | 18,012.45] 75 | * Poik.
3/21 | Diet: 418 grams boiled rice, 490 grams potatoes, 500 cc. milk
3/28 | 577| 834) 576 |.250 | 30.0} 69 | 0.86) 4,0 6,0/11.75| 79 | * Poik.
4 /2 741} 953] 605 | 334 | 35.0} 78 | 0.63) 6,2 | 11,6)11.80) 81 | * Poik.+
4/9 773| 976) 600 | 371 | 38.0} 79 | 0.70) 5,6 8,2|11.85} 82
-4/16.| 988} 1046} 588 | 448 | 42.8) 94 | 0.69) 6,8 | 10,4)11.90) 98 ;
4/23 | 1085} 1073) 576 | 492 | 45.8) 101 | 0.68) 7,4 | 20,0/11.95; 90
4 /30 | 1237) 1168) 576 | 581 | 49.7] 106 | 0.65) 8,1 8,2|/11.95} 98

* Poikilocytosis of red cells.
No previous anemia experiments on this dog.

ae” ae ae

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 227

: TABLE 42
z Blood regeneration—rice, potatoes and milk—repeat experiment. Dog 19-93. . Bull
mongrel, female, age 5 months

1A =
ge : :
Rp c
5 Pal a = a 8 % 8
wee! = 5 a 5 5 3
a es] (3 ° 2 | a 3 0 REMARKS
= > ° > ° a a = eS MJ
Ss gp & a od < ,! ” qj a F i=] Ay
- |@-8| 2 | #:} & |] 8 | Sie FP? Rane
a 2 =| ° ~s m > * i fo) e - Oo fo}
eet Ss) ge Rob sebog ey Roy | mel og
Aa |e a By a rs ss) 8 a | B ek Wee
per | per
f 4 3 fe cent | cent kgm. | ce.
: 1/16 | 1485) 1380) 742 | 616 | 44.7| 104 | 0.59) 8,9 | 18,6/10.95} 126

1/16 | Diet: Bread and milk

1/17 | Bled 345 ce.
1/18 | Bled 245 ce. No distress

1/20 | 594| 886 640 237 | 26.8| 67 | 0.76

,2|10.25] 86 |

1/20 Diet: 400 grams boiled rice, 475 grams potatoes, 500 cc. milk

* 4/27 | 768| 1084! 638 | 436 | 40.2| 71 | 0.48| 7,4 | 10,8/10.35| 105 | * Slight
2/3 | 1058| 1080| 570 | 494 | 45.7; 98 | 0.60] 8,1 | 15,2| 9.55] 113 | * Slight

Cells
small

/ 2/12 | 1040; 1063) 566 | 482 | 45.3) 98 | 0.69) 7,1 8.41 9.90) 107 | * -
- 2/19 | 1245) 1163) 570 | 582 | 50.0} 107 | 0.73) 7,3 6,4| 9.55} 122'| * Slight
2/28 | 1255} 1125] 568 | 540 | 48.0) 112 | 0.84) 6.7 5,8] 9.65) 116 | * Slight
3/7 | 1098) 1127) 568 | 542 | 48.1) 98 | 0.73) 6,7 6,0/10.35} 113 | * Slight

3/10 | Diet: Mixed diet
3/17 | 1040| 1004| 516 | 478 | 47.6| 104 | 0.70] 7,4 | 7,8|11 35} ‘9 | * Slight

3/17 | Diet: Bread and milk

3118 | Bled 251 ce.
3/19 | Bled 251 cc.’ No distress

3/21 566 916 647 | 260 | 28.4 62 | 0.84 3.7 | 12,2)11.15 82 |

*

3/21 | Diet:.400 grams boiled rice, 475 grams potatoes, 500 cc. milk -

3/28 | 896) 1028) 614 | 397 | 38.6) 87 | 0.85) 5,1 7,8/11.35) 90
4/2 908} 1085) 631 | 433 | 39.9) 84 | 0.65) 6,5 8,611.20) 97.| * Slight
4/9 858} 990) 568 | 412 | 41.6) 87 | 0.60) 7,2 6,0/11.35} 87
4/16 | 1136) 1112} 565 | 530 | 47.7| 102 | 0.67| 7,6 8,8/11.30) 98 | * Slight
4/23 | 1212) 1142) 568 | 557 | 48.8} 106 | 0.57} 9,3.) 6,8/11.35) 100

4/30 | 1313} 1198) 572 | 604 | 50.4) 110 | 0.58) 9,5 7,411.15) 107 | * Slight

* Poikilocytosis of red cells.
No previous anemia experiments on this dog.

228 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE
TABLE 43
Blood regeneration—rice, potatoes and milk—repeat experiment. Dog 19-108.
Bull mongrel, female, age 5 months
sha ro] 8 ie}
= re)
sae, 2 | 2] 2 | & sa ;
a Pee a 3 3 x a 3 - ‘REMARKS
S [bee eau be a a :
elfes 8g 81 Std Be eee
& ° 5 a = °
a4 | RoR a4 aol Be) ee ee
ce. 66: ce. i pout kgm. | ce.
1/30 | 1100) 1077| 588 | 483 | 44.9) 102 9.55} 113
2/6 978] 1020) 598 | 428 | 41.9) 96 | 0.61) 7,9 | 12,610.00) 102
2/6 | Diet : Bread and milk
2/7 | Bled 255 cc.
2/8 .| Bled 255 cc. No distress |
2/10 430 803 600 | 204 | 25.3| 53 | 0.78| 3,4 | 9,0 9.45] 85 |
2/10 | Diet: 411 grams rice, 472 grams potatoes, 500 cc. milk a
.
2/17 | 770| 854| 485 | 365 | 42.7; 90 | 0.78) 5,8 | 12,4) 8.85) 96 bs
2/26 | 996} 981) 520 | 446 | 45.5) 102 | 0.68] 7,5 | 10,8) 9.10} 108
3/3 | 13805) 1070) 491 | 574 | 52.6) 122 | 0.80) 7,6 | 8,4) 9.20) 116
3/10 | 1068) 1008) 526 | 476 | 47.3] 106 | 0.68} 7,8 7,0} 9.20) 110
3/19 | 1065} 986} 499 | 477 | 48.4) 108 | 0.72) 7,5 9,0} 9.60} 103 7
3/21 | Diet: Mixed diet. Extra food
3/31 | 1103 1103| 600 | 492 | 44.6 100 | 0.71| 7,0 | 10,6 10.65| 103 |
3/31 | Diet: Bread and milk
4/1 | Bled 276 cc.
‘4/2 | Bled 276 cc. No distress
4/3 | 444] 837] 612 | 220 | 26.3| 53 | 0.76| 3,5 | 11,6[10.40| 80 |
4/3 | Diet: 411 grams rice, 472 grams potatoes, 500 ce. milk
4/11 | 756} 964! 603 | 347 | 36.0} 79 | 0.76) 5,2 | 8,8)10-70| 90].
4/18 | 803) 957| 570 | 377 | 39.4) 84] 0.57) 7,4 | 14,6)10.55) 91
4/25 | 970} 1003) 546 | 489 | 48.8! 97 | 0.64) 7,6 | 7,2/10.65| 94
5/2 | 1077} 1057) 560 | 491 | 46.5) 102 | 0.62) 8,2 | 11,0)10.35) 102
5/9 | 1045} 1062) 592 | 460 | 43.3) 98 | 0.65) 7,5 | 14,6/10.50) 101

* Poikilocytosis of red cells.
No previous anemia experiments on this dog.

—_— oe a

a ee Pe —* - -— Ls — a ae i se SS = me a = > ¥ wet. os > c= A eo _
a i a a eal a we Pe eZ SS aaee ip, ae te 7. t J. P ‘i 4 . 7 let ee ae TT ~ ——
= $ z i a . i “ ” net ks Sa oe, —nr S
< ry D. ee i ae Fa ee ee same a, eo s ‘ 5
; = - ~ ie Se 7 . = — ea wa ue —3
6 5 . P = or ‘ = 7 ul. ‘ -
" 5 Si ed e = i ~ tine ee a a eri ”
¥ . iy ~ i aes le © ¥ pars :

Y al -

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 229

This experiment (table 44) shows admirably a reaction noted in
other experiments. ‘The bleeding reduced the hemoglobin from 123
per cent to 59 per cent and the red cells from 7,500,000 to 3,300,000.
During the 6 weeks’ observation we note a rise of only 24 per cent hemo-
globin but the red count returns to normal. The color index of course

TABLE 44

Blood regeneration—rice, potatoes and milk. Dog 19-96. Bull mongrel, female,
age 8 months

2
- = E
ag ea &
268 a Q 5 ra)
Bee! 8 | a-| a | & 6 S
Seo) 8 a g = a 4 - REMARKS
= Bel 5 : ° = a = =
S 5 , a > < o q Z a n 4 a
ns = ee 8 a s 3 is) foe} Ss) ° q a
2} s23| 6 = ° . 5 ‘ ed o °
& fx ) < ma ma ; a fa a re)
e oma) 8 < ; » re) Ps , 7 q S
—2 Ey =) Be 8 8 jen) 5 8 B B =)
per per
cc ce. ce eek li cant kgm. | cc.
3/17 | 1520) 1238) 568 | 663 | 53.6} 123) 0.82) 7,5 | 11,4/13.50} 92

3/17 | Diet: Crackermeal and milk

3/18 | Bled 310 ce.
3/19 | Bled 310 ce. No distress

3/21 | 527| 801] 636 | 246 | 27.6| 59 | 0.80] 3,3 | 14,2{12.40| 72 |

3/21 | Diet:{ 200 grams boiled rice, 200 grams potatoes, 500 cc. milk

3/28 | 616} 835) 551 | 267 | 32.0; 74 | 0.93] 4,0 | 14,8)11.55) 79
4 /2 662}. 911) 598 | 308 | 33.4) 73 | 0.65) 5,6 | 11,4/11.20) 81 | * Poik.++
4/9 784| 959} 581 | 368 | 38.4; 82 | 0.59) 6,9 | 10,4/10.75) 89 | * Poik.+ -
4/16 | 728) 908) 550 | 354 | 39.0) 80 | 0.56) 7,1 6,8}10.30} 88 | * Poik.+

4/23 | 818} 905) 532 | 368 | 40.7; 90 | 0.61] 7,4 | 10,2}10.10) 90 | * Poik.++
4/30 | 778) 938) 535 | 394 | 42.0) 83 | 0.58) 7,2 | 12,0) 9.90) 95 | * Poik.++

* Poikilocytosis of red cells.

+ Animal refused to eat larger quantities of food. Represents about 50 calories
per kilo of body weight.

Experimental history, see table 4-b.

drops from 0.93 to 0.58 and poikilocytosis is very much in evidence.
Under such conditions one feels a very strong probability of red cell
fragmentation.

This evidence (tables 40 and 44) confirms our belief that the amount
of any diet may be a considerable factor in blood regeneration. Given
a diet of a limited nature but sufficient to permit of slight gain in body

230 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE ~

weight and we may expect a certain amount of blood regeneration, at

times even a return to normal. But given a limited diet in small

amounts not sufficient for maintenance of body weight, we may con-

fidently expect a very slow blood regeneration or complete absence of

active regeneration. Under such circumstances the body may even

be unable to make up its blood cell maintenance factor and the pig-

ment volume curve may actually fall. This is a favorable time for

TABLE 45

Blood regeneration—casein, sugar, butter and lard. Dog 18-56. Bull mongrel,
. female, young adult

) ne Fy
‘ q 4
A m o

= Ss)
= =i 3 } Z o
i Z 5 = Z = & ° |
9056 B 3 5 5 5 = Ds
> a > ia 9 5 fa re) - , om
—_ () o i=]
a BA a > > > 3) 4 a . oe i)
getaes P oyak ote Se | J 5 S 3 3 i Q
a Sa9 i} a ; cf e 3 : " ic} oS
5 om a 8 < ma a xe) 2 Bs . a 8
a ry rs Py rs ea a) 8 ea e E Q
; ce. ce. cc. |per cent|per cent| — kgm. cc

10/29 | 1275 | 930] 400] 530| 57 137 | 0.89 | 7,7 | 12,6| 8.40] 111

10/29 | Diet: Bread and milk

10/30 | Bled 233 ce.
10/31 | Bled 233 ec.

1j2 | 340| 548 | 400 | 148 | a7 | 62 | 1.20 | 2,6 | 16,0 | 7.50 | Fe

11 /2 Diet: 75 grams casein, 25 grams sugar, 20 grams butter, 20 grams lard

11/9 | 428] 586| 375] 211 | 36 73 | 1.30| 2,8 | 19,6] 7.50| 73
11/16 | 786| 827| 463| 364] 44 95 | 0.99} 4,8] 60] 8.10] 102
11/23 | 725| 763} 412| 351 | 46 95 | 0.93] 5,1 | 12,0| 7.20! 106
11/28 - 908} 810] 389] 421| 52 | 112] 0.81} 6,9 | 12,2] 7.80] 111
12/5 | 1330| 985 | 384] 601] 61 | 1385/0.85| 7,9 | 13,2] 7.40] 188

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
No previous anemia experiments on this dog.

the appearance of a characteristic dietary deficiency disease which is
much like scurvy in human beings and is rapidly fatal if not energeti-
cally treated with antiscorbutic measures.

Casein and gliadin. When we consider a bread and milk diet from
the standpoint of dietary factors we are obviously dealing with many
known and unknown constituents. Two of the familiar ingredients in
this bread and milk diet are casein and gliadin, which are concerned

a
a
i
a

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 231

particularly in the following group of experiments (tables 45 to 47 in-
clusive). Casein used in these experiments was obtained ‘from a large
dairy products company in this state. It appears as a fine dry granu-
_ lar powder, pale yellow in color, and is of reasonable purity, judging
from information given us by the chemist of this company.

_ The gliadin was extracted from wheat flour in this laboratory by
use of dilute alcohol (70 per cent). The weighed amount of gliadin
was thoroughly mixed with the sugar, moistened with water and fed
to the dog by spoon. Total ingestion was readily accomplished in
this way. :

: Table 45 shows the influence of casein, sugar, lard and butter on
_ blood regeneration. The diet was sufficient to maintain body weight
and the blood regeneration was complete in 5 weeks. We must not
forget that this dog had not been used for anemia experiments pre-
vious to this time and such dogs occasionally show remarkable regener-
ative capacity on limited diets. The next- experiment, however, is
conclusive and shows the effect of casein under more carefully controlled
and less favorable conditions.

The second casein experiment (table 46) is preceded by a 2 weeks’
sugar diet period during which the expected reaction is noted. There
is the usual gain in red cell hematocrit, hemoglobin and pigment volume.
If the sugar diet had been continued we are reasonably certain that the
pigment volume would have remained stationary or even have fallen.
Casein added to the diet shows a distinct gain which is held during the
subsequent weeks when we see slight fluctuations in pigment volume
but relatively little change. There is a slight gain in weight as the.
calories in the diet are increased by the use of fats. The figures given
for the urinary nitrogen show the normal level for the sugar periods and
during the sugar and casein intervals indicate the amount of nitrogenous
metabolism. ? |

Table 47 is to be compared with a preceding experiment (table 21,
paper II). In both experiments a gliadin sugar diet is used over a
period of several weeks. This experiment shows less conclusive evi-
dence of the influence of gliadin upon blood regeneration. ‘We may
conclude that the gliadin in this experiment (table 47) was without
influence on the curve of blood regeneration. We are inclined to the

opinion, however, that sugar alone over this period in this experiment
- would have been associated with a definite loss in pigment volume by

the end of the 6-week period. This experiment shows a transient
increase in pigment volume which is lost during the last 2 weeks.

es? ee

:

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53 NO 2

SSE eee TR oR Sn SP pean ae ye Le Aree amr

peers

232

C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

TABLE 46

Blood regeneration—casein, sugar, lard and butter. Dog 1 6-158. Coach movgrel, 4

male, young adult

ig : a | 5
AE = G p
Zas a ° a 8 a 8 me
sacl a | ple | 8 wn | § g | 38
O58 5 Q p = 5 i] i) 40
fe aks (eek ele is lly le tae ie ae a | ag REMARKS
= ba : aR > i Z 3 a | af :
= zm Spi < ‘ . a 4 3 a | & | oF
< ARS a s ie) is) = oO , mq Qa <b
Sl ageS dee lidolim dog legeb ach Asch dele Bales
A |& a] me) | ol ee ad eB ee
cc 60:4, 6¢ Fatt pa kgm.) cc. | grams
10 /17-| 1817} 1367| 588) 779} 57 | 133)0.74| 8,9) 11,0)9.70) 141
10/17 | Diet: Bread and milk
10/19} Bled 342 ce.
10 /21| Bled 342 cc.
10/22) 576|. 823| 576| 247| 30 | 7ol0.95| 3,7| 11,2/9.50| 87| |
10/22; Diet: 75 grams cane sugar, 25 grams glucose, 300 cc. water
10/29} 707; 895) 573} 312) 36 | 79/0.79| 5,0) 10,6/8.60) 104) 1.81
11/5 766} 958) 575) 383) 40 | 80/0.67| 6,0} 6,0/7.90) 121) 1.61
11/5 ‘| Diet: 100 grams sugar, 50 grams casein, 300 cc. water
11/12} 856} 961) 567; 394) 41 | 89/0.73) 6,1) 3,8/7.80} 123} 4.70
11/19 | 1038} 1081) 616} 465) 43 | 96/0.67| 7,2} 5,2/7.70| 140) 6.60} Vomiting
11/21 | Diet: 100 grams sugar, 100 grams casein, 300 cc. water
11/26} 878) 944! 538} 406) 43 | 93/0.69] 6,7) 5,0/7.80} 121| 9.36
12/3 824; 970} 582) 388) 40 |. 8510.75) 5,7) 6,2/8.00} 121/11.60} ©
12/3 | Diet: 100 grams sugar, 100 grams casein, 10 grams lard, 10 grams
butter —
12/10} 876} 974} 604) 370) 38 | 90/0.57| 7,9} 9,8)/8.30} 117
12/19] 876) 952) 600) 352) 37 | 92/0.69| 6,7) 14,0/8.70} 108
12/20} Diet: 100 grams sugar, 125 grams casein, 10 grams lard, 10 grams
butter
12/26 | 40 | 960.61| 7,8| 9,0/8.90
1/2/18 38 | 93/0.55) 8,4) 7,819.10
1/9 35 | 92/0.62| 7,4) 8,6/9.30
1/17 38 | 85/0.50} 8,5) 6,8/9.10
1/23 | 975) 975) 614) 361) 37 | 100/0.58} 8,7) 6,2/8.90! 109

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA

TABLE 46-3

Experimental history. Dog 16-168

233

j BLOOD REGENER-
BEFASIMENT - DIET eben WEIGHT REMARKS
NUMBER ‘
Pigment | Blood per
volume | kilogram
kgm
Begin 9/26/16 | Meat 8.1
Bled 600 cc 7.7 | (4 bleedings)
End 11/20/16 9.5 | Complete regen-
eration of Hb.
and R.B.C.
Begin 2/12/17 | Beef heart 1523 147 9.9 | Table 56
Bled 726 cc. 385 85 9.8
End 4/11 /17 1476 152 8.8
Begin 5 /7 /17 Sugar, gliadin 1505 146 9.0 | Table 47
Bled 660 cc. Metabolism 458 84 8.4
End 6/18 /17 456 113 6.2 | R.B.C. fragment-
ed, shadow
forms
Begin 10/17/17 | Sugar 1818 141 9.7 | Table 46
Bled 684 cc. Sugar and casein| 576 87 9.5
End 12/3 /17 Metabolism 825 121 8.0
Begin 5/3/18 Sugar, glycocoll 1800 119 IF
_ Bled 698 ec. Metabolism 528 76 10.9
End 6/18/18 852 99 8.05 | Maximum regen- —
eration 3 weeks,
then drop
Begin 8/28/18 | Sugar and gela- 974 82 | 10.3
Bled 929 ce. tin 548 88 9.15 | (5 bleedings)
End 9/30/18 508 90 7.45 | Maximum regen-
eration 2 weeks
Begin 2/20/19 | Sugar and liver | 1862 111 12.35 | Table 67
Bled 934 ce. residue 478 70 11.10 | (3 bleedings)
End 3/18 /19 738 82 9.15
Begin 8/18/19 | Beet tops 1468 93 12.55 .
Bled 851 ce. Spinach 560 77 12.10 | (3 bleedings)
End 10/24/19 785 86 10.50

234 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

These experiments are not conclusive but give evidence that mod-
erate amounts of gliadin with sugar alone do not modify profoundly
the blood regeneration curve. That gliadin in combination with other
factors may have a more favorable influence on red cell production
may be granted. Also under the conditions of the experiment it ap-
pears that casein has a more favorable influence on blood regeneration
than gliadin when given with sugar only. |

TABLE 47
Blood regeneration—sugar and gliadin. Dog 16-158. Coach mongrel, male, young
adult ; |
2
-_ E ;
se ‘i 8 8
Pea] 8 |a4 8 |. 8 g 3
ofa & p 5 | A jc i
~ | Pes] 2 3 8 a a | @ REMARKS
S Beal 5 > > | Zz | : a
See es | a as He aot 8 | Sa ee
io] sO 5 ° D ° ° ; ° ° Pe o ro)
& | otal 98 < ma fm o = ma ; a |.8
a ry a Fy rs ea q 3 ef z Ez )
per per ;
ce ce. ce } aa peo kgm. | ce.
5/7 | 1505} 1320) 581 | 740 | 56 | 114 | 0.57} 10,1} 6,6) 9.00) 146

5/8 | Bled 330 cc.
5/9 | Bled 330 ce.

5/11 | 458| 705) 480 | 226 | 32| 65 | 0.64) 5,1 | 10,8) 8.40) 34 |

5/11 | Diet: 50 grams cane sugar, 25 grams dextrose, 25 grams gliadin, 300 ce.

water
5/14 | 4384) 804) 547 | 257 32 54 | 0.68} 4,0} 9,0) 8.20) 98
5/21 | 485) 791] 5388 | 253 | 32] 5510.59} 4,7} 8,6) 7.80) 101 vs
5 /28 596} 961) 519 | 346 36 62 | 0.56) 5,5) 9,8) 7.40) 129
6 /4 604; 863} 552 | 311 36 70 | 0.61} 5,7| 8,4) 6.90) 122
6/11 559| 766) 528 | 237 31 73 | 0.61] 6,0} 9,2) 6.60) 111 %
6 /18 456| 702) 477 | 225 32 65 | 0.53] 6,1) 11,6) 6.20) 113 s

* Fragmentation of red blood cells.
‘Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
Experimental history, see table 46-b.

SUMMARY

A diet of dried white bread and skim milk may cause a slow, steady _
gain in blood pigment volume from week to week. A liberal diet of —
this type sufficient to maintain or increase body weight will often suf- _
fice for complete blood regeneration. A restricted diet of bread and

BLOOD REGENERATION FOLLOWING SIMPLE. ANEMIA 235

milk barely sufficient for body maintenance will rarely permit of com-
plete blood regeneration following simple secondary anemia.
Repeat experiments done after short intervals of rest to permit com-
plete return to normal condition will show identical reactions on the
part of the hemoglobin, red cells and pigment volume. The animal
shows no increased ability to produce hemoglobin and red cells after
repeated experiments nor is there any evidence for a failure of red cell
production under these conditions.

Bile fistula dogs presenting complete exclusion of bile pigments from
the intestine show a reaction which is practically identical with that of
normal dogs.

Crackermeal (a mixture of wheat flour, barley flour and rice flour)
with milk or lard and butter, gives a blood pigment reaction following
anemia which is similar to the familiar bread and milk reaction. ~

A dietary deficiency disease may develop in these dogs kept on limited
diets for many weeks. This condition clinically resembles scurvy in
human beings and may be prevented or cured by antiscorbutic measures.
This question will be reviewed in a subsequent publication.

Splenectomy may not modify the expected reaction of red blood cells
following anemia. In certain splenectomy experiments there develops
a peculiar condition associated with spontaneous destruction or disin-
tegration of circulating red cells. This may appear following a limited
diet of several weeks and runs a very rapid course ee in death
within a few days.

Rice, potatoes and skim milk make up a diet which may be classed
with bread and milk as regards its influence upon red blood cell regenera-
tion following the unit hemorrhages. If anything, this diet is slightly
more efficient than bread and milk in promoting blood regeneration.

a 7 Casein and gliadin by themselves are not efficient factors in pro-

moting red cell regeneration but casein appears to be the more efficient
in the amounts used and under the conditions of these experiments.
Any one of these diet mixtures in proper amounts may be used to
maintain the pigment volume at a constant level following the initial
2 weeks’ blood reaction. Under such conditions any added food factor
may be measured with some accuracy as to its power of aiding in blood

___ regeneration.

BIBLIOGRAPHY

(1) Hooper anp Wutrpte: This Journal, 1917, xliii, 275.

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA

IV. InruuENcE or Manat, Liver anp Various ExrractivEs, ALONE 4
OR COMBINED WITH STANDARD DIETS

G. H. WHIPPLE, F. S. ROBSCHEIT anv C. W. HOOPER

From the George Williams Hooper Foundation for Medical Research, University of :

California Medical School, San Francisco

Received for publication April 3, 1920 -

Cooked meat and liver stand in striking contrast to the milk, bread,
potato mixtures outlined in the preceding paper of this series. Cooked
liver, lean beef or beef heart alone or in combination are very efficient
in bringing about a rapid blood regeneration following the standard
type of secondary anemia. These substances are very similar but for
the present we may say their efficiency is in the order given; that is,
cooked liver is most effective in anemia provided a sufficient amount |
(caloric value) is eaten, and cooked beef heart is least effective; but
the differences are not great and this order may be changed with the
accumulation of more data.

These three substances are efficient in ‘eatin blood regener-
ation whether given alone or in combination, or together with carbohy-.

drate or mixed diets. They all stand the severe test of promoting defi- ~

nite blood regeneration when administered after long limited diet
periods unfavorable to blood regeneration.

Meat extract (commercial) has no value in the blood regeneration
complex. Buta watery liver extract seems to exert a distinct in-
fluence on the blood regeneration. Liver residue (after the watery
and alcoholic extraction) alone exerts a definite influence on blood re-
generation. We do not wish to go into a discussion of this question of
tissue extracts until we present much more experimental data dealing
with this and other material of similar nature.

EXPERIMENTAL OBSERVATIONS

In general the experimental technige has been detailed in the first
paper of this series. All meat and. liver were cooked thoroughly in
boiling water before feeding, with the exception of “meat scraps.”

236

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA - 237

The meat scraps were obtained from the University Hospital and in-
cluded meats of various kinds cooked in different ways. A certain
amount of fat was of necessity included in this meat diet. Unless
otherwise noted these diets were completely ingested. With: an
- occasional exception noted in the tables the aoe were in uniformly
& ont condition.

TABLE 48

Blood regeneration—cooked meat scraps. Dog 17-38. Bull mongrel, female, adult

ig =
“ ; :

sha ; 8
Bee, eg} a al oe 5
Bs: 588 “RB | B 3 a | we
R 3 > 3 4 8 a a Q a
& EBa > A > mi q a : by ry
+ B68 A 3 3 3 % S) . a 8
—& |éme] 8 2 A eee 3 a S fi 8
r=) ry =) Ba rs a © ai 8 fa e E 9
cc. cc. ce per cent|per cent kgm. cc

2/12 | 1322 | 1268| 670] 591| 46.6] 104/0.71| 7,3 | 7,4] 10.90] 115

Mae. \Dict Bread and milk

2/13 | Bled 317 ce.
2/14 | Bled 317 ce.

2/16 | 550 | 1000| 714| 281] 28.1| 55 |0.75| 3,7 | 6,2| 10.85 97

2/16 ' Diet: 500 grams cooked meat scraps

2/24 | 948 | 1168 | 743°] 435 | 37.2 80 | 0.69 | 5,8 | 10,8 | 11.05). 106
3/1 1046 | 1236 | 742 | 488 | 39:2) 85 | 0.66] 6,4 5,8 | 11.50} 107
3/8 | 1437 | 1332 | 682] 636 | 47.8 | 108] 0.68} 7,9 | 9,8 | 12.10} 110 -
3/15 | 1332 | 1378 | 734 | 626 | 45.4 97 | 0.62 | 7,8 7,4 | 12.85} 107
3/22 | 1475 | 1420 | 704 | 704 49.5 | 104 | 0.62 | 84 | 8,2 | 13.20} 108

Experimental history, see table 20-b.

The first two tables show the characteristic reaction to a diet of meat
scraps. There is a prompt and rapid regeneration of hemoglobin and
red cells which brings the hematocrit and pigment volume back to
practically normal in 3 weeks. This level is sustained for the subse-
quent 2 weeks. The first experiment. (table 48) shows a rather low
initial level (104 per cent hemoglobin) but a very prompt reaction
following the anemia. ‘The hemoglobin, red cell hematocrit and pig-
ment volume return to a level slightly above the original normal level.
There was a marked gain in weight.

238 G. H. WHIPPLE, F. 8. ROBSCHEIT AND C. W. HOOPER

The second experiment (table 49) required a third bleeding to re-
duce the red cells to the usual anemia level. The regeneration of the
red cells is practically complete in 3 weeks, although the previous high
level of hemoglobin and red cell hematocrit is not reached. This
level is uniform during the next 2 weeks. In both experiments the
plasma volume is relatively constant during the entire period of
observation.

TABLE 49

Blood regeneration—cooked meat scraps. Dog 18-114. Bull mongrel, female, adult

42 =
a : F
Sea iS)
5 OO B 2 ic} = a 3
& a S > iS cS a = z
a: ERG LS / a < 2 3 e | &
ey $59 8 = 3 = j & 3 3 8
& |ota| ° < a Aa r a 2 : = 8
a |e a a Fs 2 fy 8 ei FS e F
ce ce. cc per cent|per cent kgm. ce.
2/12 | 1995 | 1705 | 766 | 931 | 54.6 117 | 0.70 | 8,4 | 10,8 | 15.50} 110
2/12 | Diet: Bread and milk ,
2/13 | Bled 425 ce.
2/14 | Bled 425 ce.
2/16 | 822 | 1232 | 818 | 403 | 32.7| 67|0.98| 36 | 7,8 | 14.30| 86
2/16 | Bled 275 ce. ‘
2/18 | Diet: 600 grams cooked meat scraps
2/24 | 1135 | 1480] 950] 504 | 34.0 77 | 0.80 | 4,8 | 10,2 | 14.25} 104
3/1 1434 | 1525 | 857 | 640 | 42.0 9410.73 | 6,4 9,0 | 14.35) 106
3/8 1580 | 1540 | 798] 726 | 47.2] 103 | 0.68 | 7,6 8,2 | 15.15) 102
3/15 | 1678 | 1678 | 874] 796 | 47.4| 100] 0.63] 7,9 7,8 | 15.50} 108
3/22 | 1787 | 1728 | 838 | 864] 50.0| 103 | 0.63] 8,2 9,4 | 15.50} 111

Experimental history, see table 12-b.

Table 50 presents a reaction very much like that noted in the two
preceding experiments. Fresh lean beef in adequate amounts was fed.
It was cooked in boiling water. This meat contained very little fat
and was purchased under the trade name of “‘chuck.”’ The dog shows
a very high level before bleeding and three bleedings did not reduce the
level to the usual anemia level. This dog had not been used previously
in anemia experiments. The blood regeneration was rapid in the first

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 239

_ 2 weeks, but there was little gain in the third week except in the red
_ count, which shows a remarkable jump. This gives a corresponding
fallin the color index. A change to a mixed diet shows but little change
until the third week of mixed diet when the original high level was

attained.
TABLE 50
‘a Blood regeneration—lean beef. Dog 18-24. Bull mongrel, male, young adult

1Z =

ae I BS

sha = & o

pHs a °

| Z bp = =I Ss s =

OBA p b < i ey ie]

roOo°o p | Me s a) I

- © > 8 ° 8 i]

= ae ro) ‘ed ° a (=) a

- Ze 8 < 7 " i a ; & Ae

- a6 a 3 S 3 @ 3 ° i a

{<3} S25 ° Dm . . 5 fo) . feo} oS [o)

e cia) § le eae oak bee 3 fe a 8

A ey re) cy a ea an) 3 a B z ry

yi Ree. ce. cc. |per cent ‘see kgm. ce.
11/14 | 1560 | 1300 | 480 | 814 | 62.6 | 120 | 0.87 8,4 | 12,0 | 13.20 99

11/12 | Diet: Crackermeal and milk

11/15 | Bled 325 ce. Slight distress
11/16 | Bled 195 ce.

111s | 582| 915 | 612] 298 | 32.6 | 64 | | | 12.70| 72

11/18 | Bled 150 ce.

11/20 | 466 | 806 | 539 | 264 | 32.7| 58 |0.81| 3,6 | 20,2 | 11.85] 68

11/20 | Diet: 567 grams cooked lean beef

5,9 | 15,8 | 12.70] 78
84] 14,9] 13.15} 81
11,9 | 15,0 | 13.40] 77

11/27 | 935 | 995-| 590 | 399 | 40.1 | 94
12/4 | 1093 | 1062 | 585 | 473 | 44.5 | 103
12/11 | 1090 | 1033 | 582 | 466 | 45.1 | 106

oo°
62s

12/13 | Diet: Mixed diet

12/20 | 1055 | 1115 | 594 | 511 | 45.8 | 95 | 0.47 | 10,1 | 25,8 | 13.50) 83
12/27 | 1010 | 1060 | 544 | 516 | 48.6 | 95 13.20) 80
1/10/19 | 1425 | 1230 | 550 | 674 | 54.8 | 116 | 0.58 | 10,1 | 7,8 | 13.20) 93

No previous anemia experiments on this dog.

Table 51 illustrates a reaction which is in no sense typical, but com-
plicated by abnormal factors. It is submitted with reservations. This
dog has been used in a variety of blood regeneration experiments (table
6-b), therefore the type of reaction is well established. But within a
few weeks following the present experiment the dog died with bilateral

240

Blood regeneration—lean beef and gelatin—lean beef and brain. Don 17-28. Bull

G. H. WHIPPLE, F. S. ROBSCHEIT AND C. W. HOOPER
TABLE 51

mongrel, female, young adult

ig a 4
Ee z :
eee gs ae Te é 2 |
= Ofal = ~ Bb < tat fe v)
en ear 4 5 3 a 5 < @ REMARKS
= a i a > > ty Z 3 ; a
= Zus < ‘ ; al ; cs &
a AAO a s o o fo ,o E a
egos} | fi] e)/ ela} s]al| a] eye
a | a a.) Re ale Ba Se ee ee
cc ce. cc perce po kgm. | ce.
12/2 | 2118|1650| 740 | 903 | 54.7} 128 | 0.58] 11,0] 9,8/15.70] 105
12/2 | Diet: Crackermeal and milk
12/3 | Bled 413 ce.
12/4 | Bled 413 ce.
12/6 | 1000|1256| 860 | 304 | 31.3] 79] | | [15.30] 82 |
12/7 | Bled 314 cc. ;
12/9 | 708|1240] 926 | 300 | 24.2| 57 | 0.79| 3,6 15,6|15.05] 82 |
12/9 | Diet: 681 grams cooked lean beef, 20 grams cooked gelatin—100 calories
per kilo
12/16] 916|1063| 636 | 388 | 36.5} 86 | 0.84} 5,1) 26,213.35 See foot-
note
12/17} Diet: 100 grams cooked brain, 580 grams lean beef—100 calories per kilo
12/23) 954)1047| 607 | 412 | 39.4) 91 | 0.53) 8,5) 19,0)12.90 See foot-
note
12/23} Diet: Mixed diet
12/30} 883/1100| 722 | 366 | 33.3] 80 13.35] 82
- 1/8/19} 1162|1263} 720 | 5381 | 42.0} 92 | 0.69] 6,7} 10,2/13.50} 94 | * Poik. ++
1/15) 1050/1290) 750 | 530 | 41.0) 82 | 0.64! 6,4] 10,6)14.05) 92 ¢
1/22} 1380/1450) 792 | 685 | 43.8] 95 | 0.64) 7,4] 17,4)14.65} 99

* Poikilocytosis of red blood cells.

December 16, 1918: Food not touched. Seems sick.. Drank considerable water
this a.m. and immediately afterward vomited it. Temperature, 38.4°C. Abdo-
men seems distended. Gave 150 grams meat and 50 grams crackermeal. |

December 23, 1918: Left 300 grams food. . Very thirsty, vomits water within 5
minutes after drinking. Temperature, 38.8°C. Inactive, slight dragging of
right hind leg. Put in metabolism cage; 400 cc. water; mixed diet.

December 26, 1918: Animal recovered. Is still thirsty.

Subsequent death. Stone in kidney.

Experimental history, see table 6-b; see autopsy, table 14.

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 241

renal calculi. It is therefore certain that this dog was suffering from
_ renal disease during this experiment (table 51). We note in this ex-
periment that the dog lost weight rapidly on the diet of beef and gela-
tin which was not eaten with relish. Finally the diet was changed as
| TABLE 52
Blood regeneration—beef heart and liver. Dog 18-116. Bull mongrel, female,
; young adult

q 1g a
:. aa E PS
4 8 : 5 i) | S g V4 5 j
4 5 8 g a a z 5 < a 8 |
Meee | ar) 3 |e] 3 | Cc a | & :
: S BES > ‘ > = B a Be
Mee peso! 2 |e] Ss | ¢ Pee le
4 § om 8 7 a a S 3 Fo a 5 3
A Fy FS Fy 2 2 fy S a Es Fs FE
or ce. cc. |, cc. |per centiper cent kgm. ce.
12/2/18} 2120 | 1720 | 805 | 907 | 52.7] 123|0.56| 11,4] 14,1 | 14.90] 115

12/2| Diet: Crackermeal and milk

12/3 Bled 430 cc.
12/4} Bled 480 cc.

12/6 939 | 1183 | 803 | 369 | 31.2] 79 | 0.57 | 6,9 | 21,2 | 14.55] 81

. 12/7 Bled 300 ce.
q 12/9] 776 | 1260 | 930| 323 | 25.6] 62|0.84| 3,7 | 17,5 | 13.95] 90

12/9 | Diet: 256 grams cooked beef heart,* 610 grams cooked beef liver*—100
calories per kilo

12/16 | 1082 | 1330 | 844 | 476 | 35.8 81 | 0.88 | 4,6] 18,2] 14.55} 91 -
:. 12 /23 | 1890 | 1685 | 860 | 818 | 48.5} 112] 0.57] 9,9] 11,5 | 15.25) 110
i 12/30 | 2270 | 1648 | 747 | 902 | 54.7 | 138 15.50} 106

12/30 | Diet: Mixed diet

72 | 831] 7,0 | 15.50) 110
.68-| 8,0 | 12,0 | 16.20; 99
71 | 8,6 | 10,6 | 15.65) 111

1/8/19} 2040 | 1715 | 785 | . 912 | 53.2] 119
1/15} 1760 | 1610 | 762 | 842 | 52.2} 109
1/22} 2120 | 1740 | 773 | 948 | 54.4] 122

ooo

" * Meat cooked, fat and connective tissue removed, and ground.
Experimental history, see table 13-b.

the beef and gelatin mixture was refused. The beef and brain mixture
was also eaten poorly, and more weight was lost. We are fortunate
in being able to refer to a fasting experiment on this same dog (table 14)
_ which shows practically an identical gain during two weeks as recorded

242 G. H. WHIPPLE, F. S. ROBSCHEIT AND C. W. HOOPER

in table 51. The distaste for the food mixture which was eaten only
in part we believe is largely responsible for this lack of blood regener-
ation. We see that the renal disease does not modify the expected
blood reaction during a fasting period (table 14) and we have no right
to assume that it might seriously modify the reaction in table 51.

TABLE 53
Blood regeneration—beef heart. Dog 19-6. Bull mongrel, male, young adult

12 =
i ; :
aha 13)
r roo] 8 2 c = ia 3 2
go cr (s) > ° Q a = a
& a8 > < F " z a : a
w a5 a = is) 3 rs iS) ” i a
= mo iS 2 a ; : ° ° : ) M4 °
< SAB 1: 8 ae : z 3 . : a g
a ry ey ry a a a) 5 fa E E Q
cc ce. cc. |per cent|per cent kgm. ce.
11/14 | 2082 | 1543 , 590 | 946 | 61.3 135 | 0.77 8,8 | 11,0 | 14.00} 110
11/14 | Diet: Crackermeal and milk
11/15 | Bled 386 ce.
11/16 | Bled 386 cc. No distress
11/18 800 | 1020 | 650 | 365|35.8| 78] | | | 18.00] 79

11/18 | Bled 200 cc.

11/20] 697 | 1026 | 710 | 317 | 30.8 | 68 |0.74| 4,6 | 16,4 | 12.80| 80

11/20 | Diet: 431 grams cooked beef heart*—100 calories per kilo

11 /27 | 1325 | 1250 | 670 | 574 | 45.9 | 106|0.79}| 6,7 | 6,0} 12.55) 100
12/4 1480 | 1170 | 568 | 601 | 51.1 | 126 | 0.64} 98] 6,7} 12.75) 92
12/11 | 1642 | 1325 | 637 | 681 | 51.4 | 124 | 0.62} 10,5 | 10,2 | 12.25; 108

12/13 | Diet: Mixed diet

12/20 | 1376 | 1188 | 588 | 588 | 49.5 | 116 | 0.58} 10,0 | 11,4] 12.50) 95.
12/27 | 1287 | 1226 | 642 | 579 | 47.2 | 105 13.15) 93
1/10/19 | 1940 | 1462 | 607 | 840 | 57.5 | 133 ]0.81 | 8,2] 7,0} 13.20) 110

* Beef heart cooked, fat and connective tissue removed, and ground.
No previous anemia experiments on this dog.

Table 52 illustrates an optimum reaction on a diet of beef heart and
beef liver. Both these substances favor a rapid blood regeneration
especially when a sufficient amount is eaten. This dog ate the mixture
with relish and gained over 1 kilo during 3 weeks. There is a truly

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 243

_ remarkable gain in red cells, hemoglobin and pigment volume. Dur-
ing 3 weeks the regeneration is complete and the anemia level (3 bleed-
ings) if anything was below the average level. Many other anemia
- experiments have been completed on this dog (table 13-b) and the type

TABLE 54
Blood regeneration—beef heart. Dog 19-84. Bull mongrel pup, female
Vg e |
as 2 PS
Oo 8 y 5 gq | & a 5 Zz 8
S jaesiais| 2] § mae :
oer aio | 8 a A | of REMARKS
S |eeale el = ry Z 3 ‘. a
= |Ssela]a| ¢ 3 % S ° by a
pas s25 ° Dm . . ti fo) A, 9 oO °
£ .|/ohal S| ¢ ma ma 2 a a 7 3 %
r=) ry mo} & rs a a) 3 8 z z )
may cc. | cc. | cc jin hate kgm. | ce.
12/2 992|780|352| 424 | 54.4) 127 | 0.53} 12,0] 10,6) 6.95} 112) R. B. C. small

12/2 | Diet: Crackermeal and milk

12 /3 | Bled 195 ce. No distress
12/4 | Bled 125 ce. No distress

12/6 | 448|401|305| 183 | 37.3| 91] 0.68| 6,7| 24,0] 6.10| 80 |
12/7 | Bled 123 ce.

12/9 428|542|360| 177 | 32.6| 79 | 1.20] 3,3| 17,1| 5.85| 93

12/9 | Diet: 228 grams beef heart*—100 calories per kilo

-12/16 | 638/608/332| 274 | 45.0) 105 | 0.86) 6,1) 10,1] 5.80} 105
12/23 | 796|676|328| 345 | 51.0) 118 | 0.59) 10,8) 15,2) 5.90) 115 | R. B. C. small
12 /30.| 968/711/323| 387 | 54.5] 136 5.90} 120

- 12/30 | Diet: Mixed diet

1/8/19} 855/750|366) 380 | 50.5) 114 | 0.71) 8,0} 23,8) 6.95) 107
1/15 | 712/818)405| 409 | 50.0; 87 | 0.61} 7,1} 10,8) 7.80) 105
1/22 | 1020/937|476| 456 | 48.7) 109 | 0.55) 10,5) 14,2) 7.50) 125
1/30 | 987|888|429} 455 | 51.2) 111 | 0.75} 7,4) 11,2) 7.50} 118

_ * Beef heart cooked, fat and connective tissue removed, ground...
No previous anemia experiments on this dog.

reaction is therefore established. It may be stated even that the
regeneration was almost complete within 2 weeks.

With the change to mixed diet we note a reaction which is not un-
common when a sudden change is made from a fixed diet to another

244 G. H. WHIPPLE, F. 8S. ROBSCHEIT AND C. W. HOOPER

very different diet. Under such conditions even when the second diet
is most favorable we may record a slight fall in red cell hematocrit,
hemoglobin and pigment volume. This experiment, too, shows a fall :
in the red count. We have no good explanation to offer, but this fact.
is to be considered in the proper interpretation of various tables.

TABLE 55

Blood regeneration—beef heart and lean meat following 3 weeks of sugar—metabolism.
Dog 16-157. Bull mongrel, male, age 10 months

Vg ; z
ao a 5 ;
pa Saal! « a 2 5 z °
E Sealine (ee). e-tite w | § 5 ;
L ;ROO| RB a a = ce 4 | | REMARKS 4
= - fo) > ° e A He} ef f 4
= cael > 8 é " ei a : a Ba ;
34 Og 4 ae = i} is) 2 3 i) a ;
ee eee ee ee ee ee mee ;
A Be Bef Be] obo oe Pe eT ao | ee ,
| ce. ce. ce. foal ena kgm. | ce.
12/14/16 | 116 7,9 6,4| 8.10
12 /14 | Diet: Sugar (3 weeks)
\«
1/5/17 546| 976 625 | 351 | 36 | 56 | 0.55] 5,1 | 7,2 6.10] 160 |
1/5 Diet: 400 grams beef heart, 100 grams sugar
1/11 rg | | | 43 | 76 | 0.58 6,6 | 11,8| 6.60 |
1/13 | Diet: Lean meat :
1/17 408| 908] 563 | 345 38 45 | 0.44) 5,1 6,4| 7.60) 119
1 /26 738| 838] 478 | 360 43 88 | 0.59! 7,5 8,2} 8.00} 105
272 838} 998} 529 | 469 47 84 | 0.57} 7,3 7,0} 8.20) 122
2/16 1130} 1202) 553: | 649 54 94 | 0.53) 8,8 9,8} 9.20) 131
2 /23 1052} 1052) 526 | 526 50 | 100 | 0.58} 8,6 | 13,4) 9.20) 115 ;
3 /2 | 1208 1220) 549 | 672 55 99 | 0.56) 8,9 9,8} 9.20) 1382

* Anisocytosis and poikilocytosis of red blood cells. .
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

Cooked beef heart is a food commonly used in experimental labora-
tories. It is usually assumed that heart muscle as a food is very like
skeletal muscle, although there may be certain differences as pointed
out by Mendel and Osborne (1). We have found that it compares
favorably with skeletal muscle as far as concerns the regeneration of
red cells and hemoglobin. Whether beef heart is actually identical
with lean beef in its effect on blood regeneration cannot be stated posi-

'

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 245

tively as we cannot at this time submit a sufficiently complete series
of controlled experiments. | :
Tables 53 and 54 are identical in all esseritial factors. Both dogs
_ were used for the first time and we must consider the unusual“ reserve”’
_ which at times may be demonstrated by such dogs. The cooked beef
4 heart was eagerly eaten, and the weight was practically stationary,
_ although the first dog lost a little during the third week. Blood re-
generation was practically complete in 3 weeks as regards red cells,

TABLE 55-3

Experimental history. Dog 16-157

BLOOD
REGENERATION
EXPERIMENT DIET WEIGHT REMARKS
Pigment |-Blood per
volume | kilogram

kgm

Begin 9/26/16 | Fasting 7.90

Bled 600 cc. 6.80 | (4 bleedings)

End 10/11/16 § 5.40 | Slight regenera-
tion of Hb. and
R: Bet

Begin 12/14/16 | Sugar, metabol- 8.10

Bled 600 cc. ism 1208 132 7.30 | (4 bleedings)

End 3/2/17 Sugar, beef heart 9.20 :

Beef heart

Begin 5 /7 /17 Gelatin, sugar 1570 130 | 10.00

Bled 648 cc. Metabolism 429 74 | .9.20

End 6/11/17 792 126 | 6.30

6/14 Killed—bled

from carotid

hemoglobin and pigment volume. Both dogs showed a distinct drop
when changed to a mixed diet,—a change recorded in red cell hemato-
crit, hemoglobin and red count. There was no change in plasma vol-
ume to explain this fluctuation and for the present we must be content.

with recording this fact without advancing any convincing explana-

tion (refer to exper. 52). A similar fall is noted in table 66 even when
the change is from a poor diet (bread and milk) to a more favorable
diet (beef heart).

246 G. H. WHIPPLE, F. 8S. ROBSCHEIT AND C. W. HOOPER

Table 55 illustrates the reaction on beef heart diet following a 3- a

week period of sugar feeding. After the meat diet is established we
note a slow but steady gain back to normal in 4 to 5 weeks. We wish
to emphasize the fact that a period of fasting or sugar feeding makes
subsequent blood regeneration much more difficult and this is a severe
test for any food substance. Only meat (including beef heart) and

TABLE 56
Blood regeneration—beef heart. Dog 16-158. Coach mongrel, male, young adult

1g r
Le : q
13 i a a i i : :
848 a b a P 4 5 ¥}
Poo. 5 a q = al | i
= eb 5 > ° oI a. = |
= RAE > > a a = 4 es oy
Ae og S S S 2 S 3 z Q
= s25 ° A ; . : 3 : Fs < °
e ee] 8) gd | fel | 8 | 8. |e
A ry a 4 8 rs a 3 a = z F)
ce. 60: cc. |per cent|per cent kgm. ce.
2/12 | 1525. | 1451 | 566] 886] 61 105 | 0.63 | 8,3 7,0 | 9.90 | 147
2/12 | Diet: Bread and milk
2/13 | Bled 363 ce.
2/14 | Bled 363 «ce.
2/15 | 385| s37| se0| 276|°33 | 46|0.70| 33 | 74|9.80| 85

2/15 | Diet: Beef heart

2/21 | 500] 980| 647] 333| 34 51.| 0.77 | 3,3 | 10,8
2/28 | 602| 885| 523| 363! 41 68 | 0.68 | 5,0 | 19,8
3/7 | 840| 1077 | 560] 517] 48 78 | 0.561 6,9 | 19,8
3/14 | 904 | 1062] 531} 531 | 50 85 | 0.57 | 7,5 | 14,0
3/21 | 1235 | 1272 | 585 | 687] 54 93 | 0.60] 7,8 | 10,0
3/28 | 1146 | 1180 | 543 | 638 | 54 97 |0.58| 83 | 80
4/5 | 1280 | 1243 | 572] 671| 54 | 103/0.56| 9,2 | 86
4/11 | 1478 | 1342 | 604] 738] 55 | 110|0.58| 9,4 | 18,6

2 3
SEBBSEBS
2

Experimental history, see table 46-b.

liver show up to advantage as compared with the mixed diet under
such conditions. Apparently the fasting or sugar feeding or other
‘limited diet causes a draining of the body’s reserve and subsequent
blood regeneration suffers because of this depletion of reserve or
impairment of function. ‘
Tables 56 and 57 are incomplete in that the amount of beef heart is
not known but the weights give assurance that a liberal amount of food

a en

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 247

q was consumed. There is only a trifling loss of weight during the last
__ week of the experiment when the blood picture had returned to normal.
_ It is obvious that the blood regeneration in these two experiments is

much slower than that recorded in experiments 53 and 54. Four to 6

weeks elapse before the hemoglobin and hematocrit return to normal

_ and 5 to 7 before the pigment volume and red cell count return to the

- original level.

TABLE 57
Blood regeneration—beef heart. Dog 17-192. Bull mongrel, male, young adult

ll =
- : ‘
Beg 2) | = 8 a S
oe kc} a B a P ra & i
mn 26 7 3 a 2 S 3 m
7 ~ ap ro) 5 {o) & =) =) 3
Bepeeet Fe lg | ee og ae pl ie Vee
cotmee, Ss | eos | 8 A a Ge ok a a
“8 | oma] 8 < z = s 3 4 . FY e
a a re) 5 ro a en 5 re E B a
ce. ce. cc. |per cent|per cent kgm.. cc
4/24 | 1157 | 1032 | 537| 496] 48 | 112] 0.78] 7,2 | 10,6] 9.80| 105
4/24 | Diet: Bread and milk
4/25 | Bled 258 cc.
4/26 | Bled 258 ce.
4/27 444 | 822 | 633 | 189 | 23 | 54 | 0.84 | 3,2 | 12,8 | 9.50 | 87
4/27 | Diet: Beef heart
5/2 | 662| 883| 609! 274! 31 75 |0.79| 4,7 | 11,6] 9.90} 989
5/9 804 | 992.| 585) 407] 41 81 | 0.67] 6,0 |. 80] 9.50] 104
5/16 | 1030 | 1050 | 567 | 483] 46 98 | 0.80 | 6,1 | 12,0} 9.50] 110°
5/23 | 1255 | 1172 | 633] 539] 46 | 107| 0.73] 7,3 | 9,0] 9.50} 123
5/30 | 1308 | 1147 | 585 | 562] 49 | 114/0.81| 7,0 | 80]|9.50]} 121
6/6 | 1360 | 1214| 607] 607| 50 | 112|0.64| 8,7 | 6819.10] 133

No previous anemia experiments. on this dog.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

Tables 58 and 59 both deal with bile fistula dogs. The experiments
were performed at the same time under identical conditions and the
reaction to the beef heart diet is strikingly uniform. It is known from

the autopsy notes that the bile was completely excluded from the in-

testine. A meat diet is not well tolerated by these bile fistula dogs
over long periods and a loss of weight is usually noted under such con-
ditions. As a result of this long period of meat feeding we note subse-
quent intoxication which resulted fatally in spite of a mixed diet régime.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 538, NO. 2 -

248 G. H. WHIPPLE, F. S.. ROBSCHEIT AND C. W. HOOPER

The beef heart was eaten with relish but the amounts given are not _

recorded. There is a distinct gain in hemoglobin, red cells and pig-

ment volume during each of the first 3 weeks. The level at the end of _

»

TABLE 58

Blood regeneration—beef heart—bile fistula. Dog 17-35. Bull mongrel, female,
young adult

SE a fi 8 5
BER og ay a ye é 3 3
. (283) 81218] 3 x | ee
ES BR al > . > = Z a ‘ 4 ie
> fy -® A 3 o iS) 3 < = a
E fenel eo | § ha led See lit) 8 ea ee
a |m E: z Fe ai | 8 a | & ES E
cc ca: cc aoe pd kgm. | cc
5 /28 | 1330) 1244) 560 | 684 | 55 | 107 | 0.74] 7,2 | 20,0) 9.10} 136
5/29 | Bled 311 ce.
5/30 | Bled 311 ec.
5/31 eel a fee: | 0.77| 3,1 | 45,6| 9.10| |
5/31 | Diet: Beef heart
6/5 540} 900} 567 | 333 | 37 | 60 | 0.79) 3,8 | 14,6) 8.20) 109
6/15 | 868) 923] 480 | 443 | 48] 94 | 0.78] 6,0 7,0} 8.40} 109
6/22 | 952) 933) 476 | 457 | 49 | 102 | 0.72] 7,1 8,4) 8.10) 115
6/29 | 800} 1000) 590 | 410 | 41 | 80 | 0.74! 5,4 6,8] 8.40} 119 | Noincrease
in bile
. pigment
7/6 649}. 729) 459 | 277 | 38 | 89 | 0.78] 5,7 7,0} 8.00} 91 :
7/9 | Diet: Mixed diet
7/13 | 694; 846) 5383 | 313 | 37 0.77| 5,3 | 20,0) 8.80) 96
7/18 | 797; 848! 500 | 348 | 41 | 94 | 0.90} 5,2 | 19,8) 9.00) 94
8/9 | Death from bile fistula intoxication

Bile pigment daily output per 6 hours before this experiment (28 day aver-
age) = 14.0 mgm.

Bile pigment daily output per 6 hours during this experiment (30 day aver-
age) = 13.4 mgm.

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

the third week is almost normal in both dogs and if the experiments

had been terminated at this point we should of necessity conclude that _

the reaction was similar to that so often observed in the normal dog.

BLOOD ‘REGENERATION FOLLOWING SIMPLE ANEMIA 249

But the fourth week in both dogs shows a decided drop in pigment
volume, hemoglobin and red cell hematocrit.

We fortunately have the daily bile pigment output figures at hand
and can say that there is no increase in bile pigment elimination during
this week of falling hemoglobin. So we may not explain this decrease
in the curve as due to some agent destructive to the red cells. It has
been established (2) that a sudden destruction of red cells in the blood
‘stream will result in an increased output of bile pigment although the
reaction is not in any degree a quantitative reaction as some observers
have claimed (3). We have no convincing explanation for these ob-
served facts but suggest that the poor quality of the red cell may be a

TABLE 58-8
Experimental history. Dog 17-35 (bile fistula)

BLOOD
REGENERATION
EXPERIMENT DIET WEIGHT REMARKS
Pigment | Blood per
volume | kilogram
kgm

Begin 12/18/16 | Lean meat 8.80 | 100 Hb.
Bled 600 ce. 8.90 | (4 bleedings)
End 1/17 /17 892 99 8.90 101 Hb.
3/8/17 Bile fistula operation :
Begin 5/28/17 | Beef heart 1330 136 9.10 | 107 Hb.
Bled 622 ce. - 9.10 .
End 7 /6/17 649 91 8.00 | Maximum regenera-
tion 3 weeks

factor. Limited diets as well as splenectomy under certain experi-
mental conditions seem to be associated with red cells which are prone
to disintegrate more readily than normal. |

The average daily bile pigment output is given in each table for a 30-
day period before the experiment and during the anemia regeneration
period. One dog shows identical figures for the mixed diet control
and beef heart period. The other dog shows a much higher output
on the mixed diet than on the beef heart diet. We believe this is to
be explained by the mixed diet which is made up of meat scraps, bones,
bread, potatoes, table scraps, etc., and is given in moderate excess.
This allows a certain choice on the part of the dog and if the animal
prefers the carbohydrate fractions we may observe the familiar reac-

250 G. H. WHIPPLE, F. S. ROBSCHEIT AND C. W. HOOPER

tion to carbohydrate feeding in the bile fistula dog, namely, an increase
in bile pigments.
TABLE 59

Blood regeneration—beef heart—bile fistula. Dog 17-155. Bull mongrel, male,
young adult

=

i] g be a

BS be 1

Sha 's So)

beas| a - } Z °

neb| § | 2 | & = ° =
Sf) § R p < ‘a fe vy
POO ~ 4 x a 5 REMARKS ; i
~ ae = ° (co) 3] Qa S S ; 4
PY RAL Pe hale & ri Z 3 a ,
- Es fo) < : z a a 3 & Pa a
- |A@-5| @ s s) re) et 1) : ea a me
<3} Sos ° nm . . . fo) . fo o ° A
Bfeeae | at) A ee et Se Le ae
a lm. a a rd rd ee o ro z = Q
nah 7
per per ‘
so Pec at On Eo iRe eg kgm. | ce }
q
5 /28} 1560/1416; 708) 708) 50 | 110 | 0.67) 8,2 | 11,2/11.50) 123 1

—_

5 /28| Diet:. Mixed diet

5 /29| Bled 354 cc.
5 /30| Bled 354 cc.

5/31} 495| 935] 645] 200| 31 | 53 | 0.63] 4,2 | 9,2{10.80| 86 | |

5 /31| Diet: Boiled beef heart

6/8 | 1030|1213] 752} 461} 38 | 85 | 0.70) 6,1 9,0}10.90) 111
6/15} 1165|1265) 645} 620} 49 | 92 | 0.66) 7,0 | 10.4|10.90} 116
6 /22} 1385|1281| 615} 666) 52 | 108 | 0.68} 7,9 9,8)10.00| 128 | °.

6/29} 946/1186] 700) 484) 41 | 80 | 0.67) 6,0 | 14,6) 9.80] 121 | Nobilepigment
increase

7/6 | 1100/1038] 571) 468) .45 | 106 | 0.73] 7,3 | 15,0|10.00) 103
7/13) 1138)1138} 683] 455); 40 | 100 | 0.86} 5,8 | 14,4] 9.90} 115 .
7 /18| 1120)1288} 824] 464) 36 | 87 | 0.84| 5,2 9,810.30} 125 | No bile pigment
. inerease

7/19| Diet: Mixed diet

8/9 | Death from bile fistula intoxication

Bile pigment daily output per 6 hours before this experiment (30 day aver-
age) = 21.8 mgm.

Bile pigment daily output per 6 hours erie: this experiment (30 day aver-
age) = 15.2 mgm.

No previous anemia experiments on this dog.

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

We may recall that the fasting bile fistula dog will react as promptly
to anemia as the normal dog (table 23) and the bile fistula dog on a
bread and milk diet also presents a normal blood regeneration curve

™. 4 +
a ee ee a , e F ee ee Te Lee Se eT a eee

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 251

(table 32). But the meat diet reaction in the bile fistula dog is not
like the reaction of the normal animal. The unfavorable clinical
reaction of the bile fistula dog to the meat diet is often conspicuous
a TABLE 60 :
Blood regeneration—liver. Dog 19-83. Bull mongrel pup male

ig Mi |
af Fst ee
Sea id & o
S BE 2\ a@ |S] 8 fe) Z .
a gah 5124 >) rw 2 e

& Posh aitoajs| #4 ~ a ne REMARKS
a Seer bagi) 8 E ie ; & =
ae B-s| €alajs| os se 3 ° = Qa
B S25 fo) D . ‘ rt ° ‘ a o fo)
+» ofa) 9} 4|* a fe) a ma ; a S
a Ey a |e | @ rs a sf rt ES z Fe)

per per
cc. | CC.| CC.| aone | cent kgm. | ce
12/2/18} 1090|1001|540/466| 46.1) 108 | 0.59} 9,1 8,8} 9.30; 109 | R. B. C. small

12/2 | Diet: Bread and milk

12/3 | Bled 250 cc.
12/4 | Bled 250 cc.

12/6 | 480| 790|586|202| 25.5| 62 | 0.63| 4,9 | 13,8| 8.80| 90
12/7 | Bled 200 cc.

* Slight

12/9 | 630 |1032|770|258 25.0] 61 | 1.20] | 13,7| 8.65 119 |
12/9 | Diet: 550 grams cooked beef livert—100 calories per kilo.
12/16 | 373| 910(536|370 40.6! 96 | 0.94] 5,1 | 11,4! 8.85! 108
12/23 | 962| 96215501407! 42.31 100 | 0.56/ 8,9 | 19,2 9.35] 103
12/30 | 1304|1100|566|530| 48.1| 118 9.90] 111

12/30 | Diet: Mixed diet

1/8/19 | 1300 123516421580 47.0} 105 | 0.80) 6,6 | 14,2)10.75 118 |
1/15 | 1095/1200/670/519| 43.2} 91 | 0.75) 6,1 | 12,8/11.40) 105
1/22 | 1355)1300|685|600} 46.2) 104 | 0.75) 6,9 8,0)11.30 115 |

*Poikilocytosis of red blood cells.
+ Beef liver cooked, fat and connective tissue removed, ground.
No previous anemia experiments on this dog.

(diarrhea and loss of weight and activity) and this may explain the
observations recorded in tables 58 and 59.

Tables 60 and 61 show the remarkable influence which cooked liver
exerts upon blood regeneration. As the sole article of food cooked
liver may not be well tolerated, but in these two experiments the dogs

252 _G. H. WHIPPLE, F. S. ROBSCHEIT AND C. W. HOOPER

ate all of the liver and gained weight. The remarkable gain in hemo-
globin, red cells and pigment volume is at once obvious at a glance.
One experiment (table 61) shows practically complete regeneration in
2 weeks from the usual anemia level and both dogs are more than

TABLE 61
Blood regeneration—liver. Dog 18-114. Bull mongrel, female, young adult

mM
Ne 2 3
at 8 <
sig al ete % 8
jc Z Bb} § 2 3 z 6 3
ofy 5 =] =) < al 53 177
5 <e) 5 a re = = a

ca) a = M4 ° a a =
& E Sa > < a ma a : & Pe
« a: 8 a s o is) fo] 's) o q a
i=] 32 3 fo) Dn . . 2 fo) . 9 S fo)
a 8 ag ia: Wd A Be er
A By m 5 re ee ee) 5 8 E 2 F)
ce. ce. cc. |per cent|per cent kgm. | ce

§

11/14 | 1640 | 1411 | 643 | 763 | 54.0 116 | 0.58 | 11,3 | 14,8 | 13.50) 105 ’

11/14 | Diet: Crackermeal and milk .

11/15 | Bled 353 ce.
11/16 | Bled 353 cc. No distress

i as

11/18 | 852 | 1077 | 695| 372 | 34.5| 79 | | | | 12.85] : 84

11/18 | Bled 270 cc. No distress

11/20 | 592 | 1015 | 724 | 280 |27.6| 58 | 0.82 | 3,5 | 14,2 | 12.50| 81

11/20 | Diet: 750 grams cooked beef liver*

ee ee) ae a

11/27 | 1305 | 1290 | 726 | 540 | 41.8] 101 | 0.74] 6,8 | 11,8 | 13.25] 97
12/4 1730 | 1480 | 740 | 720 | 48.5 | 117 | 0.76} 7,7 | 20,0 | 13.35) 111
12/11 | 1902 | 1516 | 728 | 766 | 50.5 | 125 |0.82| 7,6] 13,4 | 18.65} 111

ee oe

12/13 | Diet: Mixed diet

~~ ee

12/20 | 1479 | 1383 | 713 | 613 | 46.0 | 111 | 0.65 | 8,6 | 14,6 | 13.90} 96
12/27 | 1295 | 1276 | 670 | 594 | 46.5 | 101 13.75} 93
1/10/19 | 1625 | 1390 | 668 | 716 | 51.5} 117 | 0.68; 86] 12,8 | 14.20) 98

ttm

* Beef liver cooked, fat and connective tissue removed, ground.
Experimental history, see table 12-b.

back to normal in 3 weeks. We are able to refer to a number of
other experiments on this dog (table 61, dog 18-114, exper. history table
12-b) to give a good line on the type normal blood regeneration.
Table 62 shows another experiment with cooked beef liver but the .
amount of liver fed is much less and the rest of the food caloric value i

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 253

is made up by crackermeal and milk. We see this small amount of
liver causing a prompt rise of the hemoglobin and red cells to normal
in 3 weeks, whereas the crackermeal and milk alone would require at
least 5 to 6 weeks for complete regeneration.

TABLE 62

Blood regeneration—liver and crackermeal. Dog 19-15. Brindle bull mongrel,
female, young adult

i : , |
Eg a 8
peel ge | 2/2] é B =
ro) 5 4 5 R pb < * <] i
Peel <3 3 3. a a 3 fe REMARKS
é ae| © > : mi 2 3 a .
S |Eaal > q : es Be
(o) < . : ° 3 =
_ Q a s o is) fod o 4 a
RQ. 32 4 fo} nD e ° ‘ io} ° a oS °
a ee} } < --) rs 5 9 = Pas
aif") 8 a : : o 9 : : > a
aA | & Q Ba a a en) = r e z Q
per per
cc ce. ce peak. coe kgm. | ce.
8/9 1702} 1120} 462 | 650 | 58.0} 152 | 0.95] 7,9 6,2|10.85} 103

8/9 | Diet: Crackermeal and milk

8/12 | Bled 280 cc. No distress
8/13 | Bled 280 cc. No distress

8/15 | 663 762| 501 | 253 | 33.2| 87 | | | [10.40] 73 |

8/15 | Bled 190 cc. No distress

8/17 468| ‘669| 493 | 173 | 25.8| 70 | 0.92| 3,8 16,8|10.35| 65

~* Poik. ++

8/17 | Diet: 70 grams cooked beef liver,+ 200 grams crackermeal, 500 ce. milk

8/23 | 664| 874] 517 | 267 | 30.5] 76 | 0.88] 4,3 | 15,0/10.85] 81
8/30 | 1290] 949] 466 | 478 | 50.3] 136 | 0.90] 7,5 | 15,8|11.30] . 84
~ 9/6 | 1600] 1096] 504 | 588 | 53.6] 146 | 0.92] 7,9 | 8,2|11.20] 98
9/13 | 1700} 1089] 495 | 594 | 54.5] 156 | 0.96] 8,1 | 10,8/11.50| 95

-* Poikilocytosis of red blood cells.

‘tT Beef liver cooked, fat and connective tissue removed, ground.

No previous anemia experiments on this dog. See table 64 for subsequent
experiment.

Table 63 is not very satisfactory but is included because several in-
teresting points may be made. This dog at the beginning of the ex-
periment was very young, approximately 4 months, but the date of
birth was not positively known. The pup had a hemoglobin of 73 per
cent, which is not unusual in young dogs of this age. The amounts
bled were less than normal because of this fact, but the total reserve

254 G. H. WHIPPLE, F. 8S. ROBSCHEIT AND C. W. HOOPER

was evidently considerable, as indicated by the high figures after the
bleeding. The young dog was growing rapidly during the whole period
of the experiment, a gain of 3 kilos body weight in 6 weeks. The blood

volume shows a considerable increase during this period, almost 100

_per cent gain. The gain in hemoglobin and hematocrit is steady and

; TABLE 63
Blood regeneration—liver and bread. Dog 18-114. Bull mongrel pup, female

mM
- - 2
ae
s | 2) oS
as a A Cc °
B&B = a = & a -
S58 p fe = = « < al
cS > 3 ° i} fa Qa = |
S HE > > > ce Z ce ry
= vA tal fo) < . . - ; : § &
ant A 6 f=) s o o 2 ro) ° a-
a aoa 2". % ‘ : ; ° . a o 3
e | eta} 8 4 é . = 2 “ a 8
a ry ) oy ee r ee) 3 8 E z )
ce. ees cc. |per cent|per cent kgm. ce.
4 /24 553 | 758 | 455 291 | 38.4 73 |0.53 |. 6,9: |. 28,6 )0 7.851 > 94

4/24 | Diet: Bread and milk

4/26 | Bled 189 ce.
4/27 | Bled 90 cc. No distress

4/290 | 435 | 805 | 528| 255 | 31.7| “54|0.58| 4,7 | 146] 7.65) 105

4/30 | Diet: 200 grams bread, 500 cc. milk, 50 grams cooked beef liver,* ground |

up with bread

5/8 599 | 740] 4382] 293 | 39.6 81 | 0.56 | 7,2 8,4 | 8.00} 93

5/15 43.6 88 | 0.52 | 8,5 20,6 | 8.80

5 /22 825 | 938 514 411 | 48.8 87 | 0.54 | 8,0 16,0 | 9.50} 98
_ §/29 791 807 463 330 | 40.9 98 | 0.69 | 7,1 22,8 | 9.60) 84

5/29 | Diet: Mixed diet

6 /5 . 902 | 497 391 | 438.4 . . 10.45) 86
6/10 | 1059 | 1009 | 556 | 443 | 48.9] 105 | 0.67] 7,8 | 11,8 | 10.70} 94

* Beef liver cooked, fat and connective tissue removed, and ground up with
bread.

Experimental history, see table 12-b.

No previous anemia experiments on this dog.

comes close to the average normal at the end of the experiment, in
marked contrast to the level at the beginning of the experiment. _
Meat extract (tables 64 and 65) evidently does not add anything to
a given diet which in itself is especially favorable to blood regeneration.
We have no evidence to show that meat extract is favorable or unfa-

a

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 255

vorable to blood reconstruction. Table 64 shows the rapid blood re-
generation which we expect on a cooked liver diet and the return to
normal requires but 3 weeks. We note again in this experiment the

4 fall which often occurs following a sudden change to another diet

TABLE 64
‘Blood regeneration—liver and beef extract. Dog 19-15. Brindle bull mongrel,
female, young adult

la 2
ae : 5
= 3)
eee; 2 | a] 23) 8 atte 8 :
pee 5 3 a 2 fa 4 b>
ic} a> 5 > 3° ie ra) pe a
+4 a8 Ne < . ” a a : ey ry
« Qo s a Ss o o 3 3S o td a
omemee Ss -) G i) elie |} | 8 lo wl | el ged
A Fy FS a 2 Fy ss S 2 7 E =
: ce. ce. cc. |per cent|per cent ; ; kgm. ce
11/14 | 1615 | 1260 | 506 | 746 | 59.3] 128|0.76| 8.41] 10,8 | 12.40} 102
11/14 | Diet: Crackermeal and milk
11/15 | Bled 315 cc. No distress
11/16 | Bled 315 cc. No distress

11s | 584| 808 |550| 255|31.5| 72| | | | 11.95] 68
11/18 | Bled 202 cc.

11/20 | 508| 796 | 568 | 224] 28.1| 64|0.60| 46| 14,4 | 11.35] 70:

11/20 | Diet: 450 grams cooked beef liver* and 10 grams Liebig’s beef extract

11/27 | 808 | 917 565 | 347 | 37.9| 8s|o.76| 5,8| 12,8] 11.30! 81
42/4 | 1100 | 1002 | 555; 458 | 45.0 |- 108 | 0.66| 8,2| 15,3] 11.50| 89
12/11 | 1300 | 1084 | 560 | 518 | 47.8| 120] 0.60} 10,3} 12,2] 11.35] 95

12/13 | Diet: Mixed diet

12/20 | 912] 954 | 550 | 395 | 41.6 96 | 0.56] 86] 16,8 | 11.95 80
12/27 | 926 | 1010 | 564 | 427 | 42.2 92 11.85) 85
1/10/19} 1122 | 1060 | 540} 508 | 48.0; 106 | 0.91 | 8,0 | 13,6 | 12.00) 88

* Beef liver cooked, fat and connective tissue removed, ground.
Refer to table 62 for previous experiment.

(mixed diet) which, too, is very favorable for blood regeneration and
maintenance. It is clear that there is no fluctuation in plasma volume
which would supply an easy explanation for this phenomenon.

Meat extract (table 65) does not modify the reaction which may be
expected following a liberal bread and milk diet. The blood picture is

256 G. H. WHIPPLE, F. S. ROBSCHEIT AND C, W. HOOPER

returned almost to normal in 4 weeks after the anemia and there is a
gain in weight of 1 kilo. A change to a mixed diet gives a favorable
reaction as is usual under these circumstances. We may refer also to
a part of table 66, which gives more data on the influence of bread and

TABLE 65
Blood regeneration—meat extract—bread and milk. Dog 18-116. Bull mongrel pup,
female
Dm
=
te : 3
Ze = a =I S Z 8
RB Z p = = s = ° A
() B rs Db je p 3 ‘a 5 i
co |>esl 8 o) 3 5 a 3 - REMARKS
a ieee et 5 ft |S e513 hes
* a -8 a s rs) is) 2 i) e | Aa
I S25 ° S ‘ : p rs 5 We ic) °
& | odpm! 8 < ma ma rr} 2 ma ‘ a g
a ry Q Po a a) 5 Fe E z ry
cc cc cc tial sooth kgm cc
4/24 | 903) 962) 459 | 486 | 50.5) 94 | 0.65) 7,2 9,8| 9.75} 99) * Slight

4/24 | Diet: Bread and milk

4/25 | Bled 240 cc. from jugular vein
4/26 | Bled 190 ce. from jugular vein

4/29 | 422] 362| 594 | 259 | 30.0] 49 r 0. 57| 4,3 | 10. 2| 9. 65| 89 | * Slight

4/29 | Bled 110 ce. from jugular vein

4/30 | Diet: 200 grams bread, 500 ce. milk, 10 grams Liebig’s beef extract

5/8 717| +956) 591 | 347 | 36.3) 75 | 0.48) 7,8 | 10,0) 9.60) 99
5/15 | 882} 1297} 760 | 517 | 39.9] 68 | 0.46) 7,3 8,6} 9.50} 137
5/22 | 859} 1035) 594 | 430 | 41.5) 83 | 0.51] 8,2 | 22,8]/10.70| 97
5/29 | 865} 961] 550 | 403 | 41.8} 90 | 0.54} 8,3 | 10,2/10.85) 89

+ & & &

5/29 | Diet: Mixed diet

6/10 | 1207] 1128] 634
6/18

483 | 42.8
104

ae "

107 | 0.63] 8,5 : 30,0

* Poikilocytosis of red blood cells.
Experimental history, see table 13-b.

milk plus meat extract. The meat extract adds nothing to the reaction
which is identical with the expected reaction from the bread and oR
alone.

Tables 66 and 67 are to be considered together. The Pe on
were done at the same time under identical conditions and the results

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 257

TABLE 66

Blood regeneration—watery liver extract and sugar; bread and milk; meat extract,
bread and milk; beef heart, bread and milk. Dog 17-157. Coach mongrel, female,
young adult

a
‘7 : =
gee 8 8
ee) 2 | a | 2 | & g :
ee ee i a | 3 a

o |Pes] 2 8 a ba 4 3 o REMARKS
Beers) | is | |S Bt Sab rec podbs bas
ae eh) ae a 3 3 8 5 a a
a |sfn} 9 2 : : : 9 ; Fs ¢ 8

& | ohm] 9g < A ma 2 2 “ , a

aA |& a 3 Pa cs 8 a Es E F
cc ce. cc pF be 206 kgm. | ce.
2/20 | 1340) 1180} 567 | 606 | 51.4} 113 | 0.79} 7,2 8,2|10.35} 114

2/20 | Diet: Bread and milk

2/21 | Bled 295 ec. ‘No distress
2/22 | Bled 295 cc. Distress. Injected 50 cc. of 5 per cent sugar solution

2/24 | 420| 306| 602 | 198 | 24.6] 52| 0.96| 2,7 | 8,6 9.50 85 |

2 /26 Diet: 100 grams sugar, 10 grams watery liver extract, 250 cc. water

3/5 720} 868] 519 | 345 | 39.7} 83 | 0.90) 4,6 | 11,0} 8.95] 97
3/12 | 700] 813} 493 | 312 | 38.4) 86 | 0.88) 4,9 7,2| 8.25) 99
3/18 | 666} 730} 420 | 294 | 40.2} 91 | 0.80) 5,7 8,8) 8.05) 91

3/18 Diet: 200 grams bread, 300 cc. milk

3/26 | 824| 929| 560 | 360 | 38.7| 89 | 0.85] 5,2 | 10,0| 8.40] 110 |

3/26 | Diet: 200 grams bread, 300 cc. milk, 10 grams commercial meat extract

4 /2 756| 913] 556 | 398 | 38.1} 83 | 0.90] 4,6 | 10,8) 8.75} 104
4/8 635} 829] 522 | 298 | 36.0) 77 | 0.67| 5,7 | 14,0) 8.75} 95 | *
4/14 | 714! 876} 538 | 324 | 37.0) 82 | 0.73] 5,6 | 11,2) 8.75) 100
4/21 | 860} 984) 584 | 389 | 39.6) 87 | 0.72] 6,0 | 12,8) 8.90) 110

4/21 | Diet: 200 grams beef heart (cooked), 200 grams bread, 300 cc. milk

4/28 | 772| 942] 588 | 345 | 36.6] 82 | 0.67| 6,1 | 12,4) 9.55) 99
5/7 842} 961] 572 | 384 | 40.0} 88 | 0.73) 6,0 | 15,4/10.00} 96
5/12 | 890} 998) 590 | 397 | 39.8] 89 | 0.72) 6,2 | 15,0) 9.90) 101

5/12 | Diet: Mixed diet

* Poikilocytosis of red blood cells.

+ Watery liver extract: Beef liver cut up into small cubes, allowed to stand
in water over night in ice-chest, boiled in same water, and filtered. Filtrate con-
centrated to thick paste.

258 G.

TABLE 66-3

. WHIPPLE, F. S. ROBSCHEIT AND C. W. HOOPER

EXPERIMENT

Experimental history. Dog 17-157

BLOOD
REGENERATION

NUMBER DIET WEIGHT REMARKS
Pigment | Blood per \
volume | kilogram
kgm.
Begin 9 /3 /17 Crackermeal, 1025 115 8.40
Bled 496 cc. lard and gela- 380 83 8.20
End 10 /25 /17 tin 890 102 8.70
Begin 3/6/18 Crackermeal, 1084 99 9.80 | Table 71
Bled 488 cc.. lard, butter 614 90 9.10
End 4/9/18 and Blaud’s 984 106 8.30
pills
Begin 5/20/18 | Sugar, metabol-| 1113 120 | 10.20’
Bled 612 ce. ism, desiccated 313 75 9.50 ©
End 6/18/18 beef heart 663 100 7.60 | Maximum regen-
eration 3 weeks.
Pigment _vol-
- ume 698 cc.
Begin 8/28/18 | Hb. and sugar | 1366 102 | 10.10 | Table 78
Bled 710 cc. feeding | 433 79 9.25 | (3 bleedings)
End 11/12/18 Hb. intravenous- 886 114 | 8.15 | Pigment volume
ly and sugar 805 at end of
feeding Hb. period
Hb. intravenous-
ly and cracker-
meal + milk
Crackermeal,
milk and dried
yeast
Begin 2/20/19 | Liver extract, | 1340 114 | 10.35 | Table 66
Bled 590 cc. sugar 420 85 9.50
End 5 /12 /19 Bread, milk, 890 101 9.90 | Pigment volume
meat extract 666 cc. at end
Beef heart, bread of sugar feeding
and milk

are very suggestive. Both dogs have been observed in many other
experiments and their anemia reactions are therefore well known. One
dog was given the watery extract of beef liver and the other the liver

residue..

Sugar, 100 grams, was added to each feeding and it is clear

TABLE 67

Blood regeneration—liver residue and sugar; bread and milk; crackermeal and bread
and milk; cooked beef liver and bread and milk. Dog 16-158. Coach mongrel,
male, young adult

: : 3
oe a 8 o
BES] a = g © a |
sea} B | 2B} Bb] 8 ee 2
@ |PesSl 2 3 3 q 5 3 o REMARKS
= eRe 5 € 7 4 A 5 > Bs
= g 8 Aa Ss 3 rs) e 3 od | ra)
<3) sos ° D ° ° 3 ° ‘ Pe 1o} °
ORIG ITE ER Seon ecaevom Ut Aad Oe a | g
a |e a ry re 8 en) 5 ro E a
ce. ce. cc. ut od kgm. | cc
2/20 | 1862) 1375| 549 | 820 | 59.6] 135 | 0.77| 8,8 7,0/12.35} 111
2/20 | Diet: Bread and milk
2/21 | Bled 344 cc. No distress
2/22 | Bled 344 cc. Dyspnea; injected 50 cc. of 5 per cent sugar solution
2/24 | 823| 983| 625 | 353 | 35.9| 84 | 1.02] 4,1 | 19,6[11.40| 86 |
2/25 | Bled 246 cc.
2jo7 | 478| 778| 562 | 211 | 27.2| 61 | 0.85 3,6 | 8,a[11.10| 70]
2/27 | Diet: 100 grams sugar, 200 cc. water by stomach tube;100 grams liver
residuet
3/5 692} 867) 531 | 331 | 38.2} 80 | 0.76) 5,3 6,4/10.75| 81] *
3/12 | 876} 900) 517 | 373 | 41.5) 97 | 0.84] 5,8 5,4, 9.95) 90] * Poik.+
3/18 | 738) 754] 421 | 329 | 48.7; 98 | 0.83] 5,9 | 14,4) 9.15} 82 | * Poik.+
3/18 | Diet: 200 grams bread, 300 cc. milk
3/26 | 725| 923| 597 | 316 | 34.3| 79 | 0.77| 5,1 | 6,8|10.15| 91 | * Poik.
3/26 | Diet: 200 grams bread, 100 grams crackermeal, 300 cc. milk
4/2 748| 944! -600 | 339 | 35.9] 79 | 0.61] 6,5 9,810.50} 90 | * Poik.+
4/8 685} 966) 616 | 340 | 35.2} 71 | 0.61} 5,8 | 10,8/11.10| 87 | * Poik.
4/14 | 762) 968) 608 | 355 | 36.7| 79 | 0.59] 6,7 7,8/12.75| 76 | * Poik.
4/21 | 840} 1072) 640 | 406 | 37.9) 78 | 0.56} 7,0 | 10,6/11.20| 96
4/21 | Diet: 200 grams cooked beef liver, 200 grams bread, 300 cc. milk
—4/28°| 820} 1027] 629 | 388 | 37.8} 80] 0.73] 5,5 | 12,0)11.95) 86
5/7 | 1155) 1155} 595 | 549 | 47.5) 100 | 0.75) 6,7 9,6)11.45| 101
5/12 | 1074) 1095) 583 | 502 | 45.8) 98 | 0.66} 7,4 | 13,0)11.80| 93 | * Poik.+
5/12 | Diet: Mixed diet

¥ Poikilocytosis of red blood cells.
+ Liver residue: Residue left after water and alcoholic extraction; put in

_meat press and all liquid removed. Just before feeding residue was again washed

and brought to boiling point to remove all traces of alcohol. Dog did not always
eat full amount of food mixture. |
_ Experimental history, see table 46-b.

259

260 G. H. WHIPPLE, F.’S. ROBSCHEIT AND C. W. HOOPER

that both the liver watery extract and liver residue exert a certain in- _
fluence upon the blood regeneration which is much more than can be
accounted for by the sugar alone. The liver residue has greater in-
fluence upon the blood regeneration than does the liver watery ex-
tract but the difference is not striking.

Bread and milk feeding for 1 week subsequent to these sugar and liver
periods does not cause much reaction. There is a slight loss in hemo-
globin and red cell hematocrit in experiment 67 (liver residue).

The next 4 weeks are similar and bread or. crackermeal and milk
are the essential features of the diet. The level.of pigment volume, red
cell hematocrit and hemoglobin is constant. There is slight gain in
the red cell counts. :

We have pointed out the fact that a prolonged diet period unfavor-
able to hemoglobin regeneration will leave the dog in a condition which
may be clinically excellent but from the standpoint of formation of
hemoglobin very unfavorable. These two dogs were still anemic
although in excellent physical condition and of practically normal

weight. Under such circumstances we feel that any diet is given its
most severe test and few diets of limited nature can give a favorable
reaction as regards blood regeneration. Under ordinary circum-
stances a diet of beef heart, bread and milk (table 66) is favorable for
blood regeneration but under this severe test with unfavorable con-
ditions we note little if any blood regeneration during a period of 3
weeks.

Cooked beef liver with bread and milk even under these same unfa-
vorable conditions is able to effect a prompt blood regeneration—con-
spicuous in red cell hematocrit, red count and hemoglobin. This is
the severest test of any diet factor in its relation to blood regeneration.
Also refer to table 22 (same experiment).

DISCUSSION

The reader will observe many individual differences in the reactions
of various dogs to a unit type of secondary anemia. Some of these
vagaries we are as yet unable to explain but others are now much
clearer than in the earlier stages of this investigation. We have noted
that on occasions certain dogs made anemic for the first time presented
a most unusually rapid regeneration, even on a very limited diet.
This is not the rule, but is sufficiently common to suggest caution in
conclusions drawn from such experiments. A repeat experiment will

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 261

give the true constant reaction. How to explain this fact is not clear
to us, but a simple way out is to assume a reserve present in the body
under these conditions which permit of unusual blood regeneration
even under most unfavorable diet conditions. A knowledge of this

fact may keep the investigator from falling into error in deductions —
drawn from single experiments. With this exception the dogs will

show uniform reactions when we repeat anemia experiments under

uniform conditions.

Tt is clear that long limited diet periods following the standard
anemia may preserve the dogs in excellent physical condition as con-
cerns weight, general condition and activity. These same dogs, how-
ever, may continue to present a definite anemia. Under such condi-
tions many diet mixtures are unable to stimulate any blood regenera-
tion. Any diet factors are put to the severest test when they are
administered to dogs under such circumstances and we are inclined to
accept this as the severest test for any given diet factor. Cooked
liver gives a very favorable reaction and causes blood regeneration
even under these severe test conditions. |

Following a simple anemia many diet mixtures, if given at once, will
cause a distinct gain in hemoglobin and red cells. But if a limited
diet period intervenes between the anemia and the exhibition of the
test diet we will see a negative reaction. This may be illustrated by
bread and milk given in liberal amounts sufficient to permit a gain in
body weight or at least a maintenance of body weight. If the dog is
bled and at once placed on a bread and milk diet there will usually
appear a slow steady gain in pigment volume. If the same dog is
placed on a limited carbohydrate diet for 3 to 4 weeks before being
changed to a liberal bread and milk diet we will usually observe sub-
sequently an unchanged level of pigment volume, red cell hematocrit
and hemoglobin. Under such unfavorable conditions the bread and
milk diet is unable to give a favorable reaction for the blood regenera-
tion. In certain experiments the cooked beef heart is also unable to
modify the curve of blood regeneration under these unfavorable con-
ditions. Cooked liver is able to induce blood regeneration even under
the most unfavorable conditions. The same is true for the common
mixed diet of table scraps. It is important to keep these facts in mind
when we evaluate the reaction following the administration of a given
diet factor under different experimental conditions.

The question at once confronts us: what part of the meat or liver
substance is responsible for the favorable blood reaction? First we

262 G. H. WHIPPLE, F. S. ROBSCHEIT AND C. W. HOOPER

must investigate the pigment substances present in the meat—for —

example, the hemoglobin and myohematin. Some experiments with

hemoglobin appear in the next paper of this series, and.we hope soon
to report other experiments dealing with the myohematin pigment.

SUMMARY

Cooked lean beef and beef heart are diet factors of importance as
regards blood regeneration subsequent to simple secondary anemia.
These food substances alone or in combination with other foods will
give a rapid blood regeneration after anemia.

Anemia is produced by bleeding one-fourth of the measured blood
volume on each of 2 successive days. This anemia will be completely
repaired within 3 to 4 weeks if the dog is given a liberal diet of meat or
beef heart.

Cooked liver is as sufficient as est and may be even more efficient
in promoting complete blood regeneration subsequent to a standard
anemia. Blood regeneration may be completed in 2 to 4 weeks.

Commercial meat extract is inert and watery liver extract has but
little influence upon blood regeneration.

The meat diet reaction in the bile fistula dog is not exactly like the
reaction of the normal animal.

BIBLIOGRAPHY

(1) MENDEL AND OsBoRNE: Proc. Soc. Exper. Biol. and Med., 1918, xv, 71.
(2) WHIPPLE AND Hoopsr: This Journal, 1917, xliii, 258.
(3) Brucscu AND YosuimorTo: Zeitschr. f. exper. Path. u. Therap., 1910, viii, 639. —

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I

7 BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA

VY. Tue INFLUENCE oF BLAuD’s PILLS AND HEMOGLOBIN

C. W. HOOPER, F. 8. ROBSCHEIT anp G. H. WHIPPLE

From the George Williams Hooper Foundation for Medical Research,
University of California Medical School, San Francisco

Received for publication April 3, 1920
The familiar fact that iron in some form is very frequently given in

eases of secondary anemia made it imperative to test this drug under
a variety of experimental conditions. The outstanding fact in our

experiments is that zron given as Blaud’s pills has no influence in secondary

anemia under the conditions of these experiments. It may be objected
that the Blaud’s pills were not fresh or were not dissolved in the dog’s
intestinal tract. We obtained these Blaud’s pills from a large whole-
sale firm in this city and they were soft and easily crushed into a soft,
pasty material. ‘The pills were crushed before being given by mouth.
Further objections may be made that this drug has no influence on
the dog but does have potency when administered to human beings.
This of course is not subject to proof, but the claims for the potency of
iron in conditions of secondary anemia do not stand on firm ground.
We invite attention to the profound influence which is properly attrib-
uted to diet factors. Those who claim that iron is a potent drug must
exclude the food factors which are known to be concerned before they
can prefer too many objections to our negative results. °

The feeding of blood has at times been used in the treatment of
secondary anemia. We are able to find some experimental evidence
to support this treatment, but whole red cells or hemoglobin given by
mouth in the form of a dry powder do not appear to influence pro-
foundly the blood regeneration curve. Our experiments show that
hemoglobin does have a distinct influence on blood regeneration but
not sufficient to warrant its use in uncomplicated secondary anemia
in view of the favorable reactions due to meat and other diet factors.

The favorable reaction which seems to accompany administration
of hemoglobin by injection (intravenous and intraperitoneal) may be

263

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 2

264 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

of some value in the treatment of certain forms of anemia. It is pos- ~
sible that the reaction to this type of injection may differ from that —
associated with a transfusion and in certain diseases this procedure —
(hemoblobin injection) may stimulate rather than depress the bone —
marrow. Further experimental work is in progress. :

EXPERIMENTAL OBSERVATIONS

The same technical procedures are used in these experiments as
have been described in the first paper of this series. Blaud’s pills
were given daily. The experimental histories give the complete list
of anemia experiments on any given dog and are referred to in each
experiment. The control experiments are frequently given in the other
papers of this series, but the proper reference is appended to the ex-
periment dealing with iron or hemoglobin. We hope to report experi-
ments in the near future dealing with other drugs used in the treat-
ment of anemia. We expect to test many other pigment substances
besides hemoglobin as to their influence on blood regeneration. This
includes animal and vegetable nate as they occur in various meats,
fish and vegetables. -

Table 68 represents a long anemia experiment with Blaud’s rills

which is conclusive in showing the complete failure of this drug (car- :

bonate of iron) to influence blood regeneration under the conditions
of the experiment. Subsequent experiments will make it clear that
Blaud’s pills are inert in so far as any influence on this type of anemia
is concerned.

The experimental history of this dog (table 18—b) gives the reaction
of this animal to other diet factors and establishes the type reaction to —
secondary anemia. It is fair to say that this same type of blood re-
generation would be expected without the influence of Blaud’s pills.
This dog is more resistant than usual and tolerated this limited diet
for a long period without any symptoms of dietary deficiency disease.
The falling hemoglobin, red cell count and weight curves, however,
may indicate an impending dietary deficiency complex which did not
develop because of the change to a liberal mixed diet.

The Blaud’s pills were given daily and crushed before administra-
tion by mouth. Sufficient bread and skim milk were given to main-
tain the body weight constant until the last 2 weeks. During the first
8 weeks the red cell hematocrit is stationary but the hemoglobin rises
slowly. During this period the red count rises slowly and uniformly

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 265

hantoc* : | TABLE 68

E lood regeneration—bread, milk and Blaud’s pills. Dog 16-160. Bull mongrel,
. _ female, young adult

2
=
Ae & s
5 & = g a 8 Za 8
3 E B = is a < o g
418 2 3 lc} 2 A c - REMARKS
eee} © | = | | m a ae A GO He.
Baie Be fedes] Si-} 8 "fhe beside tae
comm! ¢ < 3 , a 3 3 ; a .
m a a a = s) re B E a
per | per
cc ce. cc pied: | come kgm. | cc
939 | 978 | 489 | 489.} 50] 96 | 0.73] 6,6 6,2| 7.70) 127
, ?

Diet: Bread and milk

| Bled 244 cc.

Bled 244 cc.

267 | 580 | 423 | 157 | 27| 46 | 0.77| 3,0 | 6,3| 7.40| 80 |
Bread, milk and 2 Blaud’s pills daily

348 | 309 | 566 | 243 | 30| 43 | 0.86] 2,5 | 10,8| 7.50] 108
375 | 694 | 493 | 201 | 29| 54|.0.71| 3,8 | 7,4] 7.40] 94
393 | 667 | 487 | 180| 27| 59 | 0.69] 4,3 | 5,6] 7.30] 91 | *
498 | 738 | 524 | 214] 29| 58 | 0.66| 4,4 | 11,0| 7.20] 104 | *
335 | 697 | 495 | 202 | 29| 48 | 0.52] 4,6 | 7,4] 6.80] 103 | *
312 | 625 | 431 | 194 | 31] 50 | 0.48] 5,2 | 10,0| 7.00] 39 | *
382 | 694 | 486 | 208 30| 55 | 0.48] 5,7 | 6,8] 6.80] 102 | *
| 368 | 681 | 470 | 211 | 31] 54|.0.46) 5,9 | 13,6] 7.00| 97 | *
500 | 725 | 471 | 254| 35 | 69'|.0.51| 6,7 | 11,2] 7.30] 99 | *
576 | 728 | 480 | 258] 35| 78| 0.51] 7,7 | 5,6] 7.20] 102 | *
560 | 700 | 455 | 245 | 35] so | 0.521 7,7 | 8,6] 7.00] 100 | *
617 | 771 | 455 | 316 | 41} 80 | 0.53] 7,6 | 12,4] 7.40] 104 | *
624 | 762 | 457 | 305 | 40| 82| 0.55] 7,4 | 9,4] 7.20] 106 | *
68s | 819 | 467 | 352| 43| 84 | 0.57] 7,3 | 6,6| 7.00| 117 | *
716 | 721 | 418 | 303 | 42] 99 | 0.59] 8,4 | 6,6| 6.80] 106 | *
830 | 847 | 491 | 355 | 42| 98 | 0.64) 7,6 | 5,4] 7.00] 121 | *
833 | 333 | 500 | 333 | 40 | 100 | 0.66] 7,6 | 5,8] 6.50] 127 | *
705 | 339 | 520 | 319 | 38 | 84 | 0.62] 6,8 | 10,0] 6.50) 129 | *
477 | 597 | 394 | 203 | 34| 80 | 0.67| 6,0 | 7,6| 6.20/ 96| *-
456 | 570 | 393 | 177| 31| 80] 0.74] 5,4 | 6,2/ 5.90| 97 | *

edg Marked fragmentation of red blood cells.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.
Experimental history, see table 18-b.

266 C. W. HOOPER, F. 8S. ROBSCHEIT AND G. H. WHIPPLE

and continues this rise to a figure even above normal. It is noted, ©
however, that there was marked fragmentation of the red cells and we ~
can scarcely account for this great increase in red cells (2,500,000 to —
8,400,000) with the red cell hematocrit showing only a rise from 27 —
per cent to 42 per cent, except on the ground of red cell fragmentation —
or abortive red cell construction. The hemoglobin does not keep pace

TABLE 69

Blood regeneration—bread, milk and Blaud’s pills. Dog 17-198. Bull mongrel,
female, young adult

la r
= es 4
BE e 5
= a a %, ro)
REE = 2 = 8 3 3
oSs] 6 3 a S 4 | i
~ |" aPl 8 3 3 2 a 3 e REMARKS
= HR a > > | a =
a | 4%] © < ae Bape S : > | ee
- |8-6| @ s is) oS i is) ’ = a
=] S25 ° et ° ‘ : ° ° Pe o °
& om! 8 < ma am 2 a a : a 8
A | ® ey ey Pe Pe ss] 5 Pe e Ps a
per | per
cc ce. ce Sida ST snes kgm. | ce.
4/24 | 1089} 1100) 517 | 584 53 99 | 0.75) 6,6 15,8) 8.40} 131

4/24 | Diet: Bread and milk

4/25 | Bled 275 ce.
4/26 | Bled 275 ce.

4/27 333 710) 547 | 163 | 23 | 47 | 0.87| 2,7 | 24,0| 8.20| 87
4/27 | Diet: Bread, milk and 2 Blaud’s pills daily

5 /2 458} 683) 492 | 191 | 28) 67 | 0.82) 4,1 9,4; 8.00} 85
5/9 636} 795} 493 | 302 | 38 | 80 |.0.75) 5,3 9,4; 8.00) 99
5/16 | 856} 1006} 533 | 473 | 47 | 85 | 0.76) 5,6 6,6} 8.00} 102
5 /23 | 1008} 1039} 561 | 478 | 46 | 97 | 0.69) 7,0 7,4) 8.00) 129
5/30 | 1041} 1021} 521 | 500 | 49 | 102 | 0.67) 7,6 | 12,4] 8.20) 124
6/6 | 1055} 1014) 527 | 487 | 48 | 104 | 0.65) 8,0 6,2} 7.90) 128 | *

* Fragmentation of red blood cells.
No previous anemia experiments on this dog.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

with the red count and a fall in the color index from 77 per cent to 46
per cent is recorded.

The high water mark for blood regeneration is noted after 33 months
and the level at this time is far from normal. Subsequently there is
a loss in red cell hematocrit, red count, hemoglobin and pigment volume.
We believe this indicated a tendency toward a dietary deficiency dis-

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 267

‘ease which would have developed had the bread and milk diet been
‘continued. The plasma volume as usual is constant throughout the
_ entire experiment with the exception of the last 2 weeks.

_ ‘The next experiment (table 69) shows a fairly complete blood re-
generation during a period of 5 weeks on a bread and milk diet plus
‘Blaud’s pills. It is to be observed that no previous anemia experi-

TABLE 70
- Blood regeneration—crackermeal, lard, butter, milk powder and Blaud’s pills.
Dog 17-205. Bull mongrel, male, young adult

4 ll : & 4
a ae Fs mi
j = ‘st a So oS
Q fe) °
3 Z rs 5 a s & 3 4
eBsl 5 <4 b < a 4 4
o fh as| 3 3 3 | ~ 3 - REMARKS
Sere |. |e |S Ba :
ah sel. 8 s 3 is) Po iS) ° e a
a 3° 3 ° wo < s + fo} : Py So °°
a bo } 2 a a | cs 4 3}
>} om) 8 e . . a 3 : . a 8
q a cy Q By a a 8) rs) rs B 3 =)
J per | per
4 cc. 60; cc. psoas 9 Fetal kgm. ce.
4 3/4 | 1182) 1055) 538 | 518 | 49 | 112 | 0.70] 8,0 | 10,8/12.60) 84

4 3/4 | Diet: 279 grams crackermeal, 10 grams lard, 10 grams butter

‘a 3/6 | Bled 274 cc.
_ 3/7 | Bled 254 ce.

3/9 605| 840| 605 | 235 | 28 | 72 | 0.82| 4,4 | 30,4|12.1 | 69 | *Anis.

3/9 | Diet: 142 grams crackermeal, 10 grams lard, 10 grams butter, 279 grams
milk powder, 2 Blaud’s pills daily

3/15 | ‘ 565) 807| 573 | 234 | 29} 70 | 0.88) 4,0 | 27,4/11.40} 71 | *Anis.
3/20 | 798) 928) 622 | 306 | 33] 86 | 0.93) 4,6 9,2/11.20} 83 | *Anis.
» 8/27) 682| 802) 545 | 256| 32] 85 | 0.79] 5,4 | 19,2/10.90) 73 | *Anis.++
. 4/3 983} 919] 597 | 323 | 35 | 107 | 0.82] 6,5 | 21,6/10.90} 84 | *Anis.++
y 4/9 795| 750) 480 | 270 | 36 | 106 | 0.79} 6,7 7,4, 9.60} 78 | *Anis.++

4/9 | Dietary deficiency disease. Recovery.

* Anisocytosis of red blood cells.
Refer to table 34 for control.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

ments had been done on this dog and the capacity for blood regen-
_ eration may be greater under such circumstances. The reaction is
not unusual, however, in view of the liberal amounts of bread and
milk which were sufficient to maintain the body weight. It can-
not be granted that the Blaud’s pills had any influence upon the blood
regeneration.

268 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

' Table 70 is of considerable interest because we are able to ref:
control period (table 34) on a similar diet but without the Blaud’s: I
If anything the control period shows slightly more bleod regeneratio:
during the first 5 weeks. The control experiment was of much lon; 1
duration (12 weeks) without dietary deficiency disease symptor ns
This may be due to the fact that this control Serta (table ;

TABLE 71

Blood regeneration—crackermeal, lard, butter and Blaud’s pills. Dog 17- é
Coach mongrel, female, young adult

n

=

“s es ” ite
ae fa 5
nae) 8 | gi] | & iS :

68a
oe) a 3 8 3 a A 3 a1
= a ° > Ss i}
a F Al > i q a a Cy
a (Beet ed ad Ot a| 8154 8ae
Ee jae Ss] 2) 4) ae} a | ge] a} 8) al g
acis FE i Pe Pe ss 8 Fe ES Ps a
cc ce. ce Eso aed kgm. | cc.
3 /4 1084| 976 | 537 | 439 45 | 111 | 0.82) 6,8 8,4) 9.80] 99

3/4 | Diet: 199 grams crackermeal, 10 grams lard, 10 grams butter —

3/6 | Bled 254 cc.
3/7 | Bled 234 ce.

3/9 614| 818 | 507 | 221 | a 75 | 1.00| 3,7 | 10,2| 9.10| 90 |

3/9 | Diet: 201 grams crackermeal, 10 grams lard, 10 grams butter, 2 Blauc
pills daily — ;

3/15 | 756} 869 | 600 | 269 | 31 | 87 | 0.99] 4,4 | 14,8] 9.00] 97 pe
3/20 | 801] 843 | 531 | 312 | 37 | 95 | 1.00] 4,7 | 11,4] 8.50] 99 | *
3/27 | 848] 848 | 517 | 331 | 39 |.100 | 0.89] 5,6 | 8,2] 8.40} 101 | *
4/3 | 943] 865 | 519 | 346 | 40 | 109 | 0.88] 6,2 | 18,8] 8.30] 104 | *
4/9 | 984) 878 | 586 | 3421 39 | 112 | 0.85] 6,6 | 11,4] 8.30] 106 | *

* Slight anisocytosis of red blood cells.
Experimental history, see table 66-b.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

on crackermeal, lard and butter was the first anemia experiment 0.
on this animal.

The control without Blaud’s pills and this secictiaaall (table 7 3
with Blaud’s pills show practically identical anemia figures for red cel. all
hematocrit and hemoglobin. The amount of red cell and hemogla pin
regeneration is practically identical in the 5 weeks in the two | oxpe

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 269

“ments. This again gives evidence that Blaud’s pills are inert .under
these controlled conditions of experimental anemia and regeneration.
_ Table 71 shows a little more blood regeneration than usual but not
~ enough to be able to attribute any of the reaction to the Blaud’s pills.
The red cell hematocrit reads 39 per cent at the end of 1 month and
this is not a normal figure. The hemoglobin was low to start with

TABLE 72

- Blood regeneration—bread, milk and Blaud’s pills—splenectomy. Dog 17-142.
q Coach mongrel, female, age 4 to 5 months

is ~ Ei

Qe &
s Ba oO .o)
a a a } °
B B p = a 3 a a 3
o8 3 pb B = he ‘al 3 4
S > rs 9 i) I ) = a
2 E ~ a > Bi > ee] a = iH es a
3 a-° a s 3 5 8 3 S q a
a=] 32 g ° a ° . N ro) f Pe bdo) °
Sopeeae Ss Pop ep ay we | ok poe Oe Shoe
Aa Bees" r=) By F | jen) 5 r e B ry
ec. ce. cc. |per cent|per cent kgm. cc
4/24 842 | 772 386 386 50 109 | 0.91 | 6,0 | ° 19,8 | 8.00 96

4/24 | Diet: Bread and milk

4/25 | Bled 193 ce.
4/26 | Bled 193 ce.

4/27 | 323| 6009 | 457 | 152 | 25 53 | 0.08 |

7 | 20,2 | 8.00| 76

bd

3 4/27 | Diet: Bread, milk and 2 Blaud’s pills daily

5/2 | 390] 600} 390] 210] 35 65 | 0.90 | 3,6 | 18,417.80 |- 76
5/9 | 760} 835| 451 | 384| 46 91 | 0.83 | 5,5 | 13,6] 7.90 | 106
| 5/16 | 8387] 790| 411| 379] 48 | 106]0.80| 66 | 7,2| 7.70} 103
5/23 | 997| 906| 453| 453| 50 | 110] 0.75! 7,3 | 6,4] 7.70| 117
5/30 | 890| 832] 416] 416] 50 | 107|0.70| 7,7 | 11,4] 8.00} 104
6/6 | 950} 863| 440] 423] 49 | 110] 0.73] 7,5 | 11,6| 7.70] 112

No previous anemia experiments on this dog.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

4 and returns to this level in 4 weeks. The type reaction of this dog is
a well established by the list of other anemia experiments given in table
66-b.

Table 72 shows a reaction to the first anemia experiment which has
_ been noted in other experiments. It is not the rule but is frequent
enough so.that we must always be on our guard in discussing the first
anemia experiment on any given dog. ‘There may be this remarkable

270 C. W. HOOPER, F. 8. ROBSCHEIT AND G. H. WHIPPLE

reserve which enables the dog to give an unusual blood regeneration
even on a most unfavorable diet. The blood regeneration is complete
in 3 to 4 weeks and thereafter is maintained for the next 3 weeks at

TABLE 73
Blood regeneration—bread, milk and Blaud’s pills—splenectomy. Dog 17-87.
Bull mongrel, female, young adult

m2
a
rg - ¢
ae
AH a o
= f o
= a ro) °
: ae, a | Boag w | 8 i
is) ~ | =|
et Bie" a S 3 2 eS 3 2 REMARKS
= as es > iy Zz S a
A ET et < 3 . a t e 4
si tages @ = 3 3 2 rs) ° td a
a) sO ° 3) . ° x fo) . Pe S °
& a ° < a a ) fa s a °
<< Te sy 3 ‘ d ae 5 . a 4
a Ey ry Ba ea a en) } a E - =)
per | per
ce ce. cc cant bee kgm. | cc.
3/26 | 927 | 850 | 459 | 391 | 46 | 109 | 0.80} 6,8 9,4/10.60 80

3/29 | Bled 212 ce. and 212 ce. (refer to table 8)

4 /23'| Diet: Bread and milk following fasting experiment of 3 weeks’ duration

4/23 | 540 | 772 | 502 | 270 | 35 | 70 | 0.66) 5,3 7,8} 6.90} 112
4/30 | 578 | 802 | 521 | 281 | 35 | 72 | 0.67) 5,4 | 11,6] 8 98
5/7 | 566 | 808 | 517 | 291 | 36] 70 | 0.71) 4,9 7,4
5 /14 | 662 | 808 | 517 | 291 | 36] 82 | 0.68) 6,0 6,8
5/21 | 656 | 830 | 531 | 299 | 36] 79 | 0.69) 5,7 5,6
5/28 | 566 | 833 | 553 | 283 | 34 | 68 | 0.68) 5,0 7,0
6/4 | 524 | 759 | 5380 | 228 | 30) 69 | 0.73) 4,7 5,2
6/11 | 528 | 754 | 529 | 226 | 30] 70 | 0.78) 4,5 | 14,6

9 90 90 G0 90 G0 GP
|S8SS8SEE SB
bo

6/11 | Diet: Bread, milk and 2 Blaud’s pills daily

6/18 | 631 | 809 | 517 | 291 | 36] 78 | 0.62) 6,3 6,4| 8.40) 96 | TF
6/25 | 514 | 858 | 626 } 231} 27] 60 | 0.79) 3,8 | 53,0) 8.30) 103 | FT
7/2 | 439 | 708 | 517 | 191 | 27} 62 | 0.79) 3,9 | 18,2) 8.40) 84] 7
7/9 | 537 | 790 | 521 | 268 | 34) 68 | 0.76) 4,5 | 13,0) 8.60) 92) TF
7/16 | 648 | 762 | 480 | 282 | 37] 85 | 0.77| 5,5 9,3} 8.40) 91} fT

* Red cells are small and much fragmented.

+ Red cells are large and fairly uniform in size.

Experimental history, see table 8—-b.

Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

the normal level. Refer to tables 74 and 75 for meat diet controls
with no previous bleeding. The fact that the dog was very young (4
to 5 months) and was without a spleen is thought to be without in-
fluence on this general reaction noted in other dogs (adult and non-

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA ye iy

splenectomized). We have no reason to suspect that the Blaud’s pills
were concerned in this reaction which has been noted in control experi-
ments on the same diet without the Blaud’s pills.

The second splenectomy experiment (table 73) shows a negative
reaction with bread and milk alone as well as with bread and milk plus
Blaud’s pills. There are peculiar fluctuations during certain weeks

TABLE 74

Blood regeneration—meat and Blaud’s pills. Dog 17-191. Bull mongrel, male,
young adult

nN

- : 2
a5 e &
a . = ‘ 1] ic>) 5 z 5
BEB! & 2 = E S 8
of 5 i b Pe 4 5 i
~“ ap fo) NS ° io} Q _ =|
S a > a > m 4 a : a
ere es | a 4 3 3 2 3 3 e a
Ba 32 an [o) mM . . “ fo) . fo) oS {o)
& om A 8 < . = 2 _ a : a 8
a yi re) my a ra en) 5 ef z 3 Fe)
CG. ce. cc. |per cent|per cent kgm. ce.

4/24 | 1352 | 1206 567 640 53 112 |.0:71 | 7,9 13,4 | 9.50} 127

4/24 | Diet: Bread and milk

4/25 | Bled 301 ce.
4/26 | Bled 301 cc.

4/27 | 382| 780| 600| 187| 24 | 49 | 0.79 | 31 | 17,6 | 9.30] 84
4/27 | Diet: Meat and 2 Blaud’s pills daily

5 /2 536 | 800 | 543] 264} 33 67 | 0.91 | 3,7 | 17,8 | 9.50) 84
5/9 1030 | 1064 | 585] 479 | 45 97 | 0.90 | 5,4 8,2 | 9.50} 112
5/16 | 1282 | 1256 | 653] 604] 48 102 | 0.73 | 7,0 | 11,8 | 9.80) 128
5/23 | 1530 | 1377 | 647 | 730} 53-.| 111 | 0.66] 8,4 8,8 | 9.70) 142
5/30 | 1571 | 1366 | 615 | 752] 55 115 | 0.65 | 8,8 | 18,6 | 10.00) 136
6/6 1510 | 1314. 591 | 419] 55 115 | 0.66 | 8,7 7,2 | 9.60} 137

No previous anemia experiments on this dog.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

in both these periods and we believe these ups and downs are referable
to the splenectomy. It is significant that the hemoglobin and red
cell hematocrit changes are not associated with any constant change
in plasma volume. The red count fluctuates with the pigment curve
and we must assume periodic constructive or destructive waves in-
fluencing the red cells in the blood stream. From data already published

(1) dealing with bile excretion in splenectomized dogs with simple

272 C. W. HOOPER, F. S. ROBSCHEIT AND G. H.. WHIPPLE

anemia we may suspect that blood destruction may be in part respon-.

sible for these irregularities in the level of the curves of pigment volume,
red cell and hemoglobin values. The color index shows no change.

There is a note to the effect that the red cells are larger and more uni-

form in size during the period of iron feeding. A single observation
of this nature is of interest but does not call for discussion at this time.
TABLE 75

Blood regeneration—meat and Blaud’s pills—splenectomy. .Dog 17-163. Bull
- mongrel, male, young adult

mM
Ng ; 4
BE. 8 8
=} 2] ° z °
aee| & B 5 2 y g 3
Pos p | c} = a 4 :
Se > rs = ) Sl a 4 J
— ° b> Q
es E Bn > * mi ra 2 ; Pe
eee Se 3 3 S e 3 - 5 a
| SO ° ° D : 9 () : 9 S °
Bojeea| $ | 4) 8 | ee be ee
A py fa cy a G is) a a
cc ce. ee per cent|per cent 3 kgm. ce.
4 [24 918 | 918 | 450] 468] 51 100 | 0.75 | 6,7 14,4 | 9.00 102
>]

4 /24 Diet: Bread and milk

4/25 | Bled 229 cc.
4/26 | Bled 229 cc.

4j27 | 340| 679| 509| 170| 25 | 50 | 1.00 | 2,5 | 17,6 | 8.00 | 76
4/27 | Diet: Meat and 2 Blaud’s pills daily

5/2 | 538| s03| 514| 280| 36 | 67|0.90| 3,7 | 19,2| 9.00] 99
5/9 | 790| 909| 509| 400] 44 | 87]|0.78| 5,6 | 19,6|8.90| 102
5/16 | 924| 880] 475| 405! 46 | 105/0.78| 67 | 82/890] 98
5/23 | 1140 | 1096 | 559| 538] 49 | 104|0.75| 69 | 12,4|8.80| 124
5/30 | 907| 863| 423] 4401 51 | 105|0.63| 83 | 9,219.00] 96
6/6 | 1080 | 1002| 491] 520] 51 | 106|0.65| 8,2 | 13,8|8.60| 117

No previous anemia experiments on this dog.
Experimental history, see table 38-b.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

The two meat feeding experiments (tables 74 and 75) give the ex-
pected reaction on this diet. The splenectomy dog reacts the same as

does the normal dog. Sufficient meat is given to maintain aconstant

body weight. Both these dogs had not been used previously for anemia
experiments; refer to table 72 which illustrates the reserve capacity
sometimes exhibited by such dogs. We have no reason to suppose
that the Blaud’s pills were in any way concerned in this reaction.”

Se ns

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA — 273

Hemoglobin experiments——Table 76 gives an experiment of much
interest and we are able to submit the control experiment (table 16).
This same dog a few months previously was made anemic and placed
upon asugardiet. After a period of 4 weeks the level of pigment volume,
hemoglobin and red cell hematocrit was practically the same as that

TABLE 76

laa en chemoulobsn and sugar—metabolism. Dog 17-28. Bull mon-
grel, female, young adult

a es =.
a z z
seg a 3 3
Oe a ei 8 3
68a} 8 R 5 s i 4 Y

peo PR = g a i) 4
; 3 ° a 4 2 REMARKS

é. a > e) = a) 5 =
S |eaal > “ i a * ; ks a
i a -8 a s o o foe is) x i] ra)
a sos ° a ° . i} f . ie} °
& fo [o} < io] is) : > fas} a Pas
< S a ps 4 ef . = fo) . . bs fe |
=@ Aa 9 Ay 8 ea) 3 8 E E =)
ce ce. cc eo Trl kgm. | cc

5/7 | 1895) 1709) 769 | 940 | 55 | 111 | 0.66) 8,4 | 13,2)12.50) 136

5 [7 Diet: Mixed food

5/8 | Bled 427 cc.
5/9 | Bled 427 ce.

5/11 | 504| ou 640 | 274 | 30 | 65 | 0.76| 4,3 | 22,0|12.20| 76 |

5/11 | Diet: 50 grams cane sugar, 25 grams glucose, 10 grams washed red. blood
‘cells

5/14 | 558} 979) 676 | 303 | 31) 57 | 0.75] 3,8 | 11,2)11.50) 85
5/21 | 676) 1055) 665 | 390 | 37] 64 | 0.70) 4,6 6,8}10.80} 98
°§/28 | 856) 1141) 673 | 468 | 41} 75 | 0.71) 5,3 9,6|10.10} 113
6 /4 887| 1137] 603 | 534 | 47 | 78 | 0.58] 6,7 9,6} 9.50) 119
6/11 | 920] 1180) 672 | 508 | 43] 78 | 0.59) 6;6 8,0} 8.90} 133
6/18 | 1085) 1277| 664 | 612 | 48 | 85 | 0.50) 8,5 | 10,0) 8.30) 154

+ 2 £%

* Marked fragmentation of red cells.

400 cc. water given by stomach tube daily.

Experimental history, see table 6-b; see table 16 for control.
Blood volume with dry oxalate. Hemoglobin by Sahli tubes.

observed immediately after the bleeding. The total blood regenera-
tion then in the control experiment was zero. There was a trifling
gain in the red count.

Under identical conditions on a sugar diet plus 10 grams washed,
dried red blood cells we see a very different reaction (table 76). The

274 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE
TABLE 76-a
Total urinary nitrogen—hemoglobin and sugar. Dog 1 7-28
DATE, 1917 phpraubolgy tide bese 2 Faw 24 WEIGHT REMARKS
grams * , ce. pounds
5/11 3.55 490 26.4 0 feces
5 /12 3.19 480 25.8 Solid black stool
5/13 361 20.5 Solid black stool
5 /14 3.50 386 25.3 0 feces
5/15 2.52 485 25.0 Slight diarrhea
5/16 2.58 358 24.7 Solid feces
5 /17 2.38 390 24.5 0 feces
5/18 2.35 480 24.4 Trace of feces
“6/19 2.30 403 24.1 Slight diarrhea
5/20 2.46 410 24.0 0 feces
5 /21 2.46 420 23.8 0 feces
5 /22 2.63 465 23.4 0 feces
5 /23 2.41 383 23.4 0 feces
5 /24 2.94 400 23.0 Diarrhea +
5 /25 2.74 376 22.4 Diarrhea +
5 [26 2.66 412 22.6 0 feces
5 /27 2.52 395 22.4 0 feces
5 [28 2.35 424 22.2 0 feces
5 /29 2.46 370 21.9 0 feces
5 /30 2.63 410 21.8 0 feces
5 /31 2.13 510 21.6 0 feces
6/1 2.32 405 21.4 0 feces
6 /2 2.18 405 21.4 0 feces
6/3 2.30 425 21.1 0 feces
6 /4 2.24 426 20.8
6 /5 2.44 425 . 20.8 0 feces
6 /6 3.00 417 20.5 Formed feces
6/7 2.24 373 20.2 Soft feces
6 /8 2.46 410 20.0 Solid feces
6/9 2.58 419 19.9 0 feces
6/10 2.60 425 19.8 0 feces
- 6/11 2.63 407 19.5 0 feces
6 /12 2.83 405 19.5 0 feces
6 /13 2.83 375 19.4 0 feces
6 /14 2.88 490 19.2 0 feces
6/15 3.00 480 18.9 0 feces
6 /16 3.28 411 18.6 Solid feces
6 /17 3.42 395 18.4 0 feces
6/18 3.02 400 18.3

ti

or

agg cae CE eget Eee

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 275

initial anemia level in the two experiments is practically: identical, also
the body weight, normal initial blood pigment, etc. The only point in
which these two experiments differ lies in the 10 grams of red cells
added to the sugar diet. At the end of 4 weeks the control shows a
gain of zero in pigment substance but the red cell feeding gives a sub-
stantial gain of 13 per cent hemoglobin, 17 per cent red cell hematocrit
and 300 units pigment volume. There is a gain of 2,400,000 in the red
cell count. The subsequent 2 weeks show a distinct gain over the level
just noted. This is in notable contrast to the expected reaction on a
sugar diet.

The urinary nitrogen and fluid excretion figures are given (table 76—a)
and show the expected values. During the last week of the experi-
ment there is a distinct rise in the nitrogen output. We have come to
look upon this as an early sign of intoxication which if not heeded may
be followed by severe clinical disturbances and death. This dog
promptly recovered when placed on a mixed diet.

It is to be noted that whole red cells were used in this experiment,
that is, hemoglobin plus red cell stroma. In the following experiments
hemoglobin alone was used. We might point out the difference in
the color index observed in these two conditions but feel that the data
are not sufficient to establish this very interesting point. We are
gradually collecting data which indicate the conditions most favorable
for stroma production and these experiments will be presented at
another time.

The next hemoglobin experiment (table 77) is not very convincing
as we do not have a suitable control of the bread and milk factors in
this dog. Moreover this is the first anemia experiment on this dog
and under such circumstances this reaction is often atypical, as has
been noted before. We may say that the blood regeneration due to
the bread and milk plus hemoglobin might be identical with a reaction
on bread and milk alone. We cannot point with certainty to any
difference which can be attributed to the hemoglobin. This experi-
ment is unlike the others of this group which give positive evidence
that hemoglobin does influence the curve of blood regeneration.
Finally, we must conclude that this experiment does not give any
evidence against the value of hemoglobin but it also gives no positive
support to the other experiments.

Table 78 presents a long experiment in which hemoglobin is given
by mouth during one period and by intravenous injection during a
subsequent period. We feel that there is good evidence that hemo-

276 C. W. HOOPER, F. 8S. ROBSCHEIT AND G. H. WHIPPLE

globin did influence favorably the blood regeneration. The anemia
level produced by three bleedings was slightly below the average. The
first period of sugar feeding plus 10 grams of hemoglobin by mouth
shows a notable gain during 3 weeks. The hemoglobin rises 50 per
cent and the pigment volume from 433 to 810. There is a correspond-

TABLE 77

Blood. regeneration—powdered hemoglobin: and bread and milk. Dog 18-124:
Terrier mongrel, female, young adult

lB
a A
eo] 3 S
= Q >
HS (<>) ° Z
R26] § 2 | 5 S Fl
eoO “ 3 3 a a | 2
00 > 5 ° ° a = = a
oS baa > > > 9) a = ‘ &
se Lees ioe | 3 3 - . 3 eI 2
i) S28 ° a f . : o ° 6 g °
fd aee | fe 3 : r iS 3 : : : S
P=) ry Q ey | a en o a B E a
cc. ce. cc. |per cent|per cent . ; kgm. ce.
5/16 515 273 234 | 45.5 5.80 89

5/26 | Diet: Bread and milk.

5/27 | 488 | 548| 362 | 241 | 43.8 | 89 | 0.53 | 84 | 10,4 | 6.10 | 90

5/28 | Bled 187 ce.
5/29 | Bled 137 ce. No distress -

5/29 | Diet: 125 grams bread (ground and dried), 300 cc. milk, 5 grams hemo- .

globin*
5 /31 207 | 398} 308 87 | 21.8 | 52 | 0.59} 44 | 12,8| 5.70} 70
6/5 370 | 481 | 348 | 129 |] 26.9] 77 | 0.80} 4,8 | 11,2|5.75| 84
6 /14 376 | 522 | 357] 160 | 30.6-} 72 | 0.61 | 5,9 8,6 | 5.385 | 98.
6 /21 421 | 513 | 325 | 1838 | 35.7] 82 | 0.60] 6,8 7,8 | 5.20} 99
6 {26 342 | 417 | 266] 145 | 34.7| 82 | 0.66) 6,2 7,2 | 5.261 7

* Hemoglobin: Defibrinated blood centrifuged, cells washed twice with N /1
salt solution, 2 volumes distilled water added, allowed to lake over night, centrif-
ugalized, stroma removed, hemoglobin dried and powdered.

No previous anemia experiments on this dog.

ing rise in the red cell hematocrit and red cell count. On sugar alone
we recall that the gain in these pigment factors is only trifling, perhaps
10 per cent in hemoglobin and corresponding amounts in the other
readings. We must hold the hemoglobin responsible at least for a
part of this favorable reaction.

Z PR
ee eee ee ee eS To) “a

“aes eS

10/11) 662

TABLE 78
Biood regeneration—sugar and hemoglobin—crackermeal, milk and hemoglobin.
- Dog 17-157. Coach mongrel, female, young adult

mM
a ; ‘i
ae a
a S fo)
eas ~ s 3 g B a
° 3} 4 5 B p 5 K 5 4 :
7 ab 9 5 3 3 é a | % REMARKS
S Sy fs = N: A > qi a a . =) Fy
i | so af ° mn . . i ° . Fa] S °
eee fe ee gained | 8b EL as
a & ey fi rf rs a0) © ee z = re)
per | per
cc ce. cc sane ‘ho Gand kgm. |} ce
8/28) 1366) 1027) 518 | 504 | 49.0) 133 | 1.00] 6,7 | 14,4/10.10| 102 | *Slight

8 /28 Diet: Crackermeal and milk

8/29) Bled 257 ee.
8/30} Bled 257 ce:

8/31] 565| 777| 560| 215 | 27.6 73; | | | 9.65| 81 |

~ §/31| Bled 194 cc.

9/3 | 433| 731| 544 | 179 | 24.5| 59 [a 93| 3,0 | 13,8| 9.25 79

*Poik.+

9/10) Diet: 75 grams sugar, 25 grams dextrose, 10 grams hemoglobinft by
stomach tube

9/10} 646) 848) 590 | 249 | 29.4! 76 | 0.84) 4,5 9,4| 8.40} 100 | *Poik.+

; Vomited
9/16} 750} 896} 593 | 294 | 32.8) 84 | 0.82) 5,1 6,8} 8.00} 113 | * Poik.+
9/25) 810) 788} 492 | 293 | 37.2} 102 | 0.86) 5,9 6,6} 7.25) 109 | * Poik.+

9 /25| Diet: 100 grams sugar, 30 cc. hemoglobin intravenously}

9/30| 625 742| 463 | 271 | 36.5] 84-| 0.70| 6,0 | 6,2] 6.95] 106 | * Poik. +

9 /30| Diet: 200 grams crackermeal, 500 cc. milk, and hemoglobin intravenouslyt

100
110 | * Slight

280
322

37.2
38.8

752
830

465 .
502

13,6| 7.55
12,8] 7.55

88 | 0.76) 5,8
97 | 0.73) 6,3

10/16) 805

10/16} Diet: 200 grams crackermeal, 500 cc. milk
10 /23 722| 902] 570 | 322 | 35.8 80 | 0.67| 6,0 | 16,0 8.00] 112 | * Poik.+

* Poikilocytosis of red blood cells.
+ Blood centrifugalized, washed once with salt solution; 2 vohunes of dis-

| tilled water added to washed, packed red blood cells. Allowed to stand 24
-hours. Centrifugalized and stroma removed. Dried and powdered.

t For injection into the vein: With aseptic technique blood is centrifugalized,

washed once with salt solution; 20 cc. distilled water added to 10 cc. washed,

packed red blood cells. Allowed to stand 4 hours, added 2.5 ce. of 10 percent salt
solution. Centrifugalized and stroma removed. Total amount injected intra-
venously daily.

Experimental history, see table 66-b.

277

278 C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

When the sugar diet is continued but the hemoglobin is given in- |

travenously, we note a fall in hemoglobin and pigment volume but no
change in the red cell hematocrit, red cell count and plasma volume.
We have no good explanation to fit these observed facts.

TABLE 79

Blood regeneration—hemoglobin injection intravenously. Dog 17-27. Bull mon-
grel, female, young adult |

cr g 8 8
pee] 8 |. a-| 3 | & g 2
o8e] 6 3 a = fs fs M
o |"*e>5] 8 ° a 5 - 3 2 REMARKS
& | Beal § > > | z 3 5 ~
ome pe ear $ Sf Br So OT oe
i=) sO a ° D - ee oS . Py ; o fo)
& | otal g $ ie . 3 3 e . = |
a ry a By rf a = 5 8 z e ro)
cc. ce. ce. ane nent kgm. | ce
8/9 | 1914] 1454] 718 | 727 | 50.0] 133 | 0.75] 8,7 | 12,0/16.15) 90°
8/9 | Diet: Bread and milk
8/12 | Bled 364 cc. No distress
8/13 | Bled 364 cc. No distress |
8/15 | 749| 1040| 732 | 297 } cel ge | | |15.58| 67 |

8/15 | Bled 260 cc.

8/17 817 1034] 718 | 311 | 30.1] 79 | 0.95| 4,1 | 24,6115 .45| 67 | * Poik.++

8/17 | Diet: Hemoglobin injection intravenously;t 75 grams sugar, 25 grams
dextrose, 200 cc. water daily by stomach tube

74
80

84
105

8/23 | 908
8/30 | 1155

1080
1100

710
647

370 | 34.3

40.2

0.76] 5,5
0.75) 7,0

14,8
5,6

14.55
13.75

* Poik. ++

9/2 | Accidental death

* Poikilocytosis of red blood cells,

+ Hemoglobin: Blood drawn from normal dog with aseptic precautions. Cen-
trifugalized, washed once with salt solution; 20 cc. distilled water added to 10
ce. washed, packed red blood cells. Allowed to stand 4 hours, added 2.5 ec. of
10 per cent salt solution. Centrifugalized and stroma removed. Total amount
injected intravenously daily.

Experimental history, see table 15-b; see sugar control, table 15.

The 2 weeks following on a crackermeal and milk diet plus hemo-
globin intravenously show a slight gain in pigment substance. Even
this slight gain may have some significance when we recall that it

4

ee ee a a

BLOOD REGENERATION FOLLOWING SIMPLE ANEMIA 279°

occurred following a long period of limited diet intake. Hemoglobin
regeneration under such unfavorable circumstances is very difficult and
becomes increasingly difficult as the limited diet periods are extended.

Table 79 gives an experiment which was unfortunately terminated

. at the end of 2 weeks by an accident. We are able to refer to a con-

trol reaction on sugar feeding alone (table 15). This table shows a
slight gain in hemoglobin, red cell hematocrit and pigment volume
during the first week on the sugar diet. The second week shows no
gain. Compare with this reaction the figures in table 79 which show a
gain in the first week which may be compared with the control but the
second week intead of the stationary level in the control shows a dis-
tinct gain in red cells, pigment volume, hemoglobin, ete. We feel
that a part of this gain is to be explained by the hemoglobin injections.

Table 80 illustrates another type of experiment in which the hemo-
globin injections are given under conditions very unfavorable for blood
regeneration. There is a 3-week period of sugar feeding during which
time there is zero gain in pigment substance. There is a slight but

distinct gain following 5 days of hemoglobin injection and further slight

gains on a crackermeal and milk diet with daily hemoglobin injections.
Some of the subsequent gains in hemoglobin, red cell hematocrit and
red cells may be due in part to the hemoglobin injections which in all
probability cannot be at once utilized. The crackermeal and milk
alone or with yeast can account for very little blood regeneration when
given subsequent to a long period of sugar feeding. The evidence for
the favorable influence of hemoglobin injections is not as strong as in
some of the other experiments which have the complete control periods. |

The last hemoglobin experiment (table 81) is to be compared with
table 76. The hemoglobin in this instance is given intraperitoneally
so that the absorption might be rapid and the elimination of slight
amounts through the urine be obviated. The influence of a different
set of phagocytic cells might well be a factor but the gross results are
much the same as regards blood regeneration. _ )

Three weeks of sugar feeding plus hemoglobin injection give an
- amount of blood regeneration which cannot be explained as due to the
sugar feeding. We note a rise of hemoglobin from 72 to 120 per cent

4 and red cell hematocrit from 27 to 45 per cent. The control sugar diet
___ figures would show only trifling gains. Subsequent weeks on a cracker-

meal and milk diet do not show much gain except in the red count.
The final period of mixed diet as usual brings the dog back to a high
normal figure. ,

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 2

Blood regeneration—hemoglobin intravenously. Dog 16-160. Bull mongrel, female,

, TABLE 80

young adult

: ; 3
Ebr a fe) 8 vs 8
seel 8 | a | 2 | & “le : |
sek re 3 5 3 a a Ss fo REMARKS
Bs bene herd cal boRad rae Bde ; |g a
me ee fi a a Reba BB BA be we -
& |otal 9 < m Bilis) pike a ¢ a |~°e
ee =) By a rs en) } a z E ry
ce. ce ce pat el kgm. | ce.
8 /28 | 1835] 1103} 456 | 654 | 58.2] 166 | 1.10} 7,6] 9,6/10.15} 109
8/28 | Diet: Bread and milk
8/29 | Bled 276 cc.
8/30 | Bled 276 ce.
9/1 | eas] 771] 524 | 243 | 31.5] 84 | fe) 9:65) send
9/1 | Bled 193 ce.
9/3 448) 696| 513 | 177 | 25.5] 64 | 0.80| 4,0| 15,4 9.50| 73 |* Poik.+-+
9 /3-| Diet: 75 grams cane sugar, 25 grams dextrose, 200 cc. water by stomach
9/26} tube (table 18)
9/25| 429| 664] 474 | 182 | 27.4 64 | 0.61] 5,2) 9,6 7.35, 90 |* Poik.+-+
9/26} Diet: Hemoglobin intravenously, t 30 cc., and 100 grams sugar by stomach
tube
9/30 | -437| 618| 421 | 194 | 31.4| 71 | 0.68] 5,2 12,4] 7.15| 86 [* Poik.++4
9/30 | Diet: Hemoglobin intravenously,{t 30 cc., and 200 grams crackermeal,
600 ec. milk
10/11} 506) 712) 477 | 227 | 31.9] 71 | 0.58) 6,1} 11,0] 7.75) 92 |* Poik.+-+
10/16] 586) 740) 495 | 237 | 32.0) 79 | 0.65) 6,1) 5,0] 7.80) .95 |* Poik.++T
10/16} Diet: Hemoglobin injection discontinued; 200 grams crackermeal, 500 cc.
milk
10 /23| 670| s26| 536 | 281 | 34.0] 81 | 0.65| 6,2| 8,4] 8.05] 103 |* Poik.++f
10/23} Diet: 1 gram dried brewer’s yeast, 200 grams crackermeal, 500 ce. milk.
10/30} 590} 808) 536 | 264 | 32.7| 73 | 0.47| 7,7| 15,2) 8.30) 97 |* Poik.++fT
11/6 | 720) 868) 547 | 317 | 36.5} 83 | 0.66} 6,3) 8,6} 8.20) 106 |* Poik.+
11/12} 784) 883) 532 | 342 | 38.7; 89 | 0.50) 8,9} 7,2] 8.25) 107 |* Poik.+
11/13} Diet: Mixed diet
11/22} 726) 853) 503 | 341 | 40.0) 85 | 0.42} 10,2} 13,6] 8.80} 97 |* Poik.+
11/29} 1122} 1020) 547 | 463 | 45.4] 110 | 0.69) 8,0} 19,0} 9.25) 105 |Excellent

condition

* Poikilocytosis of red blood cells.

+ Shadow cells.

t Hemoglobin: Blood taken with aseptic precautions, centrifugalized, washed
once with salt solution; 20 cc. of distilled water added to 10 cc. washed, packed
red blood cells. Allowed to stand 4 hours, added 2.5 ce. of 10 per cent salt solu-

tion.

nously daily.

Centrifugalized and stroma removed. Total amount injected intrave-

sy Oe I ge A oe Fe ee ASL Sa ee Wr. ees AM

ee ee ee ee ee ee ee ee

se a ee ee

ey a, on

——--

_ TABLE 81
Blood regeneration—hemoglobin intraperitoneally. Dog 17-28. Bull mongrel,
female, young adult

2
oa ; :
a. = a 5 8
sasi # | 2 | 2 | & ae ;
ree ae 2 3 = 2 A 3 e REMARKS
Sortre. |, | ce | ae ae a
4 rs] -8 a s is) 3 a 3 o = a
8 |s23! 9 Bb : ‘Aig, ae 8 : a3 =
Merine ia hed 41 Bl Site Be Le baie dS
a | a ey a a q 5 a Ea a
ce cc. ce | bait ee kgm. | ce.
8/9 | 2417) 1580) 680 | 887 | 56.1) 153 | 0.84) 9,1) 14,2/17.00) 93
8/9 Diet: Bread and milk

8 /12| Bled 395 cc. No distress

8/13 Bled 395 ce. Diet: Crackermeal and milk

/15| 1015| 1149] 750 | 387 | 33.8| so| | | |te.30| 71 |
8 /15| Bled 286 cc.

8/17} 756| 1050| 758 | 281 | 26.8| 72| 0.97| 3,7] 23,6|15.90| 66 | * Poik.+

8/19} Hemoglobint (intraperitoneal injection). Diet: 75 grams sugar, 25
grams dextrose, water

8/23) 794
8 /30| 1133

77
- 95

1030
1193

708
685

310
430

30.1
36.0

0.69
0.76

* Poik.
* Poik.+

5,6
6,2

21,4
12,6

15.40
14.55

67
82

9/4 | Hemoglobinj{ (intraperitoneal injection). Diet: 100 grams sugar in 500
cc. water

9/6 | 1670| 1385| 755 | 608 | 45.0| 120 | 0.83| 7,2

9/6 | Hemoglobin discontinued. Diet: 200 grams crackermeal, 500 cc. milk
kaolin

11,0|13.40| 103 | * Poik. ++

9/13) 1550} 1450) 876 | 566 | 39.2) 107. | 0.68] 7,9} 9,8|14.00} 103
9/19) 1433] 1310} 752 | 545 | 41.6} 109 | 0.71) 7,7) 12,2/13.90) 94 | * Poik.
9 /27| 1253) 1430} 658 | 468 | 41.4) 111 | 0.71) 7,8} 10,6/14.30) 79 | * Poik.
10/9 | 1635) 1363) 668 | 682 | 50.5) 120 | 0.77] 7,8] 12,2|14.20} 96 ,
10/18} 1836} 1386) 680 | 694 | 50.0) 132 | 0.66} 10,1] 9,0)14.45) 96
10 /25| 1550} 1352| 704 | 636 | 47.0] 115 | 0.64] 9,0} 10,8/14.60| 93

10/25} Diet: 200 grams crackermeal, 500 cc. milk, 1 gram dried, powdered brewer’s
yeast, kaolin

—_

10/31} 1415| 1400] 750 | 638 | 45..4| 101 | 0.65| 7,8| 11,8
10 /31| Diet: Mixed diet

e 1/7
B i1/13

5.00| 93 |

1975
2000

1555
1600

710
743

829
840

127
125

53.3
52.5

10,8)15.10] 103
20,2/15.05} 106

0.741 86
0.63} 9,9

—_———

* Poikilocytosis of red blood cells.

Tt Hemoglobin: 10 cc. sterile, washed, packed red blood cells and 20 ce. dis-
tilled water. Centrifugalized and stroma removed. Intraperitoneal injection.

Experimental history, see table 6-b; see table 76 for hemoglobin feeding.

281

282 -C. W. HOOPER, F. S. ROBSCHEIT AND G. H. WHIPPLE

SUMMARY

Blaud’s pills are inert when added to various diets which do or do
not favor rapid blood regeneration. We may not assume without posi-
tive proof that inorganic iron is of value in the treatment of secondary
anemia. |

Splenectomy may or may not modify this blood regeneration reaction.
Limited diets following anemia periods associated with splenectomy
- may be the cause of fluctuations in the normal expected curve of blood
regeneration. |

Hemoglobin (by mouth, intravenously or intraperitoneally) exerts
a distinctly favorable influence upon subsequent blood regeneration.

BIBLIOGRAPHY

(1) Hooper AND WuiprLE: This Journal, 1917, xliii, 275.

PHYSIOLOGIC CHANGES PRODUCED BY VARIATIONS IN
“LUNG DISTENTION ;

III. ImpaIRMENT OF THE CORONARY CIRCULATION OF THE RIGHT
VENTRICLE

RALPH HOPKINS anp FELIX P. CHILLINGWORTH

From the Physiological Laboratory, Tulane University of Louisiana, New Orleans

Received for publication June I, 1920

~ In a previous communication a method was described by which the
maximum blood pressure obtainable in the pulmonary arteries could be
measured by the increase in intrapulmonic air pressure sufficient to
prevent the passage of blood through the capillaries of the lungs (1).
The mechanical blocking of the pulmonary circulation produced under
these conditions was found to cause a maximum pulmonary arterial
pressure of 85 mm. of mercury and a coincident complete disappear-
ance of carotid pressure. In the present communication we record
the results of some experiments to determine the critical point during
progressive increase of intrapulmonic air pressure at which circulation.
of blood through the coronary arteries of the right ventricle becomes

impossible because of the maintenance of a level of blood pressure in

the pulmonary arterial and right cardiac systems equal to or higher
than that of the general systemic circulation. Distention of the lungs
to or beyond this critical point is almost immediately fatal and over-
distention even when not attaining this critical degree also interferes
with adequate oxygenation and nutrition of the heart. The necessity
for a balance in favor of the diastolic systemic pressure over the dias-
tolic right ventricular pressure has a bearing on artificial respiration
effected by increase in the intrapulmonic air pressure. Loss of this
balance through over-distention of the lungs may defeat the purpose
for which artificial respiration is givén by causing asphyxiation of the
heart muscle even while the lugs are being over-ventilated. This
observation is especially pertinent in the conditions in which, as is
usually the case when artificial respiration is resorted to, the systemic
arterial pressure is low and but little further fall is sufficient to reduce
283

284 RALPH HOPKINS AND FELIX P. CHILLINGWORTH

it below the level of pressure in the pulmonary arteries because the
latter rises during great distention of the lungs while the systemic
arterial pressure falls.

An endeavor is also made to show that even with the lungs distended
in lesser degree than the critical point referred to, death may result
owing to impairment of the coronary circulation of the right ventricle.

METHOD

The method employed to increase the pressure of the intrapulmonic
air has been described in a previous communication (2). The use of
the plethysmograph referred to facilitates the attainment of the degrees
of pressure desired and obviates difficulties that would arise from
forced expiratory efforts of the animals if the usual methods of intra-
tracheal insufflation were employed. By placing the entire animal
within the plethysmograph and connecting his trachea to the outside
air, diminution of the pressure within the plethysmograph produces the
same effects on the animal’s relation to the air within his lungs as would
result from an equal increase in the intrapulmonic air pressure while
the exterior of the animal remained under atmopheric pressure. The
decrease, therefore, in plethysmographic pressures is an exact measure
of the actual increase of intrapulmonic air pressures and in referring to
the effects of these pressures the terms are regarded as interchangeable.

A correction must be made in measuring blood pressures which are
recorded during diminished plethysmographic pressures. To the blood
pressures recorded in millimeters of mercury should be added the
difference, in millimeters of mercury, existing between the atmospheric
pressure and the pressure within the plethysmograph. The necessity
for this correction is made obvious by the fact that with diminished

plethysmographic pressures the carotid pressure may fall considerably

below its zero line. |

For recording blood pressures and the pressure within the plethysmo-
graph mercury manometers were used and the readings in millimeters
of mercury on the unreduced tracings have been multiplied by 2 when
used in the subsequent paragraphs. i

To record the pulmonary arterial pressure a branch of the left pul-
monary artery was used and artificial respiration by intratracheal
insufflation was performed after opening the chest and continued
until the animal was placed in the plethysmograph. In the intervals

es

MECHANICAL IMPAIRMENT OF CORONARY CIRCULATION . 285

between experiments, artificial respiration was maintained by rhyth-
mical changes in the plethysmographic pressure.

Recovery after distention of the lungs is rapid and many experiments
may be performed on one animal but the time factor as regards the
duration of lung distention is of importance. In obtaining the results
recorded in subsequent paragraphs no experiment exceeded one minute
in duration.

RESULTS OF EXPERIMENTS

Four typical tracings are reproduced taken from a series of 25 experi-
ments performed on 10 dogs, and a curve is plotted to show the effect
of lung distention on the carotid and pulmonary arterial pressures.
The tracings and the plot are first described and the results on the
coronary circulation are subsequently discussed.

Figure 1. Synchronous records of the carotid and pulmonary
arterial pressures are reproduced. The pressure within the plethys-
mograph is also shown. Air was exhausted from the plethysmograph
during a period of one minute effecting a gradual fall in the intra-
plethysmographic pressure to 83 mm. of mercury below the atmo-
spheric pressure. On the unreduced tracing from which this figure is
reproduced the maximum fall in carotid pressure was equal to 212 mm.
of mercury (actually 46 mm. below the carotid zero line), while the
fall in pulmonary pressure was equal to 54 mm. of mercury (9 mm. ,
below its zero line). The carotid pressure shows a corrected fall of
129 mm. (212-83), while the pulmonary pressure when corrected
shows actually a rise of 29 mm. of mercury. When corrected, the
minimum carotid and maximum pulmonary pressures are respectively
41 (preéxperimental pressure level 170 minus corrected fall 129) and
79 (preéxperimental level 50 plus rise resulting from correction 29).
Respiratory waves, following a period of apnoea which was of 18 sec-
onds’ duration, are marked in both arterial curves but show only
slightly in the plethysmographic record, since they were the result of
spontaneous but ineffectual efforts on the part of the animal. It will
be noted that during the last 14 seconds of the experiment the intra-
plethysmographic pressure rose slightly and that with this increase in
pressure there occurred a rise in carotid while the pulmonary pressure
continued to fall. It will also be noted that as the plethysmographic
pressure is progressively lowered there is a gradual disappearance of
pulse pressure in both the arterial curves. With sudden return to
atmospheric pressure within the plethysmograph there is an almost

OE ATR |

PEVUPTTeTE Tee eee ee Wed ee eee aad)

Fig.1. Experiment of March 20,1916. Dog6kilo. Ether. Time in seconds.
A, Carotid blood pressure recorded by mercury manometer; B, Zero level for
carotid blood pressure; C, Left pulmonary blood pressure recorded by mercury
manometer; D, Zero level for pulmonary pressure; EZ, Plethysmographic pressure
recorded by mercury manometer which is 68 mm, above the time record.

286

MECHANICAL IMPAIRMENT OF CORONARY CIRCULATION 287

synchronous recovery of both arterial pressures to their preéxperi-
mental pressure levels.

Figure 2. Records of carotid and pulmonary arterial pressures -
are reproduced from three parts of a typical tracing showing the effects

Fig. 2. Experiment of January 5, 1918. Dog 8.75 kilo. Ether. Time in
seconds. A, Carotid blood pressure recorded by mercury manometer; B, Zero
level for carotid blood pressure; C, Left pulmonary blood pressure recorded by
mercury manometer; D, Zero level for pulmonary pressure. This level is 20
mm. above the time record. 1, 2 and 3 show the progressive effects of increasing
lung distention. .

of distention of the lungs in varying degree. The experimental pro-
cedure was varied in the experiment on this animal and the plethysmo-
graph was not used. The lungs were inflated through the trachea
which was connected to a reservoir containing air under pressure. The
degree of pressure employed to distend the lungs is not recorded but

288 RALPH HOPKINS AND FELIX P. CHILLINGWORTH

can be estimated from the effects produced upon the arterial pressures.
Small caliber mercury manometers were used for recording blood
_ pressures. :

In the first of these tracings the lung distention is quite moderate
and is accompanied by a slight rise in both arterial pressures. The
systolic pulmonary pressure shows no rise but the mean blood pressure .
is higher due to maintenance of a higher diastolic level. Measure-
ments of the unreduced tracing show a preéxperimental mean pulmo-
nary pressure of 14 mm. of mercury which during inflation rises to 20.

In the second of this series the distention of the lungs is slightly
greater and the mean pulmonary pressure rises from a preéxperimental
level of 16 to 28 mm. of mercury. In this instance the systolic as well
as the diastolic pressure rises during distention of the lungs, but the
rise in the systolic is 8 mm. less than the rise in diastolic pressure.

In the third tracing distention of the lungs is in excess of that in
either of the previous experiments and a corresponding increase is
observed in the pulmonary arterial pressure which attains a maximum
mean pressure of 43 mm. (29 mm. above the preéxperimental level of
14). Again it is noticeable that the rise in the diastolic exceeds that in
the systolic pressure. A comparison of the mean pulmonary to the
mean carotid pressures shows that, at the point of greatest lung disten-
tion, the pulmonary pressure exceeds that in the carotid artery by 6
mm. and also that the pulmonary diastolic pressure is slightly in excess
of that of the carotid. The initial carotid pressure in this animal is
40 mm. lower than that plotted in figure 3, and as a result of this lower
preéxperimental level a comparatively small fall in carotid pressure
is sufficient to make the carotid equal to the pulmonary arterial pressure.

Figure 3. A curve is plotted from a typical tracing showing simul-
taneous carotid and pulmonary arterial pressures in millimeters of
mercury during progressive increase of intrapulmonic air pressure.
The broken line represents-the former and the unbroken line the
latter. The figures used have been corrected as explained in a pre-
vious paragraph. The ordinates indicate blood pressures and the ab-
scissae the pressures in millimeters of mercury employed to distend the
lungs. |

The carotid pressure, while distention is progressing, shows an
initial rise of 6 mm. of mercury attained when the excess of air pressure.
within the lungs is equal to 10 mm. of mercury. With increasing
distention of the lungs the carotid pressure falls progressively, the
maximum fall being attained with an increase in the intrapulmonie air

SS ee ee en ee a a ee ee a ae : > ee te

MECHANICAL IMPAIRMENT OF CORONARY CIRCULATION

289

pressure equal to 80 mm. of mercury, which was the greatest pressure
used. With the exception of the small initial rise accompanying slight
distention of the lungs,-the fall in carotid pressure below the preéxperi-
mental level of 170 mm. progresses uniformly with increasing lung

distention until the low level of 36 mm. is
reached.

The pulmonary arterial pressure, on the ,

contrary, is not noticeably affected by slight

distention of the lungs, and with increasing '
distention rises continuously until its maximum ,

is attained, which occurs when the increase in

the intrapulmonic air pressure is equal to 60 '
mm. of mercury. Beyond this point the pul- ,,

monary pressure commences to fall. The
maximum increase in the pulmonary pressure
is 40 mm. above the preéxperimental level of
50 mm. and the relatively high level of 90 mm.
is attained.

It will be noted that the pulmonary pressure
rising from a low level becomes equal to the
carotid pressure falling from a high level at a
point where the pressure in the two systems is
equalto85mm. The increase in intrapulmonic
air pressure sufficient to effect this equality is
50 mm. of mercury. With increase in air
pressure in excess of 50 the pulmonary pressure
continuing to rise attains a level 46 mm. above
that of the falling carotid pressure.

An interesting observation is, that both the
fall in carotid and the rise in pulmonary pres-
sures are, within a wide range of lung disten-
tion, approximately linear functions of the
increasing intrapulmonic air pressure.

DISCUSSION OF RESULTS

a

fo”

Fig. 3. Plotted curves
showing corrected carotid
(broken line) and pul-
monary (unbroken line)
arterial pressures during
progressive decrease of
intraplethysmographic
pressure. Ordinates in-
dicate blood pressures.
Abscissae indicate de-
crease of plethysmo-
graphic pressures in mil-
limeters of mercury.

While the pulmonary arterial pressure cannot under usual living con-
ditions be taken as an index of the right ventricular diastolic pressure,
yet, under the condition of completely blocked pulmonary capillaries

the right ventricular pressure must rise part

passu with the rise in

20 40 60 #480 100,

290 - RALPH HOPKINS AND FELIX P. CHILLINGWORTH

pressure in the pulmonary arteries and this rise must be sustained in
the ventricle even during its diastole. Blood under high pressure is
trapped between the blocked capillaries at one end of the system and
the tricuspid valve at the other. When the pressure in the pulmonary
artery equals the maximum which the right ventricle is capable of

establishing, subsequent contractions are unable to discharge blood,

and the ventricle also remains constantly full of blood under a pressure
equal to that in the pulmonary arteries. Even during diastole no relief
from this excessive pressure is obtained since the ventricle cannot,
even though relaxed, increase its capacity for holding blood. It is
even quite possible that under these conditions the pulmonary semi-

lunar valves fail to close, since the normal tendency of the blood to

regurgitate is absent when the ventricle is full of blood at the end of its
systole. In evidence of the diminished output of blood from the right
side of the heart when the pulmonary capillaries are mechanically
blocked by distention of the Jungs, a tracing (fig. 1) is shown in which
the pulse is obliterated in the record from the pulmonary artery while

the mean pulmonary pressure rises. Obliteration of the pulse waves —

occurs in the carotid as well as in the pulmonary tracing but in one
case it is due to insufficient and in the other to excessive pressure. —
When the pulmonary capillaries are completely blocked the pul-
monary arterial pressure may then be taken as a direct measure of the
diastolic right ventricular pressure and it may be assumed that with an
incomplete blocking of the capillaries there exists a rise in the diastolic
right ventricular pressure corresponding in degree to that maintained
in the pulmonary arteries. Two of the tracings shown in figure 2
are evidence of the fact that with moderate distention of the lungs the
rise in mean pulmonary arterial pressure is due to a rise in diastolic
rather than in systolic pressure. In the third tracing of this series it
will be noted that as the pulmonary blood pressure rises the output
from the ventricle diminishes. That the diminution in ventricular
discharge is not due to a diminished supply of blood to the auricle and
‘ventricle was evidenced by many post-mortem examinations which
revealed not only a dilated right ventricle but also an engorgement
limited to the right auricle and adjacent veins in those animals that
were killed by excessive increase of intrapulmonic pressure. It will be
noted in figure 3 that after attaining its maximum the pulmonary
arterial pressure commences to fall. This failure is attributable to
cardiac failure secondary to impairment of the coronary circulation.
Marked fall in carotid pressure necessarily affects the coronary circu-

MECHANICAL IMPAIRMENT OF CORONARY CIRCULATION 291

lation of the entire heart but when the right ventricle is distended dur-
ing its diastole with blood under high pressure, the impairment of
circulation through the cardiac muscles is far greater, in the right
ventricle than in other parts of the heart.

_ Taking the pulmonary arterial pressure as an index of safety during
distention of the lungs, reference to figure 3 shows that with 50 mm.
increase in the pressure of the intrapulmonic air the carotid and pul-
monary arterial pressures become equal and that with increasing air
pressure the pulmonary pressure becomes higher than that in the ca-
rotid artery. The limit of safety, therefore, in dogs lies below this
degree of pressure and death has been found not infrequently to follow
air pressures of 30 and even less.

There is no reason to believe that the pulmonary capillaries of man
are more resistant to occlusion than are those of the dog or that there
exists much difference in their relative blood pressures. Difference in
the elastic factor of thoracic expansion is not important since increase

in the intrapulmonic air pressure acts almost directly on the lung capil-

laries independently of lung expansion. The observations, therefore,
made on dogs require little or no modification to become pertinent to |
artificial respiration performed on man by any.of the methods that
increase intrapulmonic air pressure. When the lungs are rhythmically
distended an important factor of safety is the shortening of the period
of full inflation to allow the blood pressures to return to normal.

CONCLUSIONS

1. When in dogs the lungs are artificially distended by an increase
in the intrapulmonic air pressure:

a. The pulmonary arterial pressure becomes equal to that of the
general systemic arterial pressure when the excess of intrapulmonic
pressure is equal to 50 mm. of mercury.

b. With further increase in intrapulmonic pressure the pulmonary
arterial pressure may exceed that of the general system by as much as
46 mm. of mercury.

ce. Under both of the above conditions the circulation of blood
through the coronary arteries of the right heart is unfavorably
influenced.

d. With the rise in pulmonary arterial pressure there occurs a rise
in the diastolic right ventricular pressure. When the latter becomes
equal to or greater than the systemic diastolic pressure, circulation of

292 RALPH HOPKINS AND FELIX P. CHILLINGWORTH

blood through the coronary vessels of the right ventricle becomes

impossible. pir
2. Estimates of the increase in intrapulmonic air pressure sufficient |

to influence unfavorably the coronary circulation of dogs are appli-

cable also to man and should be considered in connection with methods

of artificial respiration.

We are indebted to Prof. W. E. Garrey for helpful suggestions and
criticisms.

BIBLIOGRAPHY

(1) CHILLINGWORTH AND Hopkins: This Journal, 1920, li; 289.
(2) CHILLINGWORTH AND Hopkins: Journ. Lab. Clin. Med., 1919, iv, 555.

ee ee ae oe ee ae ee aw a

ee ee a ee ee a

VAGUS AND SPLANCHNIC INFLUENCE ON THE GASTRIC
HUNGER MOVEMENTS OF THE FROG.
COMPARATIVE STUDIES IT?

‘T. L. PATTERSON

From the Hull Physiological Laboratory, The University of Chicago, and the
Physiological Laboratory, Queen’s University

Received for publication June 4, 1920

INTRODUCTION

The character of the continuous motor activity of the empty and
filled stomach in the frog has been reported (1). In the present paper
an attempt is made to determine more specifically the influence of the
vagi and splanchnic nerves on the behavior of the gastric hunger move-
ments and the gastric tonus of the empty stomach.

Much has been written concerning the excitatory and inhibitory in-
fluences of these extrinsic nerves upon the gastric motility and a rather
thorough review of the literature covering this phase of the question
has appeared in a previous paper of this series (1). In addition, the
distribution and function of the nerves innervating the visceral and
vascular systems in crocodiles and alligators was worked out by Gas-
kell (2). He found that stimulation of the peripheral end of either
vagus above or below the ganglion trunci vagi invariably led to a con-
traction of the stomach musculature. After section of the cervical
vagus above the ganglion with subsequent degeneration of its fibers
stimulation then above the ganglion almost invariably produced no
effect whatever on the esophagus and stomach, while stimulation below
_the ganglion almost invariably caused marked peristaltic contraction.
Gaskell came to the conclusion that the fibers which innervate the tho-
racic portion of the esophagus and the stomach and all probability the
intestines degenerate only in that portion whichis above the ganglion but
not in that portion below the ganglion and that the nerves for the

1 A preliminary report of this work was made before the 1917 meeting of the
American Physiological Society at Minneapolis, a brief abstract of which was
published in the Proceedings of that Society.

293

294. T. L. PATTERSON

upper part of the esophagus and the inhibitory fibers of the heart have
no connection with the nerve cells of the ganglion trunci vagi, while
the motor nerves for the rest of the esophagus andthe upper portion of
the remainder of the alimentary canal are in connection with the cells
of that ganglion—a connection by which the motor fibers proceeding
peripherally from the ganglion are prevented from degeneration but
not the motor fibers which pass to the ganglion.

Furthermore, the recent observations by Crohn and Wilensky (3) on
gastric behavior by the balloon method have shown that in atony of
the stomach the hunger contractions disappear and in advanced cases
the tonal waves also, while in purely secretory or other functional
disturbances both kinds of waves persist.

EXPERIMENTAL PROCEDURE

The studies in this series of experiments were made upon the large
bullfrog (Rana catesbiana). All the animals were provided with an arti-
ficial opening into the posterior part of the mouth or stomostomy and
the movements of the empty stomach were recorded by the balloon
method as described in a previous paper (4). A series of normal con-
tractions of the empty stomach was obtained from each animal which
extended over a period of several days and then each of these animals
was operated on a second time. In this second operation, either both
vagi or both splanchnic nerves, or both the vagi and the splanchnic
nerves together were sectioned, followed after recovery in each case by
a series of tracings from the empty stomach. The animals were an-

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esthetized. Aseptic precautions as far as possible were at first fol-.

lowed but later this was found to be unnecessary as no infection
developed in any of the animals when such procedure was not followed.
The vagi were sectioned in the region of the neck. Two oblique inci-
sions were made through the skin on either side of the median line,
ventral, about 1 cm. distant and close to the anterior tips of the shoul-
ders as represented by a line drawn from this point laterally 1 em. to
1{ cm. in length to a point slightly posterior and just internal to the
articulation of the superior and inferior maxillary bones on either side.
These two incisions exposed the cervical fascia on either side at its at-
tachment along the anterior scapulo-clavicular borders. Here there
are few blood vessels and if the fascia is carefully separated no hemor-
rhage results. As soon as this region is passed the fascial separation
becomes very easy until the thin sheet of prevertebral fascia is reached

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GASTRIC HUNGER CONTRACTIONS 295

which is about on a line of the transverse processes passing obliquely
downward and inward from the base of the skull and extending into the
thorax. ‘This latter sheet of fascial membrane is now pierced which
exposes the levator anguli scapulae muscle, over the anterior border of
which courses the vagus nerve and the internal jugular (Vena jugularis)
and musculo-cutaneous (Vena musculo-cutanea) veins. The incision
is held open by the spring of a small pair of forceps (preferably curved
points) and then by means of a small pair of mouse-toothed forceps the
nerve is carefully separated from the adjoining veins to which it is
bound by connective tissue. This is best accomplished by freeing the
nerve either between the two veins mentioned or just lateral to the in-
ternal jugular vein at the anterior border of the levator anguli scapulae
where it crosses and sectioning the nerve just below the origin of the

recurrent laryngeal branch. Section of the nerves at this point destroys

not only the gastric branches to the stomach but also the pulmonary
and cardiac branches destined for the lungs and heart. Attempts were

made at first to section only the gastric branches but as these branches

were so small and so deeply embedded in the tissues it was found to be
practically impossible with recovery of the animals. In fact, the tech-
nie as used required several months’ experience before it became per-
fected and only then did it become an. efficient procedure which, if

_ properly handled, may be called a bloodless method. Both vagi were

always sectioned at one operation and the skin incisions were closed
with five sutures.

The splanchnic nerves were sectioned in the region of the coeliac
plexus after laparotomy. An incision was made through the skin, .
the rectus abdominis muscle and the aponeuroses of the external andin-
ternal oblique muscles 25 to 3 cm. in length extending from the lower

extremity of the sternum (xiphisternum) caudalward and about 4 em

to the left of the linea alba, in order to avoid the anterior abdominal
vein (Vena abdominalis) which courses forwards along the mid-line of
the ventral body wall until opposite the liver. The stomach is with-
drawn through this opening and a larger pair of nerves, one on either
side, is found coursing along with the right and left systemic arches.
These are the third spinal nerves carrying fibers for the stomach, but
according to Steinach and Wiener (5), Dixon (6) and others, the stom-

ach also receives fibers from the fourth and fifth spinal nerves and

Waters (7) in addition includes the sixth.
Fibers from these nerves arising from both sides of the body unite
to form the coeliac plexus situated on the coeliaco-mesenteric artery

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 2

296 T. L. PATTERSON

(Ateria intestinalis communis) a few millimeters from its origin from the
left systemic arch. From this plexus arise the nerves destined for the
stomach, pancreas and duodenum. The branches for the stomach are
inbedded in the mesenteric membrane and follow closely the course of
the arterial supply of this organ. They may be best seen by raising the
stomach and allowing the light to illuminate the mesentery when they
may be picked up with mouse-toothed forceps. If they are cut close
to the plexus there are usually not more than two branches. Experi-
ence has shown that it is advisable to introduce into the stomach 5 or —
6 cc. of water previous to the operation as it tends to fill and round out
the stomach, thus making it easier to locate the plexus and the nerves.
The technic for this operation like that for double vagotomy in the frog
is very delicate, but with sufficient patience and experience it may be
developed to such a point as to be conducted without hemorrhage, and
like the former may be considered a bloodless method. The splanch-
netomized stomach is pushed back into place, the muscular incision is
closed with nine to ten sutures and the skin incision with the same num-
ber. The animals after double vagotomy or splanchnetomy are usu-
ally sufficiently recovered on the third day following the operation to
be used for experimental tests with fairly marked gastric activity,
while after a double operation consisting of the two above they are —
usually not ready for use until the fourth day following the operation.
In the decerebration experiments the balloon was not removed from the
stomach and the gastric contractions started again after a short period
of inhibition. All the tracings were recorded on a slowly moving drum
making a revolution in fifty to sixty minutes.

THE INFLUENCE ON THE GASTRIC HUNGER MOVEMENTS OF PARTIAL AND
| COMPLETE ISOLATION OF THE STOMACH FROM THE CENTRAL
NERVOUS SYSTEM

A complete knowledge of the mechanism of the gastric movements
is still uncertain. The gastric activity is regulated not only by the vagi
and the splanchnic nerves of the sympathetic system, but also by the
automatically acting plexi of Auerbach and Meissner. The most direct
and desirable method of attack on this problem is the section of the
extrinsic nerves to the stomach, although this operation abolishes not
only all direct influences from the brain of a motor or inhibitory type,
but also the central reflexes (motor or inhibitory) that may be called
into action through the sensory nerves in the stomach.

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_ The influence of these nerves on the activity of the stomach has been
studied by a number of investigators, prominent among whom have
been Cannon (8) and Carlson (9). Cannon’s observations on the gas-
tric movements of digestion in cats have shown that section of the vagi
leads to a temporary loss of tonus and a slowing and weakening of the
peristalsis, which in respect to rate is practically restored in a few days.
He infers as does Kelling (10) that their function is solely to make the
gastric muscles exert a tension (tonic state) and the result of this condi-
tion is peristalsis. Furthermore, section of the splanchnic nerves does
not affect the movements of digestion, while the combined vagi and
splanchnic section leaves the digestive movements of the stomach prac-
tically normal even shortly after the operation.

Carlson, on the other hand, has shown that section of the vagi in
dogs leaves the empty stomach on the whole permanently hypotonic,
at least for a period up to three months after the operation. Section of

the splanchnic nerves increases the gastric tonus and augments the gas-
_ trie hunger contractions, while the section of both the vagi and the
splanchnics leads to a permanent hypotonus of the stomach, except
under conditions of prolonged fasting. These discrepancies between
the results of the two investigators are probably accounted for, in that
the tonus of the vagus plays a greater réle in the movements of the
empty than in the movements of the filled stomach, or else the nerves
vary in different species of animals.

1. The effect of complete section of the splanchnic nerves. Complete
section of the splanchnic nerves on both sides in the region of the coeliac
plexus was made on twelve frogs and after recovery from the operation
records of the movements of the empty stomach were continued from two
to three weeks and compared with those from the normal stomach of
the same animal. :

When a comparative study of the records of these animals is made as
a whole it is evident that the complete section of the splanchnic nerves
with the vagi intact in frogs increases markedly the gastric tonus and
augments the movements of the empty stomach (fig.1, A andB). The
recorded contractions are small, rapid and irregular in form, and repre-
sent virtually an incomplete or hunger tetanus of the stomach. . In
other words, the stomach on the whole becomes strongly hypertonic
and more active through the destruction of the inhibitory fibers via
splanchnic nerves to the stomach, which permits the motor fibers of
the vagi to exert their full influence on the gastric motor mechanism,
thus leading to a high degree of gastric tonus much above the normal.

298 T. L. PATTERSON

This particular state of excessive tonus is evidenced not only by the
balloon in the stomach cavity but more especially by the marked con-
traction of the esophagus and stomach as is exhibited many times by
the extreme difficulty to introduce the balloon through the esophagus
into the Stomach. This hypertonic condition of the frog’s stomach
which always appears after complete section of the splanchnic nerves is
evidently more marked than Carlson (9) found it to be in dogs after
splanchnic section. Furthermore, this condition as it exists in frogs
after this type of nerve section corresponds apparently to certain clinical

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Fig. 1. Records from the empty stomach of the frog. A, normal frog after
six days’ fast; B, the same animal ten days after section of both splanchnic nerves
and twenty days’ fast. At zx, introduction of 5 ce. of 0.5 per cent solution of
hydrochloric acid directly into the stomach. Showing incomplete tetanus and
only slight inhibition of the hunger contractions by acid in the stomach after
section of the splanchnic nerves. 2x’ = termination of the aeid injection.

conditions as reported by Eppinger and Hess (11) under the term of
vagotonia. According to these observers the antagonistic influences
between the gastric branches of the vagi and the spanchnic nerves play
a very important réle in not only moderating the physiological impulses
which might reach a very marked intensity, but in addition they pre-
vent acute transitions from rest to excitation or vice versa. ‘'Thismeans
that if it were not for the above under certain conditions small stimuli
might cause large reactions either physiological or pathological. Now,

GASTRIC HUNGER CONTRACTIONS 299

if we assume that somewhere in the central nervous system there exists
a common center which controls the antagonistic actions of these two
systems, as suggested by these investigators, and that the irritability of
this center increases and decreases from time to time it is easy to un-
derstand how very weak and even transitory stimuli might act upon
such a center when in a state of increased irritability to produce the
gastric hypertonus through the fibers of the vagi. This however is
not proven, but we do know that in the condition of vagotonia there is a
functional increase of tone via vagi to the stomach and this increase of
function doubtless permits the stimuli to act more readily than if the
reversed condition existed. Furthermore, the observations of Crohn
and Wilensky (3) have shown that the hunger contractions in well-
marked cases of vagotonia exhibit an extreme degree of variability, the
contractions following one another in rapid succession and without
pause for comparatively long periods of time. The clinical findings of
these observers are apparently in accord with the results on frogs after
splanchnic section. |

The inhibition of the movements of the empty stomach of the
splanchnetomized animal when acid is introduced into the stomach cav-
ity is much less complete than in frogs with all the extrinsic’ gastric
nerves intact (fig. 1, A andB). In fact, the contractions do not cease
at all and the only effect produced is a very slight decrease in the height
of the contractions during the introduction of the acid followed by a few

contractions of a slightly longer duration and usually a slight increase in

the gastric tonus. This diminution of the inhibition following stimula-
tion of the gastric mucosa by acids after complete section of thesplanch-
nic nerves is confirmatory with the findings of Carlson (12) on dogs.

2. The effect of section of both vagi nerves. Section of both vago-sym-
pathetic nerves in the neck was made on eleven frogs and after recovery
from the operation records of the movements of the empty stomach
were continued from two to three weeks and compared with those
from the normal stomach of the same animal.

_ When all the records are compared from these animals the results
are confirmatory in showing that the contractions of the empty stomach
are only slightly changed in rate and regularity. The contractions when

viewed as a whole resemble those from the normal stomach with the
exception that they usually appear to be of a slightly slower rate, weaker
and more irregular (fig. 2, A and B). However, there is a tendency
for the contractions to increase in strength or rather amplitude up
to the amplitude of the normal contractions and some of the individual

300 T. L. PATTERSON

contractions may even exceed the normal. This is evidently produced
through a lowered tone in the gastric motor mechanism, whereby the
contractions start rather suddenly and without any marked preliminary
increase in tonus and because of this condition the air is more completely
forced out of the balloon, thus resulting in the greater contraction. In

Fig. 2. Records from the empty stomach of the frog. A, normal frog after
three days’ fast; B, the same animal nine days after section of both vagi and
sixteen days’ fast. At x introduction of 5 cc. of 0.5 per cent solution of hydro-
chloric acid directly into the stomach. Showing slightly more complete inhibi-
tion of the hunger contractions by acid in the stomach after section of the vagi
nerves.

the empty stomach of the normal animal as determined by the balloon
method, there are practically no tonus changes, or at least they are so
slight in degree as to be almost a negligible factor. In working with
these animals, extending over a period of five. years, I have never ob-
served in the normal animal an increase in gastric tone exceeding a cen-

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GASTRIC HUNGER CONTRACTIONS : 301

timeter as determined by the manometric pressure. This maximal
increase I have observed not more than three or four times in my work
during this time and is therefore rare. When tonus changes are ob-
served they usually do not exceed a quarter of a centimeter (2 to 3 mm.),
but the more common thing in the frog is to have the tonus remain con-
stant hour after hour.

Section of both vagi with the splanchnics intact leads to a sympatheti-
cotonic condition of the stomach. This means that the stomach on the
whole becomes hypotonic through the destruction of the vagal fibers
that maintain the gastric tonus, which permits the inhibitory fibers of

_ the splanchnics to exert a greater influence on the gastric motor mech-

anism, thus leading to a general diminution in the gastric tonus. How-
ever, there was a tendency in some of the animals, at least, to show a
gradual improvement in the efficiency of the local tonus mechanism as
time went on after the operation, which indicates that the hypotonic
condition of the stomach may be only temporary in the frog and not
permanent as reported by Carlson (9) in dogs, but corresponding to the
observations of Cannon (8) in cats for the movements of digestion.
This phase of the question will be discussed in a separate paper. Fur-

_ thermore, the gastric tonus on the whole is much lower than normal

as determined not only by the balloon in the gastric cavity, but also by
the ease with which the balloon may be introduced through the esoph-
agus into the stomach and inflated.

When acids are introduced directly into the empty stomach of the
vagotomized animal gastric inhibition is exhibited similar to that pro-
duced in the normal animal with the exception, on the whole, that it’
appears to be quicker and more marked than in the normal animal (fig.
2,A and 8). This is exactly contradictory to Carlson’s results on dogs
(9), yet he states that this was what he expected to find, namely—an
augmentation of the inhibition through the splanchnies after section of
the vagi.

3. The effect of complete section of the vagi tind splanchnic nerves.
Combined splanchnic and vagi sections were made on ten frogs and af-
ter recovery from the operation records of the gastric movements were
continued from two to three weeks and compared with those from the
normal stomach of the same animal. Both sets of nerves were sec-
tioned at the same operation.

After this complete isolation of the frog’s stomach from the central
nervous system the movements of the empty stomach are much the

_ same as when the vagi alone are severed. The contractions show a

302 T.. L. PATTERSON

tendency to approach or even in some cases to exceed the normal,
while at times they may even be identical in rate and character with
those of the intact stomach, but on the whole they are of a slightly
slower rate and more irregular. The stomach passes into a hypotonic
condition similar to that after section of the vagi, and therefore the
slight changes in the movements of the empty stomach after isolation
from the central nervous system must be due primarily to the persistent
hypotonus. These results are in general confirmatory with those of
Cannon (8) on cats and Carlson (9) on dogs.

The inhibition of the movements of the empty stomach by acid stim-
ulation of the gastric mucosa persists after complete isolation of the
stomach from the central nervous system,
but the inhibition like that found by
Carlson (12) in dogs is diminished in in-
tensity and duration. There is a gradual
and slow diminution, both in the rate and
amplitude of the hunger contractions but
as a rule this does not produce complete
inhibition in the frog. The inhibition is

Fig. 3.

Splanchnetomized
stomach superimposed upon the
vagotomized stomach from two
frogs of equal size, weight and

vigor. A, splanchnetomized
stomach. B, vagotomized stom-
ach. E, esophagus. P, pyloric
portion of stomach. Note the
hypertonic condition of stomach
a.

therefore primarily a local reflex deter-
mined by the local gastric mechanism
rather than by the character of the central
innervation or the central inhibition.
Since the type of gastric activity after
complete isolation of the stomach from
the central nervous system does exhibit
the typical movements of the empty

stomach, the primary stimulus to these
contractions is not to be sought in the extrinsic nerves. The extrinsic
nerves (vagi and splanchnics) must therefore be considered under normal —
conditions to play the important réle of modifying or regulating a —
primary automatic mechanism in the stomach wall.

4. Extirpation of stomachs after section of vagi and splanchnic nerves.
Early in the course of the investigation it was observed that the stom-
achs of splanchnetomized and vagotomized animals exhibited rather
wide variations in size, depending on whether the vagi or the splanch-
nic nerves had been previously severed. This phase of the problem was ~
investigated on ten splanchnetomized and ten vagotomized animals and _
the stomachs were removed from three to ten days after the operation, —
directly after the killing of the animals and while the hearts were still —

~~

GASTRIC HUNGER CONTRACTIONS 303

beating. In the selection of these animals care was taken to select —
frogs of equal size, weight and vigor. ‘The results of these experiments
are conclusive in showing that the same general influence which the
vagi and splanchnic nerves exert separately on the gastric apparatus
may be shown when the splanchnetomized stomach is superimposed
upon the vagotomized stomach from two frogs of equal size. The
latter or larger stomach represents the atonic and the former or smaller
the hypertonic, while the normal stomach takes an intermediate posi-
tion between the two (fig. 3). It may be said, therefore, that the re-
ciprocal or contrary innervation of Meltzer which may be termed an-
tagonistic tonus, may be physiological as long as it serves the purposes
of the organ in question in a beneficial manner. It is pathological as
soon as the tonus of one or the other is so exaggerated that the common
welfare of the organ is in danger, and that is exactly what happens in
the splanchnetomized frog’s stomach where the hypertonus of the vagus
leads to a state of over-excitability, or to the Eppinger-Hess condition
of vagotonia.

PSYCHIC OR REFLEX INHIBITION OF THE GASTRIC HUNGER MOVEMENTS

It was suggested to me by Doctor Rogers early in the course of this
investigation that it might be well to study certain cerebral processes
in relation to the reflex effects on gastric activity. Previous work on
other animals has demonstrated that anything which interests, annoys,
frightens or angers, leads to a temporary inhibition of the gastric hunger
contractions probably via splanchnics. Furthermore, the sight or
smell of food in the dog, at least, leads to this same temporary inhibi-
tion if not too often repeated. In order to test further the very im-
portant reflex control of the gastric hunger mechanism, as well as of the
nervous foci in the medulla, mid-brain and cerebrum concerned in the
conduction of sensory and motor hunger impulses, the effects of sound
and light stimuli were made use of in the following experiments. The
observations were made on six frogs which were later decerebrated and
the observations repeated. In the case of the sound stimuli, whistles
of different pitches were sounded for periods of from ten to twenty sec-
onds but these caused only very slight gastric inhibition which was of
short duration and after two or three repetitions it invariably became
ineffective, thus defeating the object of the experiment. Even the filing
of a glass rod on the table containing the animal was fully as ineffective
in producing inhibition although a second factor must have been intro-

304 T. L. PATTERSON

duced, that of vibration. All of these stimuli were of minimal or very
moderate intensity and evidently not of sufficient strength to produce
an effective and constant reflex, or else the central nervous mechanism
for this reflex is at a low degree of development.

Fig. 4. Records from the empty stomach of frogs. A, frog nine days after
section of both vagi with splanchnics intact; B, frog eight days after section of
both splanchnic nerves with vagi intact; C, frog nine days after section of the
vagiand splanchnic nerves. wz to 2, light and darkness shadow test showing tem-
porary inhibition in A; very slight inhibition in B; and total absence of psychic
or reflex inhibition in C, in case of the stomach isolated from the central nervous
system.

The light stimulus, on the other hand, proved to be more effective.
The room was darkened by drawing the shades and while the normal
hunger contractions were being recorded the animal was carefully un-
covered and the bottom of the window shade directly opposite drawn

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GASTRIC HUNGER CONTRACTIONS 305

back and forth quietly and at a moderate rate, thus casting light and
dark shadows upon the animal. This invariably produced temporary
inhibition of the movements of the empty stomach and there seemed
to be no diminution in the degree of the inhibition after repeated trials.
After removal of the cerebral hemispheres there was total absence of
the reflex inhibition and the contractions went on uninterrupted. It

‘would appear that the central nervous mechanism for this reflex was

more highly developed than that for sound. In other words, from the

_animal’s standpoint it may be considered an important defensive reflex .

to warn it of its avian enemies as they soar through the air, thus anxiety
and fear leading to the characteristic temporary inhibition of the gastric
hunger movements.

When the light and darkness shadow test was applied to an animal
after complete section of the vagi with the splanchnics intact it invari-
ably led to a temporary inhibition of the gastric hunger movements
via splanchnic nerves (fig. 4, A).. If the same test was applied to an
animal after complete section of the splanchnic nerves with the vagi
intact it invariably led to only a very slight inhibition of the gastric hun-
ger movements, as represented by a slight and transitory weakening of
the contractions (fig. 4, B). This slight degree of inhibition usually
in evidence after section of the splanchnic nerves is probably due to the
action of the few inhibitory fibers in the intact vagi or to some central
inhibition of vagus tonus. In the case of the stomach completely iso-
lated from the central nervous system (vagi and splanchnic nerves cut)
there is total absence of any psychic or reflex inhibition since the effer-
ent nerve pathways to the stomach have been broken by the sectioning
of all the extrinsic nerves (fig. 4, C).

CONCLUSIONS

1. Complete isolation of the frog’s stomach from the central nervous
system leads to hypotonus of the stomach with about the normal type
of gastric hunger contractions. This is in confirmation with the work
of Carlson on dogs. The automaticity of the gastric mechanism is in-
dependent of the extrinsic nerves but these nerves play an important
role in modifying or regulating the automatic mechanism in the stomach
wall,

2. Partial isolation of the stomach from the central nervous system
interrupts the normal antagonistic balance between the vagi and splanch-
nic systems which may lead to pathological reactions such as vagotonia

306 T. L. PATTERSON

after complete section of the splanchnic nerves. The stomach in this
condition becomes strongly hypertonic while after complete section of
the vagi with the splanchnics intact the stomach passes~into a hypo-
tonic condition.

3. The acid inhibition of the movements of the empty stomach ‘.
stimulation of the gastric mucosa persists after complete isolation of

the stomach from the central nervous system, but the inhibition is

diminished in intensity and duration. When the splanchnics alone
are sectioned the inhibition is even less marked, but after section of the
vagi with the splanchnics intact there is, on the whole, a slight aug-
mentation in the inhibition via these nerves. This latter statement is
contradictory to the findings of Carlson on dogs, while the other facts
above are in accord.

4. The light and darkness shadow test invariably produces psychic or —

reflex inhibition of the gastric hunger movements in the normal animal.
After decerebration or after complete isolation of the stomach from the
central nervous system there is total absence of the light reflex on the
gastric mechanism. When the vagi alone are sectioned temporary
inhibition is the result, while after section of the splanchnics with the
vagi intact there is only very slight inhibition produced.

BIBLIOGRAPHY

(1) Patrerson: This Journal, 1916, xlii, 56.
(2) GasKELL: Journ. Physiol., 1886, vii, 1
(83) CRoHN AND WiLENSKY: Arch. Int. Med., 1917, xx, 145.
_ (4) Parrerson: Journ. Lab. Clin. Med., 1920; v, 674.
(5) STEINACH AND WIENER: Pfliiger’s Arch., 1895, Ix, 593.
(6) Dixon: Journ. Physiol., 1902, xxviii, 57.
(7) Waters: Journ. Physiol.; 1885, vi, 460.
(8) Cannon: This Journal, 1911, xxix, 250; 1906, xvii, 429.
(9) Cartson: This Journal, 1913, xxxii, 369.
(10) Keune: Zeitschr. f. Biol., 1908, xliv, 161...
(11) Epprincer anp Hess: Vagotonia (transl. by Kraus and Jelliffe), New York,
1917, 8, 64.
(12) Cartson: This Journal, 1913, xxxii, 389.

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OBSERVATIONS ON THE RELATION BETWEEN EMOTIONAL
AND METABOLIC STABILITY

FREDERICK S. HAMMETT
From the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania

Received for publication June 5, 1920

The studies of Cannon (1) and his co-workers on the effects of the
major emotions on the physiological activities of the vegetative nervous
system have established and clarified certain fundamental phenomena
of general biological importance that are wide-reaching in their
application.

‘While the primary consequences of emotional stimulation have been
extensively studied, the secondary involvements such as might be
evidenced in the variability of the intermediary metabolism have yet
to be studied. The emotional ignition of the vegetative neural com-
plexes can hardly be supposed to be limited to a circumscribed reaction
but must indeed reverberate throughout the organism as a whole.

It is obvious that it is possible to divide mankind into two main
groups according.to temperament or relative emotional stability, be-
tween the extremes of which there may exist all gradations of suscepti-
bility to emotional excitation. There are those who are relatively
emotionally stable, who pursue the even tenor of their way apparently
and actually undisturbed by the surrounding daily happenings. And
there are those whose temperaments are of the hair-trigger type, whose
emotions are always on tap and who respond to the slightest stimulus
with a magnitude of reaction all out of proportion to the value of the
stimulus received. Whatever the causes of these differences of suscep-
tibility and response as evidenced in the differences of emotional stabil-
ity, the results on the recipient and effector organism must of neces-
sity be widely different. That these responses primarily involve the
vegetative nervous system has been definitely demonstrated. That
they secondarily involve the intermediary metabolism should be
expected since the processes of digestion, secretion, absorption, utiliza-
tion and excretion are all directly or indirectly bound up with the
directing or controlling influence of this part of the nervous system.
307

308 FREDERICK S. HAMMETT

During the past year opportunity was afforded the writer to study -

the chemical composition of the blood of emotionally stable and un-
stable insane and normal persons. The classification of the emotional
status of the individuals presented in this study was arrived at by
consultation with the physicians in actual contact with the patients
and by personal observation over considerable periods of time. The
analyses of the bloods were carried out as reported in a previous com-
munication (2) and for the series under discussion here were made at
weekly intervals on the seventeen subjects reported. The duration of
the periods of observation varied from three to six weeks.

TABLE 1

The coefficient of variability of the blood constituents and the total metabolic
variability of the subjects studied

NON- CREATI-| CRE- uric | AMINO TOTAL
aa es PROTEIN oN NINE | ATINE | ACID ACID aN SUGAR | VARIA-
N N Ny N .N BILITY

7.21 | 12.81 | 14.15 | 5.68 | 12.54 | 14.13 | 7.72 | 47.78 | 7.85 | 129.87
5.07 | 8.09 | 11.23 | 6.56 | 11.15 | 16.07 | 32.23 | 19.19 | 15.51 | 125.10
11.40 | 6.84 | 13.11 | 8.66 | 21.60 | 7.77 | 16.33 | 24.07 | 12.21 | 121.99
11.32 | 10.73 | 22.30 | 5.89 | 6:56 | 15.77 | 16.17 | 18.02 | 6.72 | 113.48
11.70} 8.19 | 8.67] 3.93 | 6.96 | 26.70 | 4.21 | 28.13 | 7.89 | 106.38
8.35 | 7.71.| 14.10 | 4.81 | 14.82 | 13.69 | 10.80 | 23.42 | 8.63 | 106.33
10.65 | 3.99 | 14.67 | 4.97 |.17.50 | 6.43 | 6.27 | 25.54 | 16.00 | 106.02
2.18 | 12.86 | 12.57 | 7.06 | 7.00} 4.94 | 11.71 | 23.48 | 16.91 | 98.71
9.64 | 11.33 | 18.90 | 6.57 | 6.26 | 13.47 | 12.73 | 12.16 | 7.48] 98.54
10 | 3.63] 6.22 | 9.52] 5.83 | 7.32 | 11.59 | 17.97 | 19.19 | 15.87} 97.14
11 | 9.38] 8.87 | 4°69] 1.83 | 5.54 |.13.38 | 5.77 | 39.72 | 3.98 | 92.66
12} 1.08] 2.19] 8.25] 4.31] 5.90 | 7.81 | 3.84] 45.03] 5.75] 84.16
13 | 3.56 | 8.78 | 18.05 | 4.81] 3.64] 9.33 | 3.37 | 19.47 | 12.86 | 83.87
14 | 10.10} 5.22 | 11.98 | 3.71 | 8.80 | 3.95 | 17.32 | 6.06 | 13.16 | 80.30
15 | 9.18 | 6.96 | 11.76 | 6.52] 7.28 | 6.02 | 10.96 | 7.50 | 12.72 | 78.90
16|} 8.73 | 4.18 | 1.43 | 12.61 | 5.74 | 11.00 | 10.16 | 10.84 | 13.02 | 77.71
17 | 17.35 | 4.98 | 5.95 | 4.09 ]10.06] 2.19 | 8.85 | 13.58 | 9.32 | 76.37

COIS HAW HY |

As a basis of correlation between the emotional and metabolic stabil-
ity, the coefficient of variability (3) for each blood constituent deter-
mined for each individual was calculated and the sum of these coeffi-
cients was taken as the total variability of the intermediary metabolism
of the person in question. Table 1 gives these figures for each con-

stituent determined in each subject. The subjects are arranged in the.

order of their decreasing variability.
An inspection of the table shows that while the differences in vari-

ability of one individual from the next is small, there is a very evident

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EMOTIONAL AND METABOLIC STABILITY 309

marked difference between those of highest and of lowest metabolic
instability, and this marked difference is correlated with a marked
difference in emotional reactivity if we consider the subjects seriatim
from the psychological point of view.

Number 1 is a male nurse of the small nervous type, eins upset by
minor occurrences and with a continual attitude of worry. Number 2
is a female patient, who although being completely oriented, varies in
her emotionalism from deep depression with decreased psycho-motor
activity to a wild hilarity and excitement. Number 3 is a male patient,
restless, talkative, active and excitable, showing much exhilaration and
flightiness. Number 4 is a male patient classed as an agitated depres-
sive and who has firmly fixed somatic and autopsychic delusions.
Number 5 is a male nurse, irritable, suspicious and touchy, possessing
neither decision nor attention. Number 6, the last of those showing a
total variability of over 100, is also a male patient, restless, irritable
and excitable, showing considerable emotional elation. The next two
members of the series are also of the emotionally unstable type, number
8 being a female patient showing considerable perturbation and violence
accompanied by motor activity, screaming, laughing and _ hallucina-
tions, while number 9 is a male presenting a history of hypersensitive-
ness, and who becomes apprehensive under examination, is easily
depressed and worries, although at other times he is more cheerful.
All these individuals so far described, then, can be validly considered
as persons of varying emotionalism and of obvious emotional instability.
They are also individuals whose metabolic variability is of a relatively
high grade, as can be seen from the table. Turning now to the
remainder of the subjects the next on the list, no. 10, shows a meta-
bolic variability that is practically the same as found in the latter
members of the preceding group. Yet his emotional status to all
appearances is one of relative stability as far as can be determined.
He is pleasant and agreeable and inclined to be seclusive. He is quiet —
and not irritable. An inspection of the figures obtained for the meta-
bolic stability from now on shows not only gradually decreasing values
but also values definitely and markedly lower, as a group, from those
preceding. Number 11, a male patient, is quiet and sits as though in
deep thought. He is of rather an even temperament, occasionally
becomes angry, but is usually able to control himself. Number 12 is a
male patient, quiet, maintaining a given posture for some length of
time, sits rigidly in a chair and stares vacantly into space. He is
indifferent toward his surroundings. Number 18 is a female patient

310 FREDERICK S. HAMMETT

who is emotionally apathetic and indifferent. Number 14, a male
patient, is at times excitable but is only apparently superficially dis-
turbed since he eats regularly and well. Number 15, a male patient,
was in a catatonic stupor throughout the period of observation and
obviously was not emotionally variable, to any determinable extent.
Number 16, another male patient, is never excited but is always indif-
ferent, apathetic and seclusive. Number 17, the last of the series, is a
male nurse, the emotional antithesis of number 1. He is phlegmatic
and inexcitable, paying no attention to the ordinary little vicissitudes
of life.

From the psychological point of view it is evident. that these latter
individuals present the appearance of being relatively emotionally
stable. As a group they are generally inexcitable and are not roused
to demonstrable emotional reactions by circumstances which act as
stimuli causing the marked response of the first group of high metabolic
variability.

This relation between a relatively high metabolic stability and a
low grade of emotional reaction, and between a relatively low meta-
bolic stability and a condition of temperamental excitability is by no
means claimed to be exact or quantitative. Nevertheless the data
seem to indicate such a tendency.

The logical conclusion to be drawn from this comparison is that
larger variations in intermediary metabolism are prone to accompany
conditions of ready emotional response of a marked nature to disturb-
ing stimulation, and that on the other hand the variability of the
intermediary metabolism in individuals who are less susceptible is
liable to be relatively low. .

A broader application of the tendency here demonstrated can be
made if for a moment one compares the physical condition of the
so-called emotional type of individual with his more phlegmatic and
less responsive emotional opposite. The former usually presents a
picture of deficient nutrition, the latter is in most cases well supplied
with the anabolic products of metabolism. The metabolism of the
one by its wide variability gives indications of the possibility of there
being at one time an overtaxing of the organism, and at another time
of the organism lacking a sufficient energy supply. The other type is
relatively more metabolically uniform. His metabolism consists of a
balanced give and take, in which no undue strain is put on the cata-
bolic processes nor is there a lack of sufficient material to supply the
anabolic needs. The results of these processes are shown in the end

EMOTIONAL AND METABOLIC STABILITY 311

_ products, as indicated by the figures for variability here presented,
_ and the causes are fairly attributable, other factors being absent, to
_ the relative magnitude and type of emotional response of the indi-
vidual to the incident stimuli.

Such a conception while lacking complete demonstration is never-

theless supported by correlated observations and fits in not only with
_ the observations recorded here but also with the general hypotheses of
_ Cannon (1) as to the susceptibility of the organism to respond to the
effects of emotional stimuli by disturbances of the vegetative nervous
_ system.

BIBLIOGRAPHY

1 (1) Cannon: Bodily changes in pain, hunger, fear and rage, New York, 1915.
_ (2) Hamnerr: Journ. Biol. Chem., 1920, xli, 599. |
_ (8) Davenport: Statistical methods, New York, 1904.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 2

FOUR FACTORS CAUSING CHANGES IN THE TYPE OF

RESPONSE OF THE ISOLATED INTESTINAL SEGMENT
OF THE ALBINO RAT (MUS NORVEGICUS ALBINUS) TO
SODIUM CARBONATE

S. HATAI anv F. S. HAMMETT .
From the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania

Received for publication June 12, 1920

The usual response of the isolated duodenal segment from the albino
rat intestine is that of shortening or contraction when stimulated by the
application of weak solutions of sodium carbonate. Occasionally how-
ever segments are encountered that exhibit irregularities in the type

of response. One may fail to react to the stimulating substance, an- —
other may answer by a preliminary slight contraction which is followed _

by a relaxation below the original tone level from where the response
was elicited, while a third may undergo a prompt and decided relaxa-
tion. This last is the irregularity most frequently encountered.

This occasional inconstancy in reaction of segments from apparently
normal animals which showed itself at times as a complete reversal
of the type of response demanded investigation because it was neces-

sary for our purposes to have rats on hand the intestinal segments of —
which could be relied upon to give uniform and consistent reactions to —

stimulation by weak sodium carbonate solutions.

The inconstant responses could not be attributed to variations in the —
technique of preparation of the segments nor to the carrying out of the —
tests since this was always uniform. The control of the rats up to the ~
time of killing with respect to age, sex, health, heredity and diet was —
also in the hands of the authors and consequently these factors were

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regulated. The preliminary observations which gave satisfactory —

results had been made on rats which had been for some time in the lab- ~
oratory cages. Irregularities first began to appear when animals were —

used before they had become accustomed to the laboratory cages.

Donaldson (1) having reported that the domesticated albino rat is q
extremely sensitive to changes in environment, we began to think that 2
possibly the change from the colony house to the laboratory had in- —

312

INTESTINAL RESPONSE TO SODIUM CARBONATE 313

duced an excitement causing the variations. This idea that excitement
was the cause of the irregularities was considerably fortified when the
segment taken from a rat which had resisted capture and had become
quite agitated gave a marked relaxation on stimulation by carbonate.
The test of this hypothesis was easy.

The method of procedure for the preparation of the segments for
testing was as follows. The rat was first put under light ether anesthesia
and then killed by crushing the cord in the cervical region. The duo-
denal portion of the intestinal tract was then removed without stretch-
ing, and cleaned of mesenteric fat. A segment about 1.5 cm. in length
was cut from the gastric end of the duodenum and suspended by silk
threads in a glass cell containing 4 cc. of Tyrode’s solution kept at
body temperature and through which oxygen was continually passing.
One end of the segment was attached by a thread to a support within the
cell and the other end was connected with a light lever writing on a
slowly moving drum. Rhythmical contractions immediately appeared
and were recorded for one revolution of the drum (about 10 minutes)
by which time the tone level had become uniform and consistently
parallel with the base line.

With segments prepared in this way the addition of 0.25 or 0.50 cc.

_of an M/10 solution of sodium carbonate to the Tyrode’s solution in

the cell usually caused a shortening of the segment with a consequent
rise of the curve of rhythmical contractions as is shown in the tracings.
It was possible to obtain this type of reaction from one and the same

- segment for several successive applications of the carbonate. After each

application the cell and segment were thoroughly washed with oxy-_
genated Tyrode’s solution kept at body temperature. After washing
the segment as a rule came back to the original tone level within one
revolution of the drum and was then ready for another test. The tests
as presented in figures 1 to 5 were obtained in this manner.

In order to determine whether the excitement of the rat brought
about either by change of environment or by other means was a factor
in causing the irregularities previously described, a number of male —
rats of the same age and on the same diet were brought to the laboratory
cages and allowed to become accustomed to the new environment dur-
ing three or four days. When a segment from one of the lot was pre-

- pared as usual and tracings of the effect of the sodium carbonate stimu-

lation recorded the normal reaction was invariably obtained. When
others of the same lot were annoyed by various methods just before
killing and similar tracings made the irregularity of reaction occurred

S. HATAI AND F. 8. HAMMETT

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INTESTINAL RESPONSE TO SODIUM CARBONATE 315

in practically every case and usually took the form of a reversal of the
type of response as shown in figure 1. Occasionally however an animal
was found which did not appear to be susceptible to the preliminary
excitation. Such an one is shown in figure 2, rat C. Nevertheless the
relative frequency of the irregularity was such as to establish the hy-
pothesis that excitement is one of the factors causing variation in the
response of the isolated intestinal segment to sodium carbonate stimu-
lation. In this connection it should be noted that although relaxa-
tion occurs in the segments from excited animals, this type of response
gives way to the normal type after several applications of the sodium
carbonate.

The results described in the iniines paragraphs were obtained from
rats close to one hundred days old. Desirous of not limiting our ma-
terial to animals of this age, we attempted to continue the observations
on rats one hundred and fifty days old. Here it was found that while
evidences of irregularities were present in rhythm, amplitude and base
line level, the type of response was-usually a normal contraction. When
we used a series of rats some two hundred days old it was found that
these older animals were quite insusceptible to the effects of the pre-
liminary excitation as indicated by the response of the duodenal seg-
ment to carbonate stimulation. This is plainly shown in figure 3.
There were no exceptions. It is accordingly evident that the disturb-
ing effect of excitement is modified by age and that immaturity is one
of the factors causing susceptibility to excitement with resulting irregu-
larities of response.

The exclusive use of male rats has certain obvious disadvantages.
With this point in mind several series-of tests were made using as con-
trols undisturbed males and comparing with them females of like age
and conditions of environment and diet. It was found that-when fe-
male rats were used the response of the segment to the sodium carbon-
ate stimulation was normal if the animals were not menstruating.
When menstruating, however, as evidenced by congestion of the uterus,
variability of response was uniformly obtained and of the same type as
that given by segments from the young disturbed male rats. This is
shown in figure 4. Hence menstruation is a factor preventing the uni-
form response to carbonate stimulation and the test demonstrates
that female rats during sexual activity are not suitable material for
general use.

The observations up to this point contribute further support to Can-
non’s (2) theory of the influence of emotional reactions on the vegetative

HAMMETT

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HATAI AND F.

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INTESTINAL RESPONSE TO.SODIUM CARBONATE 319

system. They also bear out with more exactness the observation made
by Alvarez (3) that the intestine of younger animals (rabbits in his
experiments) tends to be more unstable than does that of the older.

It seemed desirable to determine the immediate cause of these phe-
nomena. In view of Cannon’s (2) studies, the conception of an endo-
crine origin of the differences has a certain plausibility. Against this
notion is our observation that we were unable to obtain any but a
normal response to carbonate stimulation from the segment from the
normal animal after application zn vitro of extracts of the homologous
adrenals, hypophysis or thyroid. The dependence of the normal mode
of movement of the intestine on the integrity of Auerbach’s plexus as
demonstrated by Magnus (4) and the hypothesis that emotional dis-
turbances spread to the vegetative system, in part at least, through the
splanchnics, led us to study the effect of stimulating the superior splanch-
nic. The rats used were all males of the same age and living in the
same conditions. The tracings obtained are given in figure 5. The
tracing A was made by the isolated segment of an undisurbed rat when
stimulated by M/10 sodium carbonate. It is normal. The tracing B’
was made by the segment from a rat that had been annoyed and shows
the characteristic effect of excitement on the response. Tracing C
was made bt the segment from an undisturbed rat which had been opened
immediately after killing and the splanchnic nerve electrically stimu-
lated before removal of the segment. It is quite evident that in this
case the preliminary splanchnic excitation has induced changes in the
intestine of a nature that has caused the segment to respond as do seg-
ments obtained from young emotionally excited males. Splanchnic
irritation is therefore a fourth factor in causing irregularity in response
to sodium carbonate stimulation. The unusual height to which the
curve rises is noteworthy although no explanation can be offered for
the intense response after the preliminary relaxations. This relaxa-
tion on the application of the sodium carbonate solution to the duodenal
segment isolated after splanchnic stimulation is similar in type to the
relaxation of the intestinal segment of the intact animal after splanch-
nic stimulation alone, as obtained by Bayliss and Starling (5), and is
very suggestive. |

Of the four factors so far investigated it seems as if the last revealed
the path for the expression of the other three. There is at present no
reason to suppose that the splanchnic stimulation could so affect the
adrenals as to cause a secondary reaction through the mediation of
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INTESTINAL RESPONSE TO SODIUM CARBONATE 321

tion at the time stimulation began. And further, we showed earlier
that adrenalin did not modify the response to sodium carbonate. Con-
sequently we are inclined to the opinion that the irregularities of re-
sponse observed are due to disturbances of the intestinal plexus.

SUMMARY

Whese experiments demonstrate that the normal response of an iso-
lated duodenal segment of the albino rat to sodium carbonate stimula-
tion is one of shortening or contraction but that certain factors may
modify the type of response.

These irregularities of response of the segments from young, healthy
male rats are due to a condition of excitement induced in the animal by
change of environment or rough handling just before its use for experi-
mental purposes.

‘The susceptibility of the intestinal segment to such external disturb-
ances is modified by age in that material from rats of two hundred days
fails to show the irregularities after excitation.

Female rats are not suitable subjects for general studies of this nature
inasmuch as the act of menstruation sets up such changes in the intes-
tinal segment as to cause it to respond in a manner analogous to the
segment from young excited male rats.

The electrical stimulation of the splanchnic nerve of a normal undis-
turbed male rat before the removal of the segment results in the produc-

, tion of a similar type of response to carbonate stimulation as is that

obtained from the segment of the excited rat of the same age. This
inclines us toward the hypothesis that the irregularities of response

here observed are due to disturbances of the intestinal plexus.

CONCLUSION

It is possible to obtain an intestinal segment which will give a uni-

form and consistent response to sodium carbonate stimulation. The

gastric end of a duodenal segment satisfies these requirements when
taken from healthy male adult albino rats eighty to two hundred days
old some fifteen hours after the last feeding and in which no emotional

disturbance has been induced by recent changes of environment or

rough handling. Excitement, age, menstruation and electric stimula-
tion of the splanchnic nerves are factors tending to cause changes in
the type of response of the segment to sodium carbonate.

(2) Cannon: Bodily shacks) in pain, a] mia and re
(3) ALVAREZ: This Journal, 1914, ani 177.

THE ADJUSTMENT OF BLOOD VOLUME AFTER INJECTION
OF ISOTONIC SOLUTIONS OF VARIED COMPOSITION!

ARTHUR H. SMITH ano LAFAYETTE B. MENDEL

From the Sheffield Laboratory of Physiological Chemistry, Yale University,
3 New Haven

Received for publication June 12, 1920

Water plays such a large part in the physical regulation of funda-

mental physiological processes as well as in the chemical transforma-

tions in the body, that the lack of suitable amounts or the abnormal

- distribution of fluid in the body may be correlated with distinctly path-

ological conditions. Volume changes in the body take place only
through the movement of water. .

The various types of edema illustrate what may happen when there
is an abnormal distribution of water in the body. Besides the edema
associated with cardiac and renal diseases we may add another type
known as nutritional edema. The etiology of neither. of these condi-
tions is clear. |

There are also conditions in which water is lost from the body to
such an.extent that desiccation takes place and the blood becomes ex-
ceedingly concentrated. Such a condition prevails in Asiatic cholera
in fatal cases of which Rogers (1) has reported a serum loss of 62 per
cent. Intravenous injection of isotonic saline solution in such cases is
at once followed by diarrhea with the resulting loss of all the added
water. In war gas poisoning Underhill (2) has described the marked
loss of water from the blood and the movement of water into the lungs.

The mechanism of the loss of heat from the body is largely physical
in character. Balcar, Sansum and Woodyatt (3) have reported experi-
ments in which they were able to produce fever at will by intravenous

injections of hypertonic glucose solutions provided diuresis ensued.

Temperatures of 109°,111° and 126° were thus obtained in dogs. These
authors have suggested that in the body the water may be either
“free” or “‘combined.” The “‘free’’ water is available for evaporation

1 The data in this paper are taken from a dissertation presented by Arthur H.
Smith for the degree of Doctor of Philosophy, Yale University, 1920.

323

324 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL

with the consequent heat loss while the water combined with the col-
loids of the body is not available for heat regulation. Any condition
which increases the “combined” water or decreases the ‘free’ water
leads to a rise in temperature due to the absence of proper heat loss.
Lussky and Friedstein (4) reported that in cases of pneumonia ending

in crisis in infants, there was a decrease in body weight with. crisis due —

to loss of water.

The large amount of raters in the cell may aid considerably in main-
taining the optimum temperature of the cell, for water has a high specific
heat. The large percentage of water in tissues in which oxidation is
most intense, may be corrélated wih this unique property of acting as
a heat buffer.

Another evidence of water movement in the body has recently been
reported by Weed and McKibben (5). They found that the brain in-
creased in volume after injection of hypotonic salt solutions or distilled
water and decreased in volume after hypertonic solutions—these
changes being independent of vascular changes and of the cerebro-
spinal fluid pressure. The remarkable clinical study of Cushing (6) on
the alteration of brain volume after administration of hypertonic salt
solution per os indicates that the brain is peculiarly sensitive to the
movement of water in the tissues.

The question naturally arises as to the physical character of water
in the body. By drying blood one finds that it contains nearly 81 per
cent of water. Butis this water in the form with which we are familiar?
In health-some of this water is in such a form that it can be given off as
vapor in the lungs and can be appropriated by the sweat glands and
given off as water. When one considers, however, that in the blood and
in the cells there is really a solution of protein and lipoid in aqueous so-
lution of salts, it becomes apparent at once that the physical chemical
relationships of water to the other components of the body fluids are
exceedingly complex. The hydrophilous colloids probably take up
water by adsorption on the internal or disperse phase. This adsorbed
water then dissolves in the substance of the particles of the colloid.
Large amounts of water are thus taken up by gums, proteins and other
emulsoid colloids with the result that swelling takes place.

This imbibition of water is dependent on a variety of conditions such
as hydrogen ion concentration and nature of the salt present. It is
to the study of some of these conditions on the passage of water out of
the circulation that the present investigation is devoted.

a ee ee ae ee

ON Re Te Re ETS a Se eT Se eee EES ieee

ADJUSTMENT OF BLOOD VOLUME 325

in the study of the application of physical laws to the behavior of colloids,
much importance has been attached to the early work of Hofmeister and his
pupils. Lewith (7) studied the efficiency of various ions in precipitating the
serum proteins. The arrangement of his series in the ability to precipitate the
proteins was as follows: nitrate<chloride<acetate<sulfate. Hofmeister (8)
studied the comparative efficiency of anions in salting out egg globulin and
obtained the following series: sulfate>phosphate>acetate>citrate>tartrate>
bicarbonate>chromate>nitrate>chlorate. In a later series of experiments (9)
he found that the order of efficiency in precipitating gelatin was sulfate> citrate
>tartrate>acetate>chloride>nitrate>chlorate. For the precipitation of col-
loidal iron the series was practically the same. When he studied the swelling of
gelatin discs in various solutions, Hofmeister (10) obtained the following order
of efficiency: sulfate, tartrate, citrate<acetate<chloride<chlorate, nitrate,
bromide, in prompting the swelling of the discs.

Pauli (11) found that different salts raised the coagulation temperature of
proteins to different degrees and the order of efficiency was citrate, sulfate<
chloride< iodide <acetate<chlorate<nitrate<bromide. Lillie (12) reported
that the order of salts in decreasing the osmotic pressure of gelatin and egg albu-
men was sulfate, tartrate, citrate, phosphate, ferricyanide> fluoride, chloride,
nitrate, chlorate>bromide>iodide> sulfocyanate.

Studies of the same series of salts applied to ‘ ‘living”’ colloids, i.e., to the
protoplasm or membrane of cells, have in many cases shown a somewhat similar
order of activity. Hd6ber (13) found that the order of anions for stimulating frog
muscle was sulfate<chloride<nitrate <bromide<iodide<sulfocyanate. Like-
wise the order of efficiency of the anions in hemolyzing blood cells in slightly
hypertonic solution was sulfate < chloride <bromide<nitrate<iodide. Lillie (14)
has pointed out that those salts that are good activators for Arbacia eggs, increase
the surface permeability of the egg and the order of efficiency of the salts is
chloride < bromide <nitrate<sulfocyanate<iodide. Héber (15) studied the ab-
sorption of salts from the small intestine. Sodium chloride was absorbed faster
than sodium sulfate and for the series he found Stand Hae een tOch
sulfate in speed of absorption.

Reviewing the evidence it seems that the sulfate, citrate and tartrate iad
always to decrease the permeability, to increase the precipitability of protein,
to lessen the imbibition of water, while chloride and acetate assume a middle
position and nitrate, bromide, iodide and sulfocyanate tend to increase the per-
meability, to increase the imbibition of water and to lessen the precipitability of
protein. In other words, as Spaeth (16) has pointed out, the order of dispersion
of hydrophilous colloids by anions is sulfate<tartrate<citrate<acetate< chlo-
ride < chlorate < bromide <iodide<sulfocyanate which is the same order in which
these ions produce their deleterious effect on cells.

The method employed in the following series of experiments consisted
in the sudden increase of the blood volume to twice the normal volume
and the measure of subsequent volume changes by means of hemoglobin
determinations. The injected fluid was an isotonic solution of the
salt under consideration or, if the toxicity of the substance prohibited,
the maximum non-lethal dose made isotonic with sodium chloride.

326 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL

Healthy rabbits in good nutritive condition were used in all the ex-
periments. The solutions were isotonic with rabbit blood, the osmotie
pressure being controlled by freezing point determinations, They were
kept at the proper temperature in thermos bottles and injected at 37°C.
The injection was made during ether anesthesia through a cannula into
the left jugular vein from a graduated cylinder by means of pressure at
such a rate that in two minutes a volume of fluid equal to the estimated
blood volume of the animal was introduced. In some cases second and
third injections were made. In the early experiments the figure 50 ce.
per kilo (ef. Boycott, 17) was used as the normal blood volume of the
rabbit but later the value a of the body weight was used.

From a cannula in the right carotid, samples of blood were taken
before injection, immediately after injection and thereafter at five-

minute intervals for one half hour followed by ten-minute intervals .

until the end of the experiment. The few drops of blood required for
the analysis were caught in depressions of a paraffin plate from which
measured volumes were immediately taken for the hemoglobin deter-
minations. In the early part of the work these were made by the
method of Haldane (18) using a standard whose oxygen capacity was
determined by the method of Barcroft (19). The hemoglobin deter-
minations made later were done by the method of Cohen and Smith (20).

The bladder of the rabbit was emptied by squeezing before the ex-
periment and all of the urine voided during the experiment and that
remaining in the bladder on autopsy was collected and measured. At
the conclusion of the experiment the animal was killed and autopsy
made. ast :

The term relative blood volume is used to mean the ratio of the hemo-
globin percentage after the injection to that before the injection. The
method of calculating it is as follows: assuming the original volume to
be 100, if the hemoglobin value before the injection is 80 and after it is

50, the relative blood volume after injection is = xX 100 = 160. These

figures for relative blood volume, then, are really the percentage of the
normal blood volume based on the hemoglobin content.

The use of hemoglobin determinations as an index to the blood volume changes
requires special consideration. This is‘a valid index only if, under the conditions
of the experiment, there is a negligible variation in the erythrocyte count due to
extrusion of new red cells into the blood stream. Increases in the red count and
hemoglobin have been reported in a variety of conditions. Muscular contrac-

EC ee ee ee ee

—— i a

_ gated the factors producing polycythemia.

TABLE 1

ADJUSTMENT OF BLOOD VOLUME

327

tion (21), exercise with perspiration (22), increase in blood pressure (23), and
_ decrease in atmospheric pressure (24), have all been reported as conditions under
_ which there is an increased red count and hemoglobin. Lamson (25) has investi-
He found that in some cases hemor-

_ rhage will produce an increased blood count shortly after the blood is lost.

Comparison of variation in hemoglobin with that of total solids before and after
; injection of isotonic salt solution

: aprons | | aren | mppvoren
q per cent per cent per cent
_ Experiment 30
4 MN ce ee i a eo re 90.0 73.0. 81.0
sc pitas ais hey Ve cv ale ka kgs es 16.0 13.3 83.0 -
oe Gmlormes Of whole blood.................... 0.42 0.36 | 86.0
_ Experiment 46 | |
Sensehiay.:, S20 ik uivls.. Gals lah side 67.0 56.0 16.4
) I Seceibiies tha Gwraddh's Hs « <'- Faws es +» 13.8 11.9 13.4
_ Experiment 48
a EE Te Si ON a a 75.0 64.0 14.6
A aS de el a a ee 14.7 12.9 12.2
TABLE 2
The effect of repeated small hemorrhages on hemoglobin percentage
TIME INTERVAL FOR EXPERIMENT NUMBER
REMOVAL OF
SAMPLES CA. 1-2 cc. 40 50 51
minutes
Normal 67 89 65
5 65 90 65
10 62 90 65
15 60 90 66
20 59 90 65
25 59 90 66
30 59 90 67
40 59 89 66
50 59 90 65
60 59 89 55

_ might be questioned.

_ ‘THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 2

a Among other factors he mentions emotional excitement and anesthesia. If these
_ factors were to play a part in our experiments the validity of the results obtained

Lamson quotes figures illustrating the increase in red count in a cat after fright.

328 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL

ately after anesthesia with ether the red count is above norma] but that it grad-
ually falls: It is conceivable that there may be an increase in red count without
an increase in hemoglobin.

Experiments were carried out to determine whether or not the hemo-
globin could be used as an index to blood volume under the conditions
of the procedure outlined above and also whether the hemoglobin per-.
centage changed as a result of excitement and anesthesia in these experi-

ments. The results (tables 1 and 2) show that not only did anesthesia, © ;

fright and withdrawal of successive small volumes of blood not change
the hemoglobin percentage, but that the hemoglobin percentage varied
in a parallel manner with the total solids. It seems reasonably certain,
then, that under the experimental conditions outlined, the hemoglobin
is a valid and useful index to blood volume changes. :
- Inasmuch as glycosuria has been observed following intravenous in-
jections of salt solutions, it seemed of importance to determine whether
or not there was hyperglycemia which, of itself, might exert some in-
fluence on the dilution of the blood. In the present experiments there
was no glycosuria following the injections except with colloidal silver.
In one experiment (exper. 24) when sodium sulfocyanate was injected,
the blood sugar was 0.075 per cent before anesthesia, 0.112 per cent
after anesthesia and 0.209 per cent one half hour after the injection.
At this time the relative blood volume had returned to normal so that
it may be said that such hyperglycemia as follows intravenous injection
of salt solutions, exerts no appreciable effect on blood volume. This
point needs, however, further investigation. Me
Experiments with salt solutions. The following solutions were used:
Sodium chloride 0.98 per cent, sodium acetate 1.23 per cent, sodium
bromide, 1.8 per cent, sodium nitrate 1.5 per cent, sodium sulfocyanate
1.36 per cent, sodium sulfate 2.0 per cent, sodium tartrate 1.5 per cent
in 0.45 per cent sodium chloride and sodium citrate 0.26 per cent in 0.9
per cent sodium chloride. These solutions were isotonic with rabbits
blood. The injection was rapid—a volume equal to the blood volume
being introduced in two minutes. In the case of the chloride, bromide
and sulfate in some of the experiments, the respiration was slower and
more labored during the injection. In the ease of tartrate the respira-
tion was quickened. With the citrate there were muscular spasms.
From table 3 it will be seen that the salts differ somewhat in their
influence on the passage of water out of the circulation. With citrate,
tartrate and sulfate the relative blood volume stayed above normal —
longer than in any other case; with the chloride the return to normal

ADJUSTMENT OF BLOOD VOLUME . Bag

was more rapid while with the bromide, acetate and nitrate the return

to normal blood volume was most rapid. Except in the case of the
citrate, tartrate and sulfate, however, the differences were negligible.
- In all of these experiments the larger part of the injected fluid dis-
appears within the first five minutes and a considerable part of it leaves
the circulation during the injection (17). Immediately after the in-
jection there is an enormous increase in the filtration pressure in the
capillaries. This increased pressure accounts for the rough parallelism
_ in volume changes between all the salts within the first five minutes.
after injection.

TABLE 3

5 Regulation of blood volume after injection of solutions of various sodium salts;
average results of all experiments; relative blood volume

SODIUM SALT
TIME INTERVAL ;
Acetate | Nitrate | SUMO" | Bromide Chloride] Tartrate| Sulfate | Titrate
minutes
Normal 100 100 100 100 100 100 100 100
Immediately 164 166 158 142 141 » 148 148 137
5 116 130 128 119 | 118 123 124 119
10 108 118 117 111 7 114 118 116 120
15 104 108 111 109 112 115 112 116
20 104 108 107 105 109 110 112 115
25 104 106 106 104 106 110 114
30 101 103 104 104 105 | 111 112 (114
40 100 100 102 101 102 111 112 114
50. 100 102 102 111 109
60 . 107

The differences in behavior between the citrate, tartrate and sulfate
and the rest of the salts became apparent later in the experiment, i.e.,
“ twenty-five or thirty minutes after the injection. The effect of the
given salt on the diffusion of fluid is then seen. On the basis of the
present experiments the salts can be arranged in the order of their ef-
fect in permitting the passage of fluid out of the circulation, as follows:
_ —acetate, nitrate, sulfocyanate, bromide > chloride > tartrate, sulfate,

citrate.
_ The physical chemical systems in the blood vessels immediately after
- introducing the injection fluid are very complex. In the blood the

_ plasma proteins, the red corpuscles and lipoids occur suspended in, or

_ dissolved in, a water solution of various inorganic salts. It is obvious

330 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL

that the injection fluid will first react with the substances in the blood
insofar as it is capable of producing any change in the equilibrium there.
Since proteins are ionized and obey the law of mass action (26), (27) a
chemical readjustment in the blood stream after the injection of the
salt solutions may be expected. Hence the character of the fluid thus
established in this newly constituted system may be sufficient to account
for the differences of rate with which fluid leaves the circulation after
the injeetions of various salt solutions.

Again, although we were very careful to have the solutions isotonic
when injected, it does not follow that the osmotic conditions in the
blood stream remain unchanged after the addition of the salts. There
may have been some degree of ion-colloid readjustment or even double
decomposition between inorganic salts which might change the osmotic
pressure of the blood temporarily. The calcium precipitants, especially,
might act in this way.

A third possible factor in regulating the speed with which the added
fluid leaves the circulation is the “permeability” of the membrane form-
ing the capillary wall. It is obvious that most of the investigations of
the permeability of isolated plant and animal cells can not be applied
to the capillary endothelial membrane except in a very general way, for
the proteins and especially the salts of the plasma constitute, as can be
inferred from the above discussion, a very effective system for main-
taining not only a constant reaction but also a constant balance of salts
and colloids. It is because of this regulating mechanism that the dif-
ference between the salts used in these experiments was so small when
judged by their effect on the movement of fluid out of the circulation.
The sulfate, tartrate and citrate action was more pronounced because
of their double effect: they may have reduced the water-holding ca-
pacity of serum proteins either through formation of less ionized com-
binations or through purely physical means; and secondly, they may
have decreased the permeability of the capillary membranes so that the
free movement of surplus fluid out of the circulation observed with
the chloride, for instance, was interfered with. It is possible that the
combination of the above mentioned factors resulted in the diminished
speed of return of the augmented blood volume observed after injection
of solutions containing sulfate, tartrate or citrate.

The analogy between the action of the various salts on the capillary
membranes and on other membranes is suggestive. Magnesium sul-
fate, sodium sulfate, magnesium citrate and sodium potassium tartrate,
saline cathartics containing anions which appear at one end of our series

ee ee ee ee ™_.

ADJUSTMENT OF BLOOD VOLUME 331

Relative
Bhod Volume
Flood Volume
/60 lat egulalion in Habbils

afler Lnyection of
Various Salf Solutions

| \ Sodium N. Irale
Sddium Suffocyanas

j .
gis
/20 ‘
WIS wae
/ A
$odsumLrbad Fat ag

te eae WE a as

}
! Sodium Ack tate 7 ie ‘Go PK

— 105 |

wl

Se
Se
es —>
aS 70.6) 18 BO. ZR GOn., FS FO

7ime in Minules
Chart 1

332

ARTHUR H. SMITH AND LAFAYETTE B. MENDEL

| frelalive

Blood Volume

/60

/55

/50

Blood Val ine

Ftegulation in Habbits
afler [nyeclion oft
Various Salt Solutions

1/435

/40

1/35

/30

/R5S

/20\

S15

410

/05

- 100

7ime inMnutes

Chart 2

| | 8) ZL Sadium Crtrale
| yf ooyen Su/fple
CPtica,
“vorium Janfrale
desopt SO IO AG 20 I. SO” Sa, re

ADJUSTMENT OF BLOOD VOLUME 333

do. not easily diffuse through the gut wall into the blood stream, and so
they act osmotically to draw water from the blood stream to flush the
intestine. Goldschmidt and Dayton (28) have shown that this is pre-
cisely what happens in the colon. There was free passage of water with
practically no diffusion of sulfate in their experiments. Hdéber (15) has
studied the absorption of salts from the small intestine and finds that
the anions of the salts arrange themselves in the same order as in the
present experiments, sulfate being absorbed least rapidly and chloride
most rapidly. Wallace and Cushny (29), studying the intestinal
absorption of saline cathartics, found that the salts arrange them-
selves in order of the speed of absorption as follows:—chloride, bro-
mide, iodide > sulfate > phosphate, tartrate, citrate.

The effect of calcium. Calcium was studied in these experiments first be-
cause, as Loeb (30) and Osterhout (31) have pointed out, it exhibits an antago-
nism toward sodium and secondly because of its alleged action on the permea-
bility of membranes. In experiments on the prevention of pleural exudates in
dogs, Chiari and Januschke (32) reported that calcium injected subcutaneously
- inhibited the transudation after treatments which in control dogs caused a
copious amount of fluid. The whole scheme of experimentation was rather severe
in this investigation. The appearance of icterus after the injection of fluo- ;
rescin was found by Rosenow (33) to be distinctly inhibited by calcium chloride.

Small amounts of concentrated sodium chloride injected intravenously in
rabbits elicit a hyperglycemia which is prevented by adding calcium chloride to
the salt solution (84). Slow injections of large volumes of sixth molecular sodium
chloride into rabbits produce a glycosuria which is prevented by the addition
of three-eighths molecular calcium chloride in the ratio 975 ee. NaCl to 25 ee.
CaCl, (35). MacCallum (36) found that saline purgatives injected intravenously
produced active peristalsis but that the action was inhibited by calcium chloride.
Calcium antagonizes not only sodium but also magnesium (37) and potassium

(88), (39), (40), (41).

In the present experiments calcium chloride was injected with sodium
chloride in isotonic solution to ascertain whether or not the calcium
would hinder the movement of fluid out of the capillaries. As will be
seen from table 4, instead of inhibiting the return to normal relative blood
volume, the presence of the calcium seemed, if anything, to hasten the
outward passage of the injected fluid. Yanagawa (42) had reported
that calcium chloride does not reduce the permeability of the capillaries
under normal conditions.

- In the second injection of experiment 31, 0.25 gram calcium chloride
dissolved in isotonic sucrose solution was injected and the animal died
with tetanic convulsions. The left ventricle was in strong contraction

334 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL

and there was a clot in the right auricle. When a higher concentra-
tion of calcium chloride in sodium chloride solution was injected there
were no untoward effects. This seems to indicate that the sodium
exerted an antagonism not shown by the sucrose.

TABLE 4

Regulation of blood volume after injection of sodium chloride containing calcium
chloride, relative blood volume

EXPERIMENT NUMBER

TIME INTERVAL _ 32 (First injection) 32 (Second injection) 33 (First injection)

250 ce. 0.9 per cent NaCl | 250 ce 0.9 percent NaCl | 250 cc. 0.85 per cent NaCl

6.4 cc. 3M CaCle 12.8 cc. 3M CaCle 20 cc. 3M CaCle
minutes
Normal 100 100 100
Immediately 123 141 (134
5 - 108 113 110
10 102 105 100
15 100 105 100
20 100 100 100
25 100 100 100
30

40

50

60

The effect of colloidal silver. Colloidal silver is a suspensoid colloid
while the blood proteins are emulsoid colloids. Spiro (43) has reported
that the imbibition of water by gelatin is accelerated by the presence
of colloidal iron oxide. The present experiments were carried out to
determine the effect of the interaction of the two types of colloids upon
the movement of water out of the circulation. The technic was the
same as used before in the present experiments. The silver preparation
used (Solargentum, Squibb)? is said to contain about 20 per cent silver
It was dissolved in 0.97 per cent sodium chloride solution and in three
cases the dosage was 50, 100 and 200 mgm. respectively dissolved in
100 ce. of the salt solution. It caused no depression of the freezing point

and gave a golden brown solution which did not interfere with the —

hemoglobin determinations.

2 This preparation was kindly furnished. ii? Ae Dr. I. F. Harris by E. R.

Squibb & Sons.

ADJUSTMENT OF BLOOD VOLUME 335

Table 5 shows the results of injecting colloidal silver solutions. When
the amount injected was 50 or 200 mgm. there was no appreciable effect
on the return to normal of the relative blood volume. When 100 mgm.
were injected the relative blood volume remained above normal. This
injection was, however, the second dose of colloidal silver that rabbit
had received. It appears that there may be a cumulative effect of the
’ colloidal silver solution. There was glycosuria in all cases after the
injection of colloidal silver. This point will be investigated further.

TABLE 5

Regulation of blood volume after injection of colloidal silver solution (Solargentum
Squibb); relative blood volume

EXPERIMENT NUMBER
TIME INTERVAL 44 (First injection) 44 (Second injection) 45
100 ec. 0.97 per cent NaCl |100 cc. 0.97 per cent NaCl |100 cc. 0.97 per cent NaCl
50 mgm. silver 100 mgm. silver 200 mgm. silver
minutes ;
Normal 100 100 100
Immediately - 143 132 142
5 "122 118 129
10 106 113 106
15 104 113 : 106
20 106 7 101
25 104 111 101
30 104 111 101
40 104 111 100
50
60

The effect of acacia. Since varying accounts of the value of acacia
in shock have been reported, it seemed of interest to try its effect in
normal animals under the conditions of our experiments. In circula-
tory shock saline solution or Ringer’s solution given intravenously
leaves the circulation so rapidly that it helps little. Bayliss (44),
(45), (46) introduced the use of gum acacia and has been its foremost
champion. The value of acacia lies in the fact that it possesses not only
viscosity but also a small osmotic pressure thus simulating the plasma
proteins. He recommends the intravenous use of 7 per cent acacia in
0.9 per cent sodium chloride solution in the treatment of shock on the
theory that if the blood pressure and aeration are kept up acidosis need
not be feared.

336 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL’

A study of the blood volume regulation in normal animals after the
injection of acacia was made in the present investigation according to
the technic already described, a solution of 7 per cent acacia in 0.9 per

cent sodium chloride ( A = 0.59) solution being used. The A due +

the acacia alone was 0.07.

Table 6 shows that considerable fluid passed out of the ciroultiiod
during the injection. The acacia maintained the augmented blood
volume in a way not observed with any other substance used in these
experiments. It held the relative blood volume one-third above nor-
mal for more than an hour. 3

TABLE 6

Regulation of blood volume after injection of acacia-sodium chloride solution;
relative blood volume

EXPERIMENT NUMBER
TIME INTERVAL
33 (Second injection) 52 53
minutes

Normal 100 100 100
Immediately 153 141 144
5 141 137
10 146 141 137
15 146 . 141 137
20 141 137
25 136 141 137
30 134 - 141 134
40 — 133 141 131
50 | 133 135 131
60 133 ; 129 oe

Moore (47) attributes the value of acacia to the restoration of the hydrophilous
colloids to the blood. That it exerts a small persistent osmotic pressure and thus
maintains the blood volume has been asserted by Gasser, Erlanger and Meek

(48). Kruse (49), however, has suggested that the value of acacia in maintaining
blood volume may be due to its adsorption on the capillary walls, whereby the —

exit of the fluid from the vessels is impeded.

The effect of acid. The effect of acid on the swelling of colloids and on the per-

meability of membranes has been widely studied. Spiro (43) reported that +f,
hydrochloric acid caused gelatin discs to swell more than did water, while Chiari
(50) showed that increased swelling was obtained when carbon dioxide was present
in conductivity water. Fischer (51) demonstrated the swelling of muscle in acid.
Osterhout (52) studying diffusion, found that acid first decreased the permea-

bility of the cell membrane but later increased it. Harvey (53) showed that all — =

acids excepting benzoic and salicylic encounter resistance to diffusion at the
surface of the living cell.

ae a ee.

ADJUSTMENT OF BLOOD VOLUME Sot

It might appear a priori that injection of acid would increase the
water-holding capacity of the plasma colloids or would decrease the
_ permeability of the capillary membrane to such an extent that the pas-
‘sage of fluid out of the circulation after injection would be inhibited.’
On the other hand, the body has such remarkable ability to take care
of increased acid and to maintain its constant reaction (45) that it would
appear problematical whether or not one could introduce enough acid

to change the physical chemical equilibria without killing the animal.

TABLE 7

Regulation of blood volume after injection of hydrochloric acid-sodium ‘chloride
solutions; relative blood volume

EXPERIMENT NUMBER
34 alge 54 56
TIME INTERVAL (Second injection)
, se HClin =| ~ # HClin *M. HClin }t HCl in
0.7 per cent NaCl | 0.6 percent NaCl | 0.6 percent NaCl | 0.6 per cent NaCl
minutes
Normal 100 100 100 100
Immediately 117 124 121 138
5 111 111 108 115
10 108 108 . 109
15 104 105 104 cmt 108
20 104 102 ; 108 103
25 102 104 101 .
30 Tt) ieee 108 104 103.
40 105 102 101
50 104 101
60 104 103

To test this point with reference to changes within the blood vessels,
zo and 75 hydrochloric acid made isotonic with 0.7 per cent and 0.6
per cent sodium chloride respectively were injected as in experiments
already recorded. js acid seemed to be the limit of tolerance under
the conditions of the experiment. Several of the rabbits died during
the injection and on autopsy showed pulmonary edema. During in-
fusion there were in every case dyspnea and spasmodic contractions of
the voluntary muscles.

From table 7 it will be seen that in the concentrations used hydro-
chloric acid failed to decrease the rate of passage of fluid out of the
blood vessels. The relative blood volume returned to normal as rap-
idly when acid-sodium chloride solutions were used as with the sodium
chloride alone.

338 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL

Relative
Blood Volume

Blood Volume
/60 Fregulation in [rabbis
after [nyection of

«158

Various Sall and Colloidal
150 Solulions Pett:
a

145

/40 ae . 7
| \
135 UR .
I \ ae A Py SN q
| iN ? | a \Chloride 4
/30 uN
| \ \ |
\

125 | \ q
| Aw {
' \ .

by
/20 \ is .

\ Co//ordal

H \ Silver + Sodium Chloride 4
| \ ;
re fo) i
h ¥ a
} \
} . \ a
//0 %

3

2 4

Choride Sedim N R ie Ags op 4

Chloride )
100

ek. 8 10 IS) 2B Ze eae. ae

Time in Minales
Chart 3

Oe ee ee ee

ADJUSTMENT OF BLOOD VOLUME 339

The effect of sucrose. To determine the effect of a crystalline non-
electrolyte on the removal of fluid added to the blood 10.9 per cent
sucrose solution (A = 0.59) was injected in one experiment. From
table 8 it will be seen that the relative blood volume returned to normal
as rapidly as with saline solution.

_ TABLE 8

Regulation of blood volume after injection of sucrose solution; relative blood volume

TIME INTERVAL EXPERIMENT NUMBER 31
minutes
Normal 100
Immediately 158
5 : 146
10 119
15 109
20 104
25 101
30 100
40 100
50
60

Fate of the injected fluid. From the preceding experiments it is evi-
dent that with the exception of the citrate, tartrate, sulfate and acacia
none of the substances injected decreased the rate of loss of fluid from

_ the circulation as judged by the variation in blood volume. In the

majority of cases a volume of fluid equal to the blood volume diffused
out of the circulation in less than one half-hour. In speculating upon,
and, if possible, determining the fate of this fluid, attention is directed
to the possible paths of elimination of this excess of volume. It may
be excreted through the kidneys, it may accumulate as edema, fluid, it
may form serous exudates, it may pass into the tissue spaces or it may
be excreted into the gastro-intestinal tract.

Haldane and Priestley (54) have shown in a striking manner that
copious water drinking results in a large urine output. The effect of
water drinking upon the circulation must be analogous to that produced
by the injection of isotonic solutions in our experiments. Magnus
(55), (56) concluded that the cause of the diuretic action of salts lies in
the composition of the blood and not in the increased capillary pressure.
He found that sulfate solutions of the same osmotic pressure as chloride
solutions produced greater diuresis.

340 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL

TABLE 9

Urine volume as percentage of injected solution

EXPERI- > EOS Sot th
SOLUTION MENT PER CENT AVERAGE a
NUMBER Te
q : er
; ’ 7 64
Sodium sulfates... ysis... sissies dalwmager set eees 20 51 .
27 110 70
4 28
5 66
Sodium nitrate.......... abs ¢ ose Sandee Re pecatt 4 17 63
{ae 37 43
Pict 44 16 ee
Cobibidal'eibege. * Dhys. 2.2. serena 45 29 23
| 36 28
Souiumy eitwite.. . ft oo. ios co pel recs 37 21... 7a
38 14 21
3 27
Sodium sulfocyanate:....... 0... 00 obey ewik yy eae 14 17
. . 23 13 19
, . 15 15 - )
Sodium acetate............ Ay tif wis 54 RM 29 23 19
x St : , é 32 14
Calcium chloride-sodium chloride............... 33 20 17
: } |, 46. 26
Bodinm, tartrate... «0 dcksid ssi 5:4 oe dare ea ee 48 8 Pe
. 48 7 13
34 10
Hydrochloric acid-sodium chloride. ............. 54 16 RTH
55 8 ll
Be 6
OGM OMIOPICG o,f oe cas goce os ea sa de eaeees 11 12
18 8 9
10 8
Ode DIOUMIE 6 5 oie cans Va va ke) va a _ 12. 8
13 12 9

a a ae

a a

ee a ee

ADJUSTMENT OF BLOOD VOLUME 341

Table 9 shows the average figures for urine volumes excreted during
the various experiments and calculated as percentages of the injected
volume. Sulfate and nitrate induced the largest secretion of urine ob-
served with any of the salts or substances investigated. This corre-
sponds with the statements in the literature on the comparative diuretic
effect of these salts. However, even in the sulfate experiments where
the salt caused the greatest diuresis the urine volume did not account
for the fluid which had left the circulation.

The nature of edema and the factors involved in its production have
long been matters of discussion. Cohnheim and Lichtenheim (57) in-
jected salt solution until they had given 46 per cent of its weight to a
rabbit and 64 per cent to a dog yet they failed to observe subcutaneous
edema. On infusing salt solution after arsenic poisoning, Magnus (58)
produced edema although in normal animals plethora elicited no edema.
It is evident from the literature that hydremic plethora will not produce
the edema which is characteristic of nephritis. In the present experi-
ments no patent edema was observed in any of the animals.

The pleural and peritoneal cavities offer considerable free space for
the accumulation of pathological fluids. After injection of saline solu-
tion Cohnheim and Lichtenheim (57) found fluid in the peritoneal cavity
but the pleural cavity was dry. Of the fluid injected into rabbits,
Dastre and Loye (59) found 75 per cent in the tissue spaces and serous
cavities. In the present experiments the pleural cavity was, as a rule,
normal in appearance. The peritoneal cavity usually contained fluid but
there was never more than 5 cc. of transudate which usually contained
protein and the salt injected and would clot. It is certain that the fluid
in the peritoneal cavity accounted for very little of that which diffused
out of the blood vessels.

The indefinite area known as “‘tissue spaces” constitutes a reservoir
of fluid in the animal body. After hemorrhage the volume of blood is
brought back toward normal by the passage of lymph from the muscle
and other tissues into the circulation. Hypertonic solutions draw part
of the fluid which renders them isotonic from the tissue spaces. Engels
(60) reported that 68 per cent of the fluid injected into a dog was con-
tained in the muscles. In the present investigation experiments were
carried out to determine whether or not the amount of water contained
in the muscle was demonstrably increased. From table 10 it will be
seen that the percentage of water did not vary after the injection.
This was rather surprising after the statements made in the literature.
All of the older experiments dealt with slow infusions extending over a

342 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL —

long period of time. ‘Perhaps the manner in which we carried out the
experiment altered the distribution of the fluid.

The excretory function of the small intestine has been given oonplile
erable attention in the literature. In the course of their experiments on ~
hydremic plethora, Cohnheim and Lichtenheim (57) observed that the
stomach and intestine became swollen. Albu (61) reported that after
injection the intestinal contents became a thin paste. J. B. MacCal-
lum (62) injected large volumes of normal saline solution intravenously;

and from a cannula tied in the intestine he obtained fluid which
amounted to 15 per cent, 9 per cent and 10 per cent of the ington
volume i in three different experiments.

TABLE 10

The water content of muscles before and after injection of isotonic salt solutions

GASTROC- TIBIALIS
NEMIUS ANTICUS
per cent per cent
Experiment 41 4 diel
Melore ini@etion } i656 © 48) Ao. 3 eA Laea 78.0 (7.8
PIER WA OCEOE fo 1s 06 5. ns te lh Ne ee ae & he: 77.5
Experiment 42 :
Peeters MiGewIOn. . se s e s bee sae cake eee: 76.0 76.5
After injection? *..02.05.0). 2.05 0. a TE aE 76.2 76.2
Experiment 43
Detere injection «.s = (ees ssny dpe Re eee 79.5 79.5
Miter intention: 6.6 ici - 5k eee ae 79.3 79.4

In the present experiments abnormal distention was repeatedly no-
ticed which, on autopsy, was seen to be due to the injection of the stom-
ach, small intestine and cecum. The mass of material in these organs
was a thin paste and on section fluid streamed from the stomach. These
observations indicate that, under the conditions of our experiments,
some fluid was excreted into the gastro-intestinal tract.

From the above discussion it appears that the part of the injected
fluid which diffused out of the blood stream in these experiments can be
accounted for in the urine, in the exudations into the serous cavities
and in the excretion into the intestinal tract. In these experiments
there was, however, no trace of edema nor was there detectable increase
in the water content of the skeletal muscles.

ae
.

ADJUSTMENT OF BLOOD VOLUME 343

SUMMARY

When isotonic solutions of the acetate, nitrate, sulfocyanate, brom-

ide, chloride, tartrate, sulfate or citrate of sodium are injected intra-

venously at such a rate that a volume equal to the estimated blood vol-
ume is introduced in two minutes, the rate at which the added fluid
escapes from the circulation, as measured by the relative blood volume
at successive subsequent intervals, is decreased to a slight extent by the
sulfate, tartrate and citrate.

When calcium chloride, hydrochloric aia or colloidal silver was
dissolved in sodium chloride solution and was injected intravenously
there was no alteration of the rate of return to normal blood volume.

When acacia-sodium chloride solution was used there was a marked
and long sustained increase in the relative blood volume.

Suerose in isotonic solution did not delay the passage of fluid from the
blood vessels.

The fluid which leaves the circulation in the restoration of blood vol-
ume after the injection could not be accounted for-by the passage into
the muscles or by edema fluid. The volume of urine, exudate into the
serous cavities and the excretion into the intestine and stomach proba-
bly are concerned in the disposal of the fluid leaving the circulation.

BIBLIOGRAPHY

(1) Roarrs: Phil. Journ. Sei., 1909, iv-B, 99.

(2) UNpERHILL: Arch. Int. Med., 1919, xxiii, 753.

(8) Batcar, SANSUM AND WoopyatT: Arch. Int. Med., 1919, xxiv, 116.
(4) Lussky AND FrigepstTEIN: Amer. Journ. Dis. Child., 1920, xix, 337.
(5) Weep anp McKissen: This Journal, 1919, xlviii, 512.

(6) Cusnine: Report at New Haven Meeting, Soc. Exper. Biol. and Med., 1920.
(7) Lewitu: Arch. exper. Path. u. Pharm., 1888, xxiv, 1.

(8) HormetsterR: Arch. exper. Path. u. Siar 1888, xxiv, 247.

(9) Hormeister: Arch. exper. Path. u. Pharm., 1888, xxv, 1.

(10) Hormerster: Arch. exper. Path. u. Pharm., 1891, xxviii, 210.

(11) Pauur: Arch. gesammt. Physiol., 1899, Ixxviii, 315.

(12) Linure: This Journal, 1907, xx, 127.

(13) Héser: Biochem. Zeitschr., 1908, xiv, 209.

(14) Linture: This Journal, 1917, xliii, 48.

(15) Héser: Arch. gesammt. Physiol., 1898, lxx, 624.

(16) Spartu: Science, N. S., 1916, xliii, 502.

(17) Boycorr: Journ. Path. and Bact., 1918, xviii, 11.

(18) Hatpane: Journ. Physiol., 1901, xxvi, 497.

(19) Barcrort: The respiratory function of the blood, Cambridge, 1914.
(20) Conen AND Smrtru: Journ. Biol. Chem., 1919, xxxix, 489,

344 ARTHUR H. SMITH AND LAFAYETTE B. MENDEL |

(21) Scorr, HERMANN AND SNELL: This Journal, 1917, xliv, 313.

(22) BootuBy AND Berry: This Journal, 1915, xxxvii, 378.

(23) Scorr: This Journal, 1917, xliv, 298. Pei

(24) ScHNEIDER AND Havens: This Journal, 1914, xxxvi, 380. = Beye

(25) Lamson: Journ. Pharm. Exper. Therap., 1915, vii, 169. 2 aa

(26) Lors: Journ. Biol. Chem., 1918, xxxiv, 395. ee

(27) Lons: Journ. Gen. Physiol., 1919, i, 39.

(28) GoLpscHMIDT AND Dayton: This Journal, 1919, xlviii, 450.

(29) WALLACE AND CusHny: This Journal, 1898, i, 411.

(30) Lors: Journ. Biol. Chem., 1917, xxxi, 343.

(31) OsreRHovT: Science, N. S., 1917, ane 97.

(32) CHIARI AND JANUSCHKE: Wie klin. WodhenseHs’ 1910, xxiii, 497)

(33) RosEnow: Zeitschr. gesammt. exper. Med., 1914, iv, 427.

(384) Witenko: Arch. exper. Path. u. Pharm., 1911, Ixvi, 143.

(35) UNDERHILL AND Ciosson: This Tenant. 1906, xv, 321.

(36) MacCatuium: This Journal, 1904, x, 101.

(37) MreutzeR AND AvER: This Journal, 1908, xxi, 400.

(388) MacCaLuuM AND VoEGTLIN: Johns Hopkins Hosp. Bull., 1908, xix, ot.

(39) Logs: Journ. Biol. Chem., 1915, xxiii, 139.

(40) Howruu: This Journal, 1898, ii, 47.

(41) GreENE: This Journal, 1898, te 82.

(42) YanaGawa: Journ. Pharm. Exper. Therap., 1916, ix, 75.

(43) Sprro: Beitr. chem. Physiol. u. Path., 1904, v, 276.

(44) Bayxiss: Intravenous injection in wound shock, 1918,

(45) Bayuiss: Journ. Physiol., 1919, liii, 162.

(46) Bayuiss: Journ. Pharm. Exper. Therap., 1920, xv, 29.

(47) Moore: Brit. Med. Journ., 1919, no, 3068, 490.

(48) Gasser, ERLANGER AND Merk: This Journal, 1919 1, 31.

(49) Kruse: This Journal, Proceedings, 1920, li, 195.

(50) Cutart: Biochem. Zeitschr., 1911, xxxiii, 167.

(51) Fiscuer: Edema and nephritis, 1915.

(52) OsteRHOUT: Journ. Biol. Chem., 1914, xix, 493.

(53) Harvey: Science, N. S., 1914, xxxix, 947.

(54) HaLpDANE AND PrigstTLey: Journ. Physiol., 1916, 1, 296.

(55) Maanus: Arch. exper. Path. u. Pharm., 1900, xliv, 68, 396.

(56) Maanus: Arch. exper. Path. u. Pharm., 1901, xlv, 210. ban

(57) CounHEIM AND LicutenuEmM: Arch. pakh: Anat. u. Physiol. u. klin. Me
1877, lxix, 106.

(58) Maanus: Arch. exper. Path. u. Pharm., 1899, xlii, 250.

(59) Dastre anD Loys: Arch. d. Physiol. et Path., 1888, iv, 93.

(60) Enenus: Arch. exper. Path. u. Pharm., 1904, Si 346.

(61) Aupu: Arch. path. Anat. u. Physiol. u. klin. Med., 1901, elxvi, 87.

(62) MacCatuium: Univ. of Cal. Publ. Physiol., 1, “no. 14, 425,

aor eee
rahe at .
¢ n

344 ~

THE AMERICAN
J OURNAL OF P HYSIOLOGY

VOL. 53 OCTOBER 1, 1920 : No. 3

ANTAGONISM OF INHIBITORY ACTION OF ADRENALIN
AND DEPRESSION OF CARDIAC VAGUS BY A
CONSTITUENT OF CERTAIN TISSUE
EXTRACTS

J. B. COLLIP

From the Department of Biochemistry and Physiology of the University of Alberta,
Edmonton, Canada

Received for publication June 1, 1920
INTRODUCTION

The presence of the so-called ‘‘peristaltic hormones’’ in various -
tissues has frequently been demonstrated. Enriquez and Hallian (1)
found that extracts of the gastric and intestinal mucosa contained a
powerful stimulant for the intestinal musculature. Fawcett, Rahe,
Hackett and Rogers (2) found that protein-free aqueous extracts of
the liver, pancreas and spleen, and of the pituitary, pineal, thyroid,
parathyroid; thymus and adrenal glands caused a characteristic stimu-
latory effect upon the unstriated muscle fibers of the cat’s uterus.
This stimulatory effect they found to be antagonized by adrenalin.
Abel, Pincoffs and Rouiller (3) have prepared from the intestinal
mucosa of the pig a water-soluble powder which showed great physio-
logical activity in stimulating an intestinal or uterine strip or in caus-
ing the pancreas to secrete freely.

Stern and Rothlin (4) have recently shown that water extracts of
liver, kidney, thyroid, lung, muscle, thymus, bone-marrow, bile and
blood, contain two kinds of substances, one causing contraction. of
smooth muscle, the other relaxation. The former substances only,
they hold, are present in lymph glands, adrenal bodies and spleen.
They found that the constrictor substances were soluble in alcohol
and insoluble in ether, while the depressor substances were insoluble

343 E

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 3

a
344 J. B. COLLIP

in alcohol but soluble in ether. Both types of substances resisted
boiling, but the former were destroyed by alkali while the latter were
not. Matsumoto and Macht (5) found that the vas deferens of labora-
tory animals responds with contractions only to large doses of gland
extracts. Cow (6) has found that the inhibitory action of adrenalin
upon the uterus of the guinea pig, rat and non-pregnant cat is reversed
by previous treatment with pituitrin.

In view of the fact that associated with shock following severe

trauma there is always the possibility of certain constituents of dam- —

aged tissue entering the blood stream, thus giving a condition some-
what analogous to the intravenous injection of tissue extract, a further

investigation of the effects of extracts of various tissues, prepared in a
variety of ways, upon isolated organs was undertaken.

_ The general observations that aqueous extracts of various tissues
contain certain principles which increase the tonus of smooth muscle

were confirmed. Certain additional phenomena of considerable

. interest were manifested in several experiments; it is these latter that:

will be dealt with subsequently.

EXPERIMENTAL

The action upon isolated mammalian -intestine and uterus, and

on the heart of the terrapin, of tissue extracts prepared in the following
ways was studied. Extracts made by grinding fresh tissue in the
mortar with pure sand and normal Ringer-Locke’s solution and sub-
sequently centrifuging. Extracts made by boiling fresh tissue, pre-
viously macerated, with several volumes of distilled water, filtering
and concentrating the filtrate on the water-bath to an aliquot volume
of the original tissue used, thus rendering the extract practically iso-
tonic. Extracts made by extracting the water-bath dried, water-
soluble fraction with 98 per cent alcohol, filtering and evaporating the
filtrate to dryness on the water-bath and taking up the residue in
Ringer-Locke’s solution. Extracts made from tissue previously dried

at 110°C. to 120°C. Extracts made by boiling desiccated glandular 4

tissue supplied by Armour & Company, Chicago, with distilled water
in amount equal to the water content of the original tissue, and filter-
ing. The desiccated thyroid body, parathyroid gland, thymus gland,
pancreas, testes, corpus luteum and pituitary body (anterior and
posterior lobes) were treated. in this manner.

The uterus and intestine of the rat. were used most extensively in -

this investigation, but preparations were also made from the guinea

ee oe

ANTAGONISM BETWEEN ADRENALIN AND TISSUE EXTRACTS 345

pig, rabbit, dog, mouse and cat. Preparations were suspended in
normal Ringer-Locke’s solution contained in a glass perfusion chamber
of 40 cc. capacity which was immersed in a water-bath kept at 38°C.
Oxygen was continuously bubbled through the perfusion fluid. Move-
ments of the isolated smooth muscle strip were recorded on the drum
by means of a Harvard heart lever. The extracts were brought to
body temperature previous to being added to the saline bath. The
effect of tissue extracts upon the heart of the terrapin was determined
in one of two ways. The brain of the animal was pithed, the heart
exposed, and the apex of the ventricle connected by a silk thread to a
heart lever. Both vagi were then dissected free in the neck and their
response to rapid make and break shocks determined. The extract
was then injected directly into the ventricle or else it was added to
Ringer-Locke’s fluid and the heart perfused by means of a cannula
placed in the left abdominal vein, the fluid escaping through the cut .
aorta. Intravenous injections of certain extracts were also made
into dogs to determine the effect upon the cardiac vagus and the re-
sponse to adrenalin administered by the intravenous route. _

RESULTS

It was found, of the tissues investigated—heart, lung, spleen, liver,
brain, cord, posterior spinal root ganglia, pancreas, skeletal muscle,
testes, small intestine, gastric mucosa, corpus luteum, and thyroid,
parathyroid, pituitary (anterior and posterior lobe), and thymus
glands—that all contained in greater or less amount substances which
caused increased tonus, amplitude and rate of contractions of strips of
duodenum, small intestine, and virgin and gravid uterus. The
substances were not destroyed by boiling and were soluble in alcohol.
_ Depressor substances were also present but the usual effect of the

addition of 1 or 2 ce. of an extract to the Ringer-Locke’s solution in the
bath was an immediate increase in the tonus of the smooth muscle
strip taken from the duodenum, jejunum or ileum. It was more
difficult to demonstrate stimulatory action in the case of the uterus
owing to the fact that so often excellent tonus and rhythmic contrac-
tions were manifest prior to the addition of the extract. In the case
of the extract of pancreas the immediate effect upon the duodenum
was a decrease in the general tonus which, however, was quickly recov-
ered and definite stimulation was manifested (fig. 1, a). Pancreatic
juice obtained from a cannula placed in the pancreatic duct of a dog
did not have this effect. Again in the case of aqueous extracts of oven-

346 J. B. COLLIP

dried tissues studied (brain, cord, posterior root ganglia, duodenum,
heart, pancreas, spleen, skeletal muscle) a deviation from the general
rule was noticed. The primary effect of such extracts upon the intes-
tinal musculature was typically that of adrenalin (fig. 1, 6). The pri-
mary depression which was caused by the addition of such an extract

was, however, followed at once by an increase in the general tonus. —
These latter extracts had little effect upon the uterus unless used in on
fairly large amount, in which case decreased tonus resulted (fig. 1, ¢).

It was found that extracts prepared from the heart, pancreas, corpus

luteum, pituitary body (both anterior and posterior lobes), testes, —
and thyroid, parathyroid and thymus glands in addition to causing —
intense stimulation of the uterus antagonized the inhibitory action of —

\

adrenalin on such uteri as are normally inhibited by this latter sub-—

stance, as in the case of the rat and guinea pig (both virgin and gravid), —
and the virgin rabbit and dog (figs. 1, d; 1, e; 2, a; 2,b; 3, a). This —

phenomenon is strikingly similar to that, recorded by Cow (6), of the ©

reversal of the inhibitory effect of adrenalin on the uterus of certain

animals by previous sensitization with pituitary extract. The results —

of Cow (6) were in part confirmed by the writer. Extracts of tissues -
studied other than those mentioned above, were not as effective in this -

regard. Partial antagonism was obtained by extracts of lung, gastric

mucosa and liver, but in these instances the amounts of extracts used
were relatively quite large. This antagonism of the inhibitory action —

of adrenalin by extracts of certain tissues was effective only as long as

the extract was actually present in the saline bath. The inhibitory

response to adrenalin returned in each case after the uterine strip was"

washed free from the extract (fig. 3,a). It was difficult to determi a

absolutely whether the stimulant action of adrenalin on the gravid —
uterus of an animal such as the rabbit was antagonized by extracts | 4
like that of the spleen, as the tonus produced by the addition of the .-
latter was practically maximal; however, the writer is of the opinion | a
that the stimulatory effect of adrenalin is not antagonized on such

uteri as are normally stimulated by it.

The antagonism of the inhibitory action of adrenalin on ‘cone 4
uteri by extracts above noted was not demonstrated in the same deci-
sive manner in the case of the intestinal musculature. Partial antag-

onism was, however, clearly demonstrated on different occasions by
the raising of the threshold for adrenalin inhibition of the duodenum
or jejunum of the rat by extract of spleen or heart (fig. 4, a).

347

ADRENALIN AND TISSUE EXTRACTS

ANTAGONISM BETWEEN

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ANTAGONISM BETWEEN ADRENALIN AND TISSUE EXTRACTS 351

It was found, when splenic extract was injected intravenously into
a dog, that the rise in blood pressure produced by a definite dose of
adrenalin was slightly augmented, and also the high pressure was
longer maintained (fig. 4, d). Electrical excitation of the vagus which,
prior to the injection of the extract into the ventricle or the perfusion
of the same through the heart by way of the anterior abdominal vein
caused complete inhibition of the heart, was found to be without effect
after treatment with the extract (figs. 4, b and 4, c). The extract
caused at first considerable depression of the myocardium but spon-
taneous beats continued and a later effect was in some instances
increased amplitude of contraction with little change in the rate. The
response of the heart to excitation of the vagus did not return till some
time after the injection of the extract. If however the extract was
washed away by perfusing the heart with a quantity of modified
Ringer-Locke’s solution, the heart as a rule responded to vagus stimu-
lation after a period of from fifteen to thirty minutes’ washing. When
the stimulation of the vagus with the induced current from the sec-
ondary coil of a Harvard inductorium set at zero with a 6-volt current -
running in the primary failed to produce inhibition, injection of pilo-
carpine into the heart caused partial inhibition marked by a decrease
in the rate of contraction. The injection of 30 cc. of heart extract,
made by boiling the macerated tissue with distilled water and filtering .
and concentrating the filtrate till it was practically isotonic with blood
serum, into the left jugular vein of a 12 kilo female dog under ether
anesthesia made complete vagus inhibition temporarily impossible to
obtain (fig. 4, e). Depression of the vagus in the intact dog by splenic
extract was not so obtained.

The stimulating action of splenic extract upon uterine muscle was
manifested after the tissue had been atropinized (figs. 2, ¢ and 3, b).
Adrenalin caused complete inhibition of the atropinized gravid uterus
of the rat, but this was antagonized by splenic extract and further
treatment with adrenalin was without effect (fig. 3, b). It has been
found that the water-alcohol soluble fraction of the splenic extract
remains active as regards its ability to antagonize the inhibitory action
-of adrenalin on certain uteri and to depress the cardiac vagus of the
terrapin after it has been freed from certain constituents by treat-
ment with basic lead acetate and silver nitrate in alkaline medium.
Also the lead and silver precipitates, after removal of the lead and
silver respectively, dissolved in Ringer-Locke’s solution failed to pro-
duce any such effect. ott

352 J. B. COLLIP

An acid-alcohol extract of the water-bath dried lipoid-free .water-
soluble fraction of ox spleen, treated with anhydrous ether, gave a
brownish-red precipitate. This was freed from ether and dissolved in
Ringer-Locke’s fluid. It was found to have no stimulating action on
the uterus of the rat and it did not antagonize adrenalin. A slight
stimulatory effect was produced upon the isolated duodenum of the
rat by this extract. Crawford (7) found that the active principle of
the pituitary gland was removed from acid-alcohol extracts by an-
hydrous ether. | ee }

DISCUSSION

The fact that extracts of several tissues antagonize the inhibitory
action of adrenalin on strips of isolated uterus which are normally
inhibited by it, and also depress the cardiac vagus of the terrapin,
suggests that the same constituent or constituents are present in each
of these tissues. The point of action in each case is undoubtedly in
the peripheral inhibitory nervous mechanism of the organ concerned,
probably the myoneural junction itself. Nicotine is without effect
after the action of adrenalin on the uterus has been abolished and
after the vagus has been completely depressed in the heart of the
terrapin. Pilocarpine may have an appreciably stimulatory effect on
the uterus after the addition of certain extracts and it is still mildly
active on the heart after electrical excitation of the vagus fails to
inhibit. Atropine causes slight depression of the uterus after heart or
splenic extract have acted and it abolishes the slight action of pilo-
carpine on the heart of the terrapin with vagus depressed.

As the stimulatory action of splenic extract is still manifested on the
atropinized uterus of the rabbit and rat, it is probable that the stimula-
tory effect is due to the direct action upon the smooth muscle fiber.
It is also true that some of the stimulatory effect of certain tissue
extracts upon strips of smooth muscle may be due to a depression of
the local inhibitory mechanism.

The depression of the inhibitory mechanism which is so clear-cut
in the case of certain uterine preparations and of the heart of the terra-
pin is of interest in another way. The inhibitory nerves of the heart
are of the parasympathetic type while those to the uterus are mixed,
but in those instances where adrenalin inhibits at all times it is to be.
supposed that the inhibitory fibers: are largely of true sympathetic
derivation. Here then (granting that it is the same type of principle
which is active in each case) is an instance of certain tissue constit-

ANTAGONISM BETWEEN ADRENALIN AND TISSUE EXTRACTS 353

uents acting selectively upon the inhibitory nervous apparatus of a
_ tissue irrespective of whether it is of the sympathetic or parasympa-
thetic type.

As the intravenous injection of splenic extract augments the pressor
effect of a definite amount of adrenalin, it may be that some part
-of the vasodilator mechanism which is stimulated by adrenalin (8) is
depressed by the splenic extract, and hence a greater pressor response
ensues when adrenalin is injected than would otherwise be the case.

As such a heterogeneous group of tissues has been found to contain
substances which act in a similar manner in antagonizing the inhibitory
action of adrenalin upon certain uteri, and to a lesser degree depressing
the cardiac vagus of the terrapin, it is possible that all tissues contain
these principles in certain amount. It would in any case seem clear
that these particular substances should not be regarded as ‘‘hormones.”’ |

SUMMARY

1. Extracts of various tissues were found to have definite stimulatory
effect upon isolated intestinal and uterine musculature.

2. The primary effect of pancreatic extracts is depression in the
tonus of the duodenal musculature.

3. The primary effect of extracts of tissue dried at 110°C. to 120°C.
on the intestine is inhibition. This is followed immediately by definite
stimulation.

4, Extracts of heart, spleen, pancreas, testes, anterior and posterior
lobe of the pituitary body and thymus, thyroid and parathyroid glands
in addition to stimulating the uterus of the rat, guinea pig, virgin dog
and cat, antagonized the inhibitory action of adrenalin on these organs.

5. Extracts of heart and spleen caused the complete abolition of
the response of the cardiac vagus to electrical stimulation.

6. The action of pilocarpine on the heart of the terrapin was par-
tially but not completely antagonized by extracts of heart and spleen.

7. The substance or substances which antagonize the inhibitory
action of adrenalin on the uterus and depress the cardiac vagus are
soluble in alcohol and resist boiling.

8.. Intravenous injection of extract of spleen augments the pressor
response to adrenalin.

9. Intravenous injection of extract of heart raises the threshold for
vagus inhibition by electrical stimulation of the peripheral end of the
cut vagus.

10. It is suggested that substances similar to those found in extracts
of various tissues may be present in greater or less amount in all tissues.

(1) ENRIQUEZ AND HaAuion: Compt. nk ‘Soe. B Biol., 1904, ‘vi,

(2) Fawcert, RAE, Hacxerr ae Rogers: This Seika dogg 1915, x Xxx x

5

(4) Grant AND Riéentin: Toute. plibaisile
(5) Matsumoto AnD Macut: Journ. Nissho, 1919, ait 70.
(6) Cow: Journ. Physiol., 1919, lii, 301. i, ak
(7) Crawrorp: Journ. Pharm. Exper. Therap., 1920, xy, 81

(8) HARTMAN AND Fraser: This Journal, 1917, xiv, 353. spi

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RECIPROCAL REACTIONS IN THE CARDIO-VASCULAR
SYSTEM

ETHEL W. WICKWIRE
From the Department of Physiology, Columbia University, New York

Received for publication June 9, 1920

INTRODUCTION

Sherrington (1) in his Integrative Action of the Nervous System, has
enunciated the doctrine of reciprocal nervous relations existing in the
living organism. Isolated facts pertaining to any biological mechanism
or to any of its parts are valuable and useful if we can weave from them
the whole fabric of truth. But we cannot escape the fact that func-
tional systems, even in lower forms, are closely interrelated and inter-
dependent. As Burdon-Sanderson has expressed it, the processes in
the organism go on in the interest of the organism. In man the most

- complex of willed movements are possible because of the reciprocal in-

nervation of many parts and systems, together with a central seat of
integration for his sensory and motor mechanisms. Intake of food,
digestion, metabolism and elimination, important as they are, cannot
avail much if the blood supply to a part is interfered with by mechanical
means, by the blocking out of nervous impulses experimentally, or by
accident or disease.

One might expect to find a reciprocal functional relationship between
the constituent parts of the cardio-vascular system. This research |
was undertaken with the object of arriving at a clearer understanding
of the reciprocal relationships, whose existence one might be led to sus-
pect, between the cardiac and the vascular nervous mechanisms, through
experimental procedures which involved changes in the rate of the heart
beat and the magnitude of arterial blood pressure.

From a simple functioning mechanism possibly, in some forms or at
some stage of development, independent of nervous connections, the
heart has been gradually developed phylogenetically into a complex dif-
ferentiated organ. Throughout its history its activity has been de-
pendent upon and modified by its increasingly complex physico-chemi-

: 355

356 ETHEL W. WICKWIRE

calenvironment. At first.apparently devoid of nervous relations, later
it appeared with nerve fibers connecting it with a more or less simple
central nervous system, and.it became subject to nervous control. As
the central nervous system became more and more complex and highly
organized in some of its levels, but less highly organized in others, the

nervous connections of the heart multiplied the number of impulses—

modifying the inherent quality of rhythmicity possessed by the cardiac
muscle tissue. :

In the course of this development the automatism, originally common
to all cardiac muscle, gradually disappeared from some parts, notably
the apex. In the higher mammals, therefore, we find a heart with its
automatic rhythmicity more and more under the influence of an in-
creasingly complex physico-chemical environment and dependent for
its normal activity upon nerve impulses receivéd from the central nerv-
ous system. )

In the normal animal the rate of the heart beat is closely connected |

with the blood pressure. If there is a large vasoconstriction, as in cold
weather, the Accompanying rise of blood pressure is associated with a
slowing of the heart beat. Conversely, a vascular relaxation and lower
blood pressure are accompanied by acceleration of the heart beat.

From observation of these and other related facts Marey. (2), in 1860,

stated that the heart beat varies inversely as the blood pressure. In
the slowing of the heart beat the vagus nerves are of course active,
although in certain conditions, for example in muscular exercise, the
increased blood pressure is not accompanied by a slower heart rate. It
is hoped that the results of experiments recorded in this paper may give
some information regarding the nervous mechanism for the adjustment
of heart rate to changing blood pressure.

METHOD

The experiments were done on cats. The routine experimental pro-
cedure was ether anesthesia, tracheotomy and connection with a mer-
cury manometer by means of a cannula placed in the left carotid artery.
Blood-pressure and time tracings were made on a Hiirthle kymograph

and the heart rate was computed in beats per minute. The stage of —

anesthesia was determined principally -by the presence or absence of

the corneal reflex, light anesthesia, however, being always sufficient to |

abolish any sense of pain. In seeking some way of bringing about
changes in blood pressure that could be readily measured without pro-

RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 357

ducing any permanent lesions in the blood vessels and with these
changes in pressure the consequent or accompanying changes in heart
rate, it was found that digital compression of the abdominal aorta was
a convenient method. Compression of the abdominal aorta was made
just below the diaphragm and yet high enough to avoid any effect by
pressure upon the splanchnic nerves. This was accomplished by plac-
ing the fingers under the vertebral column, the animal being in the back-
downward position on the board, and sliding the thumb in just below
the last pair of ribs and dorsal to the viscera until the groove on the
ventral surface of the vertebral column is felt. The abdominal aorta
lies in this groove, and can be compressed by pressure with the thumb.
An alternative method is to slide the second and third fingers in from
the side and compress the aorta by pressure with their tips.. In order
to be sure that no part of the effect was due to pressure on the splanch-
nic nerves, control experiments were made with a ligature placed around
the thoracic aorta by means of a large aneurysm needle passed between
the twelfth and thirteenth pairs of ribs. Results of compression of
the aorta by means of this ligature coincided.with those by means of
digital compression. Compression of the descending aorta increases the
volume of blood in the anterior portion of the cardio-vascular system
and thus raises blood pressure and also increases pressure and velocity
of blood flow in the coronary circuit and in the bulbar nerve centers.

Some of the experiments were made with the extrinsic cardiac nervous
mechanism intact; others with this mechanism interfered with by cer-
tain experimental lesions. These lesions consisted in severing the vagi, ©
removing the stellate ganglia, thus abolishing the influence of the ac-
celerator mechanism, dividing the dorsal roots of the spinal nerves,
and dividing both dorsal and ventral roots of the thoracic nerves.
Some experiments were also made on the relation of heart rate and blood
pressure to hemorrhage. Details of the various types of experiments
are given under the respective headings.

RESPONSE OF HEART RATE AND BLOOD PRESSURE TO COMPRESSION OF
THE ABDOMINAL AORTA

1. With cardio-vascular nervous mechanism intact. It was desired first
to determine the nature of the response of heart rate to changing blood
pressures with the cardio-vascular nervous mechanism intact. This
was done by compressing the abdominal aorta when the animal was
under different stages of ether anesthesia, and also when there had been

358 ETHEL W. WICKWIRE

interference with the blood supply to the bulbar nerve centers. The
data are presented in table 1.

By reference to table 1 it is seen that under light anesthesia compres-
sion of the abdominal aorta causes the carotid blood pressure to rise
immediately; during the continuance of the compression there is a
gradual but marked fall in carotid pressure until the aorta is released.
Then the pressure falls immediately to a point below its initial level.
Subsequently the pressure continues to fall, but soon rises to the level
before compression. The high carotid pressure produced by compres-
sion of the aorta is accompanied by a marked decrease in heart rate.
When the aorta is released and pressure falls the heart rate increases,
but does not immediately reach the initial rate. From the averages of

TABLE 1

Response of heart rate and blood pressure to compression of the abdominal aorta,
with cardio-vascular nervous mechanism intact. Heart rate in beats per
minute; blood pressure in mm. Hg, carotid artery

BEFORE
COMPRES- |DURING COMPRESSION] AFTER COMPRESSION.
SION

CONDITION

NO. OF EXPERIMENTS

2/8 | 3 2
: uh : Blood pressure a Blood pressure
$') 83) 3 g
ia) 2 ss
Light anesthesia............... 50 | 206} 124| 178} 197-166 | 193} 110—76-108

197| 101} 192} 164-155 | 191} 102-82-87
161} 56| 158) 103-97-100 | 147; 55-46-43
149| 107; 164| 145-149-140) 182) 103-84-101

a)
Or

Deep anesthesia...............
Restricted blood supply.......
Reversed reaction.............

ie)
“NI GO

fifty compressions upon thirty-five different animals under light anes-
thesia, there was thus a compensatory response of heart rate to changing
blood pressures and Marey’s law of inverse ratio of heart rate and blood
pressure applies.

Under anesthesia deep enough to abolish the corneal reflex the initial
blood pressure and heart rate are lower than under light anesthesia.
Compression of the aorta does not raise the carotid pressure as high
as under light anesthesia and there is not the marked fall in carotid
pressure during continuance of the compression. When the aorta is
released carotid pressure falls immediately to about the initial level. -
Subsequently it falls more gradually than under light anesthesia, then
rises very slowly. The high carotid pressure produced by compressing

RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 359

the aorta is usually accompanied by a very slight decrease in heart rate,
but this decrease is wanting in very deep anesthesia. When the aorta
is released and pressure falls the rate remains about the same or de-
creases slightly. From averages of twenty-five compressions on ani-
mals under deep anesthesia, it is thus seen that there is usually a slight
compensatory response of heart rate to blood pressure when the pres-
sure is high in the medulla oblongata; when the pressure is low, as upon
release of the aorta, this compensatory mechanism is seriously interfered
with and the return to the initial rate and pressure is either perma-
nently lost or very much delayed.

When the blood supply to the medulla oblongata is restricted, as in
hemorrhage or in some instances of partial ligation of the head arteries,
blood pressure falls and heart rate decreases. Now when the abdomi-
nal aorta is compressed the carotid blood pressure rises immediately,
as in light anesthesia, but not to the same extent. During the continu-
ance of the compression, blood pressure at first falls and then rises
slightly. When the aorta is released the carotid pressure immediately
falls to about the initial level, and then continues gradually downward.
When carotid pressure is high during compression of the aorta, the heart
rate decreases somewhat, but when the aorta is released and carotid
pressure falls the heart rate decreases with the falling pressure. If the
anemia of the medulla be severe or long continued, the return to the
initial heart rate and blood pressure is lost.

Under certain conditions there appears what may be termed a ‘‘re-
versed reaction.” This is sometimes observed if the abdominal aorta
is compressed: a, during the early period of recovery following exces-
sively deep anesthesia; b, during recovery from pronounced asphyxia;
c, shortly after the shat ickmnient of the blood supply to the medulla
oblongata after hemorrhage; and d, during restricted blood supply
from ligation of the head arteries. Instead of a compensatory slowing
of heart rate in response to the increased blood pressure, the rate in-
creases during the period of high pressure and continues to increase
even after release of the aorta and consequent fall of pressure.

After observing the occurrence of this so-called “‘reversed reaction’’
many times in the earlier experiments of this research and noting that
it appeared under the conditions above mentioned, it was decided to
try to reproduce this type of response under controlled conditions.
By referring to reactions 15 and 16 in the protocol of December 23, 1919,
(p. 360) it will be seen that this duplication was possible. This reac-
tion follows the recovery of the bulbar centers from anemia produced

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362 ETHEL W. WICKWIRE

by ligating the head arteries even when the anemia has been severe
enough to necessitate artificial respiration. The first of these compres-

sions (reaction 15) was made when the heart rate and blood pressure ~

' were falling; the second (reaction 16) shortly after and at a higher level

of heart rate and blood pressure brought about by the increased pres-
sure and velocity of blood flow in the medulla oblongata by the previous
compression of the abdominal aorta.

The most salient points of the facts above mentioned, when the cardio-
vascular nervous mechanism is intact, are: a, Under light anesthesia
there is a compensatory response of heart rate to changing blood pres-
sures. 6b, Under deep anesthesia and also when the blood supply to the
medulla is severely restricted there is a slight compensatory response of
rate to pressure when pressure is raised, but when pressure falls this
response is completely abolished. c, There seems to be a certain level
to which the functional activity of the bulbar cardio-vascular mechan-
ism may sink before permanent injury is done to these central cells, a
level at which subsequent increase of blood pressure and velocity of
blood flow will bring back their function, a previously decreasing heart
rate being accelerated and a blood pressure being restored equal to or
nearly equal to that before the insult to the bulbar mechanisms occurred.

2. Response of heart rate and blood pressure to compression of the ab-—

dominal aorta, with experimental lesions. The behavior of the cardio-
vascular mechanism to changes in blood pressure when experimental
lesions have been produced by sectioning some or all of the extrinsic car-
diac nerves is shown in tables 2 and 3. In these tables averages are
given to show the type of response of heart rate to changes in blood
pressure when the mechanism is intact, for comparison with the types of
response when various lesions in the mechanism have been .produced
experimentally. :;

In those experiments in which section of the extrinsic cardiac nerves
was ultimately desired, the routine procedure was ether anesthesia,
_ tracheotomy, exposure of vagi and dissection for exposure of the stellate

ganglia by Anderson’s method as described in Sherrington’s Mammalian

Physiology (3).

It will be observed that changes in heart rate and blood pressure due
to the operative procedures alone are not great. Marey’s law of inverse
ratio of rate to pressure holds upon compression of the abdominal aorta
after dissection for the exposure of both the vagi and the stellate ganglia,
but, in the case of the latter, at a slightly lower level of pressure and rate.

ae
eee

es
Ps ~

RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 363

The specific differences in the types of response, when lesions have
been produced experimentally in the extrinsic cardiac nerves, are here
set forth. :

a. Division of vagi, accelerators intact. If the abdominal aorta is
compressed after bilateral vagotomy (table 2), when the accelerators
are intact, the compensatory response of a lower heart rate to the in-
ereased blood pressure remains, but the degree of compensation is much
less than before division of the nerves. The level of pressure is about
the same as, and the rate slightly higher than, before section of the vagi.
When the aorta is released and pressure falls, the compensatory response
of a higher rate to a falling pressure is lost and a small decrease in rate
accompanies the fall of pressure.

TABLE 2

Response of heart rate and blood pressure to compression of the abdominal aorta,
after certain experimental lesions. Heart rate in beats per minute; blood pressure
in mm. Hg, carotid artery. Averages from experiments of November 12, 14, 19
and 26, 1919

BEFORE COMPRESSION| DURING COMPRESSION AFTER COMPRESSION
CONDITIONS IN SEQUENCE
OF OCCURRENCE
Mate, f paseure |> rate’. | -presoured/| ° ee mee emare
After tracheotomy...| 223 127 183 202-178 214 104-130
After dissection for
stellate ganglia....| 184 102 166 173-159 171 99-66-81
After bilateral yvagot-
gs ede Oi ar ee IBF = *90 181 171-168 | 178 | 103-75-76
After excision of ;
stellate ganglia....| 161 78° 156 135-133 149 87-61-68

b. Division of vagi followed by excision of accelerators. If the abdomi-
nal aorta is compressed after excision of the stellate ganglia, subsequent
to division of the vagi, there remains the compensatory response of a

decreasing heart rate to an increased blood pressure. Both rate and

pressure are at a lower level than when the stellate ganglia are intact,
the level of pressure being comparatively lower than that of rate.
When the aorta is released and blood pressure falls the heart rate does
not increase but decreases with the falling pressure.

c. Excision of accelerators followed by division of vagi. The deport-
ment under the conditions a and b may be contrasted with what occurs
when the accelerators are removed first (table 3). Here, immediately

364 ETHEL W. WICKWIRE

after removal of the ganglia, the pressure is found to have fallen greatly
and the rate to have decreased somewhat. During compression of the
abdominal aorta the cardio-vascular mechanism, after the first sudden
rise of pressure, compensates, not by the usual gradual fall, but by a
gradual rise of pressure which continues during the whole time of com-
pression; compensation of rate to pressure is greatly diminished, this
compensation being entirely lost after release of the aorta and the re-
sulting fall of pressure. Bilateral vagotomy itself, after the accelera-
tors are removed, causes a further small fall in blood pressure but an
increase in heart rate. Compression of the abdominal aorta now brings
the blood pressure higher, with the same inability on the part of the

TABLE 3

Response of heart rate and blood pressure to compression of the abdominal aorta,
after certain experimental lesions. Heart rate in beats per minute; blood pressure
in mm. Hg, carotid artery. Averages from experiments of October 24, 28 and 29,
1919

BEFORE COMPRESSION! DURING COMPRESSION AFTER COMPRESSION
CONDITIONS IN hah er
OF OCCURR Cc
fate’ | preesure| fater| (preeeure | tote” nn
After tracheotomy... 238 155 171 215-184 212 | 185-112-137
After dissection for
stellate ganglia.... 198 115 182 181-156 190 | 105~-75-106
After excision of stel- :
late ganglia........ 167 63 164 81-91 162 67-53-49
After bilateral vagot-
GY. Pee 178 52 177 79-101 170 79-61-49 ~

cardio-vascular mechanism to bring about a fall of pressure during the
maintenance of compression. Instead, the blood pressure continues
to rise and, when the aorta is released, falls more gradually than before.
The compensation of rate to pressure is almost lost during compression
and entirely lost when the pressure falls after release.

It is thus seen that when all the extrinsic cardiac nerves are severed
either the heart rate falls somewhat and blood pressure considerably, or
there may be a greater fall in heart rate and only a moderate fall in
blood pressure. The final result seems to depend on whether the vagi
or the accelerators are sectioned first. After division of the vagi, when ~
the accelerators are intact, the heart rate usually increases somewhat
though the blood pressure may remain the same or may fall slightly.

RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 365

While there is not sufficient increase in rate to influence pressure, the
maintenance of pressure seems in some way to be connected with the
accelerators (see Hunt, 4), as is shown by what happens when the stel-
late ganglia are subsequently removed, for then both the rate and pres-
sure fall. | |

Removal of the accelerators alone results in a moderate decrease in
heart rate and a great fall in blood pressure. Now when the vagi are
divided subsequent to excision of the stellate ganglia and the impulses ©
inhibiting the heart rate are removed, the rate increases somewhat
though it does not reach the rate it maintained before the accelerators
were removed, while the pressure continues to fall. After an interval
the latter sometimes rises, which rise may possibly be due to the re-
moval of the ‘‘tonic’’ effect of the cardiac nerves over the heart muscu-
lature (5), (6), (7), and a greater volume of blood entering the heart.

The’ following summary will serve to make more clear the above
statements:

Averages from experiments of November 12, 14 and 26, 1919

Rate - Pressure
Before vagi are sectioned............... 3,0: 0icKE MDs. Geena 183 98
After vagi are divided................. tealege ioe ates 188 ~ 96-98
After stellate ganglia are removed...................0.4 161 > 80
Percentage fall when vagi are divided first.............. 12 19.2

ee from experiments of October 4, 28 and 29, 1919

Rate Pressure
Before stellate ganglia are removed...................000- 195 116
meiner SLCLLOLeS DITO POMIOVE” .. 0... oni es cee ane nie nin tae waa 167 63
MMR URIS CIVRICU SY) foie. oo os phe eb a elu 178 “51
Percentage fall when stellate ganglia are removed first... 8.7. . 55

We might add that when the extrinsic cardiac nerves are divided the
type of response to compression of the abdominal aorta also seems to
depend on whether the vagi or ecopleratoxs are divided first, as we have
seen above (a, b and c).

With all the extrinsic cardiac nerves cut the heart rate is not so sus-
ceptible to the effects of low blood pressure as when the nerves are in-
tact. Thisis shown by observations made after the injection of sodium
nitrite and after hemorrhage. In both of these cases the heart rate is
relatively high even when blood pressure is approaching the base line.
This is to be contrasted with the gradual simultaneous fading-out of
rate and pressure when the nerves are intact.

366 ETHEL W. WICKWIRE

In this research it has been observed also that the left ventricle is not
firmly contracted after section of the cardiac nerves. This is in ac-

cordance with previous observations of other structures. It has been.

found by Bierfreund (cited by Pike, 8) that the onset of post-mortem
rigidity is delayed by the hemisection of the spinal cord. This occurs

on the hemisected side. Sherrington also found later that the onset of ©

rigor mortis in the hamstring muscles is delayed by section of the
appropriate dorsal spinal nerve roots of the same side.

TABLE 4

Response of heart rate and blood pressure to compression of the abdominal aorta,
after certain experimental lesions. Heart rate in beats per minute; blood pres-
sure in mm. Hg, carotid artery. Averages from experiments of June 6, 11, 16
and 17, 1919

BEFORE COMPRESSION] DURING COMPRESSION AFTER COMPRESSION
CONDITiONS IN SEQUENCE
ll aati ha Heart Blood Heart Blood Heart Blood
rate pressure rate pressure rate pressure
After tracheotomy and
laminectomy ........ 240 ‘108
After section of dorsal
spital roots..:.t25.. 230 85 196 | 152-142 200 72-61
After bilateral vagot- a
ro. 21 Wy Seepage “Was any a ae ol ORE 222 58 214 121-114 201 59-52
‘After removal of stel-
late ganglia.......... 187 34 183 71-82-81} 165 35-26
After section of dorsal roots
Light anesthesia... .. 223 78 189 | 155-134 201 | 88-64-61
Deep anesthesia. .... 191 58 191 | 112-101 186 | 55-47

d. Section of dorsal roots of spinal nerves..: The data obtained on the
response of the cardio-vascular mechanism to changes in blood pressure
after section of the dorsal spinal roots in a region as extensive as cervical
6-7 to lumbar 1-2 are presented in table 4.

Section of the dorsal spinal roots in this region does not abolish the
compensation of heart rate to high and low blood pressures. The law
of inverse ratio of rate to pressure still holds true and to practically the

same extent during compression of the aorta and the resulting increased _

pressure. This is shown also by the following brief summary from the
data of tables 1 and 4. | |

at> ae
Pee Tome

RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 367

During compression of the abdominal aorta and high blood pressure.
After tracheotomy, decrease in heart rate = 13.6 per cent.
After laminectomy, decrease in heart rate = 15.8 per cent.
After section of dorsal roots, decrease in heart rate = 14.7 per cent.

When the aorta is released and pressure falls, the degree of increased
heart rate to the falling pressure is less after section of the dorsal roots
than before:

After release of the abdominal aorta and falling blood pressure.
Before section of dorsal roots, increase in heart rate = 7.7 per cent.
After section of dorsal roots, increase in heart rate = 2.0 per cent.

The similarity of response after section of the cardiac nerves before
and after section of the dorsal roots is to be noted. Bilateral vagot-
omy after section of the dorsal roots produces a somewhat lower level
of pressure than it does when these roots are intact. Removal of the
stellates, subsequent to bilateral vagotomy after section of the dorsal
roots, results in a much lower level of rate and pressurethan occurs when
these cardiac nerves are divided before the dorsal roots are cut.

Previously it has been noted that after section of the extrinsic cardiac
nerves compensation of rate to pressure holds to some extent during
_ high pressure, as during compression of the abdominal aorta, but is
lost after release of the aorta“and a falling pressure. This is also true
when the extrinsic cardiac nerves are divided after section of the dorsal
roots, although the fall in rate after release of the aorta is greater than
when the dorsal roots are intact. Sherrington (9) notes the general
maintenance of arterial] pressure after section of the dorsal spinal
roots in the thoracic region.

There seems to be a greater sensitivity of the cardio-vascular mech-
anism to deep anesthesia after the dorsal roots are sectioned than before.
This is shown not only by the complete lack of compensation of rate to
pressure when the abdominal aorta is compressed, but by both rate and
pressure falling to a much lower level than when the roots are intact.

e. Section of dorsal and ventral roots of thoracic spinal nerves followed
by stimulation of sciatic nerve. The usual response of an increased heart
rate and blood pressure to stimulation of the sciatic nerve is shown in
figure 1 and table 5. Here it is also seen that this familiar response
to sciatic stimulation seems to be abolished after section of the spinal
nerve roots, thoracic 1-5, independently of whether weak or strong
stimuli are used.

368 ETHEL W. WICKWIRE

It is impossible to say, on the basis of the number of experiments
done, whether the above lack of response to sciatic stimulation is a
necessary effect of section of these roots, or whether it is due to some
error of technique which has not yet been detected. Section of these
roots, which lie in the region of the emergence of the cardiac acceler-
ators does not, however, abolish the compensatory ratio of rate to pres-
sure upon compression of the abdominal aorta but does alter the type
of response of the cardio-vascular mechanism to changes in pressure from
that seen when the accelerators alone areremoved. Instead of a steady
climb of pressure upward, when the accelerators only are removed, after
root section pressure first rises and then steadily falls. And there re-
mains a slight compensation of an increased rate to the falling pressure

After Stim- Stimu- After
laminectomy lation lation |bilateral vagotomy
and before After section of of -|following section

°
section of sciatic |spinel nerve roots,| Sciatic | of spinal nerve
spinal roots nerve Thoracic 1-5 nerve

200
ovy

150

100

50

fal
Vv

Fig. 1. Response of heart rate and blood pressure to stimulation of the sci-
atic nerve and to compression of the abdominal aorta, after certain experimental
lesions. Lower curves represent the pressure in the carotid artery; figures at the
left indicate actual pressures in mm. Hg. Upper curves represent the rate of
the heart; figures at the right indicate rates per minute. A = point of compres-
sion of abdominal aorta; C = point of release of aorta.

when the aorta is released, which is totally abolished when the stellates
only are excised. An increased rate follows bilateral vagotomy after
section of these spinal roots as it does when the vagi are sectioned after
removal of the stellates. But when the aorta is compressed, after root
section and bilateral vagotomy, the pressure rises steadily and much
higher than when the cardiac nerves alone are cut, and the heart rate
falls with the falling pressure after release of the aorta.

Further investigation of the results of section of these spinal roots in -

combination with lesions in other parts of the cardio-vascular mechan-
ism will probably prove of much interest.

ee a

RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 369

TABLE 5

Response of heart rate and blood pressure to stimulation of the sciatic nerve and
to compression of the abdominal aorta, after certain experimental lesions.
Heart rate in beats per minute; blood pressure in mm. Hg, carotid artery

BEFORE COMPRESSION DURING COMPRESSION AFTER COMPRESSION
' CONDITIONS IN SEQUENCE :
ac aiding oe Heart Blood Heart Blood Heart Blood
rate pressure rate pressure rate pressure
After laminectomy..... 203 107 170 | 157-155 179 |86—75-83
Stimulation of sciatic
os ee aa aa ee 205 107-113
After section of spinal
nerves, thoracic 1-5.. 147 58 131 | 80-109-91 136 53-45-50
Stimulation of sciatic
MORO ics. is. se 6 cay No effect
After lateral vagot-
BO Bic s oo nn ie 169 67 160 |82-117-152| 150 |80-58-—50

RESPONSE OF HEART RATE AND BLOOD PRESSURE TO HEMORRHAGE

The réle played by the accelerators is well shown in one particular
group of experiments, for when severe hemorrhage is followed by divi-
sion of the vagi there is a conspicuous increase in heart rate, though not
enough to rescue the slightly falling pressure. The accelerators un-
doubtedly respond to the emergency of a falling blood pressure. The sum-
mary below presents the data of three experiments.

HEART RATE vat nga
February 10, 1919. Before hemorrhage............... 183 150
After hemorrhage................ 156 56
After bilateral vagotomy......... 160 60
February 17, 1919. Before hemorrhage.......... rae 163 162
After hemorrhage ...... Tia com 156 54
After bilateral vagotomy......... 204 56
February 21, 1919. Before hemorrhage............... 148 120
After hemorrhage................ 125 34
After bilateral vagotomy......... 141 22
Averages: Before hemorrhage....................-+45- 165 144
Mier MOMMOTIOAge...... «2... eee ene aey 146 48
After bilateral vagotomy.................. 168 46

370 ETHEL W. WICKWIRE

The réle of the accelerators is further elucidated by an examination
of the data obtained when hemorrhage follows excision of the accelera-
tors on one or both sides with or without bilateral vagotomy (table 6).

The above table presents some interesting comparisons. A hemor-

_rhage of sufficient volume to cause a great fall in blood pressure before _

a break in the heart rate occurs, when the cardio-vascular mechanism is
intact (1), causes a somewhat greater fall in pressure and nearly a 50
per cent decrease in rate when the accelerators are excised (2).. With

TABLE 6

Response of heart rate and blood pressure to hemorrhage in connection with cer-
tain experimental lesions in extrinsic cardiac nerves. Heart rate in beats
per minute; blood pressure in mm. Hg, carotid artery

HEART RATE BLOOD PRESSURE
Fem alata deat Before After Before After
? hemor- | hemor- | hemor- | hemor-
rhage rhage rhage rhage
1. Experiment April 1, 1919.
Cardiac néfves ntaets. od. a: Gok 180 180 100 24
Percentage change. .« i. -).< n44 5 esi ekass a 75
2. Experiment December 9, 1919
Accelerators excised (vagi intact)....... 158 90 _ 80 16
Percentage change; . 2s 0.5.6 0s is pneigst a 48 80
3. Experiments October 29 and December
17, 1919
Cardiac nerves excised (accelerators
out first + bilateral vagotomy)...... 160 150 71 16
Percentage change. ...........2.90535.4 6.2 77.4
4. Experiment December 13, 1919
Excision of left stellate; bilateral va-
gotomy (right stellate intact).......... 216 210 126 28
Percentage ehangeé... oi... 64.4.) 7teoe 1.8 77.7

all the extrinsic cardiac nerves severed (3), the fall in pressure is nearly
as great but with only a slightly lower rate (6.2 per cent decrease) than
before hemorrhage occurred. With the accelerators of one side intact
(4) the fall in pressure is about the same as when all the cardiac nerves
are severed but the decrease in rate is somewhat less (1.8 per cent).

In the last two instances it is again seen that the rate is not so susceptible

to low pressure when all the extrinsic cardiac nerves are cut.
Various writers have called attention to the fact that the difference in
response to stimulation of the accelerators depends principally on the

/
RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 371

frequency of rhythm before excitation. It seems also that this differ-
ence in response depends upon the level of blood pressure during excita-
tion. This is shown by results obtained when the accelerators are
stimulated during periods of a diminishing blood volume produced by
a series of hemorrhages. When the pressure is low, but the rate yet
high, it is found that there is not as great an increase in rate from stimu-
lation as when the pressure has fallen lower from a greater loss of blood
and the rate too begins to decrease. It would thus appear that there is
a level of pressure at which the effect upon rate from stimulation of the
accelerators is greatest, other factors being equal. It may also be said
that the accelerators respond to artificial stimulation when the blood
pressure has fallen much below this level of greatest response to excita-
tion. »

DISCUSSION

In the beginning of this paper it was stated that this work was under-
taken with the object of arriving at an understanding of the reciprocal
functional relationships existing between the cardiac and vascular ner-
vous mechanisms. That this relationship exists can hardly be doubted
when we remember that both heart and blood vessels have their own
particular functionally antagonistic innervations. To the heart go
efferent fibers that carry impulses from the central nervous system
which increase or diminish its rate, and to the blood vessels go efferent
fibers that bring about changes in blood pressure by dilating or con-
tracting their vascular walls. The nervous centers specifically con-
cerned with the regulation of this cardio-vascular mechanism, located
in the region of the medulla oblongata, have a wide relation with af-
ferent nerve fibers from all parts of the body, including the heart itself.
This makes it possible for the heart and blood vessels to be directly or
reflexly influenced in their behavior. Changes in rate and amplitude of
the heart beat, differences in volume and tone of the heart musculature,
and changes in the peripheral resistance may all be brought about by
nervous impulses or by changes in the character of the blood flow
through the medulla oblongata. With this in mind and remembering
that heart rate is closely related to blood pressure and that conditions
which affect one influence the other, we can now consider the signifi-
cance of the differences shown in the behavior of this mechanism when
various parts of its regulatory system have been abolished.

The differences, we may say at the outset, are closely related to con-
ditions in the bulbar mechanism. When it is interfered with by partial

372 ETHEL W. WICKWIRE
+

or complete anemia, asphyxia or too deep anesthesia, the effect on rate
and pressure following section of the cardiac nerves is not as great as
when the blood supply to the medulla is normal. And we can say also,
from observation of many experiments, that the vasomotor center
seems more susceptible to such changing conditions when part of the
nervous mechanism of the cardio-vascular system is eliminated. When
bulbar conditions are favorable there is a reciprocal response on the
part of the vasomotors, and blood pressure gradually rises after its first
fall following section of the cardiac nerves. If the accelerator branches
from the stellate ganglia have been sectioned this rise of pressure is ac-
companied by a marked increase in heart rate, which is possibly, even
probably, due to accelerator impulses that reach the heart through the
cervical ganglia (10), (11). But if, instead of a mere section of the ac-
celerator branches, the stellate ganglia have been removed this rise of
pressure is unaccompanied by a change in rate. In many other cases
of a steadily falling pressure and presumable failure of the vasomotors,
pressure can be raised by manipulation of the lower extremities and
abdomen and a redistribution of the blood.

The ultimate heart rate and blood pressure after section of the car-

diac nerves seems to depend on whether the accelerators or vagi are
divided first. In each case the partially intact mechanism apparently
adjusts itself to the altered conditions, but the manner of adjustment
differs with the sequence of section. With the accelerators removed
and the vagi intact, the rise of pressure during manual compression of
the aorta is not as great as when the impulses coming over the vagi are
also blocked out. It is as if this inhibitory mechanism were standing
guard against a rising tide of pressure that might do violence to the
best interests of the organism. When the vagi are divided, with the
accelerators intact, pressure from compression of the aorta rises higher
than when the accelerators are excised. The fall in pressure, after re-
lease of the aorta, continues longer and is greater than before excision of
the accelerators. With the accelerators removed it is as if the cardio-
vascular mechanism had lost part of its efficient protection against a
falling blood pressure and the resultant harm to the multitude of tissue
cells. .
These results would lead one to the conclusion that when an increased
heart rate occurs in the organism through the mediation of the cardio-
vascular nervous system, it is due to a positive activity on the part of
an accelerating mechanism and not to some mechanism that inhibits the
activity of the inhibitors.

a a” — a
2 .
oo

RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 373

This idea of a mechanism of inhibition of inhibition for the regulation
of heart rate and blood pressure in the cardio-vascular system may be
said to begin with the discovery in 1845, by the Weber brothers, that

_ stimulation of the vagi inhibits the heart rate. E.H. Weber extended

the notion of inhibition to the increased spinal activity after ablation of
the brain. Following the Webers came Setschenow, who found that
stimulation of the midbrain and bulb prolongs reflex time and also that —
a frog draws its foot from acidulated water much later if the brain be
stimulated. But it was not until after his work, m 1863, that ‘this
notion of the inhibition of activity of one part of the central nervous
by the activity of another became a working physiological thesis’’ (12).

Later it was shown by L. N. Simonoff (1881) and by A. Herzen (1884)

that inhibition of spinal reflexes was obtained from other foci in the
brain and cord itself. This was done by sensory stimulation or by
direct stimulation of the foci themselves. ‘But,’ as Pike has said,
“in all this infatuation with the hypothesis of inhibition, its devotees
lost sight of the accompanying positive motor phenomena. Until some
reason is advanced for this oversight, the hypothesis of inhibition is

- open to suspicion of being overworked.”

In the light of later work on the general life processes of biological
mechanisms, it seems that Setschenow’s interpretation took color, as so
often happens, from the depressing political and economic conditions
of his time. If the degree of activity of the inhibiting center is itself
dependent upon inhibition, and if the processes in the organism go on
in the interests of the organism, as Burdon-Sanderson phrased it, why
is there not then this activity of inhibition of inhibition when the ac-
celerators are removed so that the heart rate and blood pressure may be
kept up? The activity of the inhibiting vagi, however, is not stopped
until the impulses coming over the vagi have been blocked out by double
vagotomy or by atropine, or if it is stopped, the results are not of the
same magnitude as when the accelerators are present.

It seems rather difficult to apply an hypothesis of inhibition of inhi-
bition for the regulation of heart rate and blood pressure in the cardio-
vascular mechanism in view of results obtained in this series of experi-
ments and the facts just stated.

My results however, do, I believe, provide sufficient evidence to
postulate a simpler mode of action of the inhibitor-accelerator nervous
mechanism, This nervous mechanism consists of: a, An inhibitor
mechanism acting positively in the best interests of the organism; i.e.,
to prevent overwork by a too rapid heart rate and to bring about suf-

374 ETHEL W. WICKWIRE . 4

ficient periods of rest for metabolic repair of the heart musculature.
The exercise of this function of the mechanism is not regularly medi-
ated by means of inhibitory influences acting from without upon the
mechanism itself; b, an accelerator mechanism acting positively in the
best interests of the organism and intimately connected with the main-
tenance of blood pressure. These two functionally different parts of
this mechanism possess different thresholds of sensitivity to the same
forms of stimuli and varying degrees of sensitivity to each of the various
physico-chemical changes occurring within the organism.

Such an hypothesis is in complete accord with the physiological and
pharmacological antagonism of other systems innervated, as is this
cardio-vascular system, through the cranio-sacral and thoracico-lumbar
divisions of the autonomic nervous system.

One further point comes out with regard to the organization and
general deportment of the cardio-vascular nervous mechanism, sug-
gested by Prof. F. H. Pike. Hee

The degree of organization, to use Hughlings Jackson’s expression,
of the neural portion of the cardio-vascular mechanism is high; the
responses are, in general, very specific in character, and marked by the

absence of any great variety of combinations. Marey’s law expresses —

a definite and relatively unvarying relationship existing between two
very definite conditions. The result may be interpreted as being in
the interests of the organism. The organization of the central mechan-
ism is not, however, so rigid as to prevent dissociation of these two
reciprocal responses of heart rate and blood pressure when the condi-
tions are such that it is in the interest of the organism for this disso-
ciation to occur. As an illustration of this we may cite the deportment
of the cardio-vascular mechanism when the blood supply to the medul-
lary portion of it is reduced. If the central mechanism demands a cer-
tain definite set of conditions in the way of volume of blood flow and
blood pressure for the maintenance of its function, it is obvious that
such conditions can be kept uniform only if the whole mechanism has
certain powers of adaptation to changing conditions. The adaptation
expressed in the sequence of events described by Marey’s law is an
adaptation to one set of conditions. If conditions should change in
such a way as to demand a high systemic blood pressure for the fulfill-
ment of the requirements of the central mechanism in the way of volume

of blood flow and blood pressure, a falling heart rate in response to such —

a high systemic blood pressure would be distinctly not in the interest of
the organism. Haldane has suggested that the rigidity of the deport-

——

j

RECIPROCAL REACTIONS IN CARDIO-VASCULAR SYSTEM 375.

ment of the respiratory mechanism’ may, under certain conditions, act
contrary to the interest of the organism. On that view, the dissocig-—
tion of the normal response to high systemic blood pressure. is ‘to: be
regarded as an adaptation to restriction of the volume’ of blood ‘flow
through the medulla. It would appear that in the case of the cardio-
vascular mechanism as well as in the case of the respiratory mechanism,
it is the conditions in the central mechanism itself whale in part, at :
least, determine its degree and state of activity (13). (gt (2)
This dissociation of the usual reactions of the sddicsetamoula edd

anism is analogous to the dissociation of res sponses in vertigo, as pointe

out by Wilson and Pike.

ir] J ao. |
ta 4
! ‘)

To Prof. F.'H.'Pike I offer grateful eevee reakeh)

SUMMARY

1. Marey’s law of inverse ratio of rate and pressure holds in light
anesthesia (deep enough, however, to insure complete anesthesia).

2. This compensation of rate and pressure is lost or seriously inter-
fered with in deep anesthesia; also, in restricted blood supply to the
medulla. |

3. What may be termed a ‘‘reversed reaction” appears under certain
conditions of anemia of the bulbar mechanism. There is dissociation
of the usual reactions of the cardio-vascular mechanism.

4. The accelerators undoubtedly respond to the emergency of a fall-
ing blood pressure.

5. When the accelerators are excised there is a greater percentage
change in rate and pressure both falling lower after hemorrhage than
when the accelerators and vagi are cut.

6. These experiments demonstrate the tonic activity of the acceler-
ators.

7. The ultimate heart rate and blood pressure, after section of the ex- -
trinsic cardiac nerves, depends on whether the vagi or accelerators are
excised first.

8. Section of the dorsal spinal roots, cervical 6-7 to lumbar 1-2, does
not abolish the compensatory response of heart rate to high and low
blood pressures.

9. When the extrinsic cardiac nerves are divided the musculature of
the left ventricle remains more flaccid after death than when the car-
diac nerves are intact.

_ 10. There is an inhibitor-accelerator nervous mechanism acting posi-
tively for the control of the cardio-vascular system.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 3 \

376 ETHEL W. WICKWIRE

BIBLIOGRAPHY

(1) SHERRINGTON: The integrative action of the nervous system, 1916.
(2) Marey: Journ. d. la physiologie de l’homme et des animaux, 1860.
(3) SHzeRRINGTON: Mammalian physiology, 1919.
(4) Hunt: Journ. Exper. Med., 1897, ii, 2.
(5) Lucrant: Human physiology, 1911, i.
(6) Stewart: A manual of physiology, 1918.
(7) Tigmrstept: A text-book of human physiology, 1906.
(8) Pike: Quart. Journ. Exper. Physiol., 1913, vii, 1.
(9) SHeRRINGTON: Schafer’s text book of physiology, 1900, ii, 869.
(10) Ranson: Journ. Comp. Neurol., 1918, xx.
(11) Spapouinti: Arch. d. Fisiol., 1916, xv, 70.
(12) ScnarFrEeR: Text-book of physiology, 1900, ii.
(13) Pixr, Coomss anp Hastines: This Journal, 1919, xlix, 125.

FURTHER OBSERVATIONS ON THE RELATION OF INITIAL
LENGTH AND INITIAL TENSION OF AURICULAR
FIBER ON MYO- AND CARDIODYNAMICS

ROBERT GESELL

From the Department of Physiology of Washington University Medical School and
of the University of California

Received for publication June 24, 1920
INTRODUCTION

In a previous paper (1) the relation of initial length and initial ten-
sion of auricular muscle to strength of muscular contraction was pointed
out in a qualitative way. The present paper represents a more exact
and quantitative study of the same problem.

Blix (2) demonstrated in the case of striated muscle of the frog that
tension developed is a linear function of initial length of muscle fiber.
Evans and Hill (3), employing the same muscle, find heat and tension
developed to increase in direct proportion to the increase in length of
muscle through an extension of the original unloaded length amounting
to approximately 15 per cent. These changes in length, according to
Evans and Hill, approximate those normally occurring in situ.

The auricle of the turtle may undergo far greater changes in length
of fiber and consequently is a suitable structure not only for determining
whether cardiac muscle follows the same laws as striated muscle but
also whether the linear function of tension developed holds for these
great changes in length of fiber.

It was demonstrated (1) that an increased auricular volume was
accompanied by a striking increase in strength of contraction. Though
the results indicated at that time that length of fiber was by far the most
important factor influencing the strength of contraction yet some re-
sults were obtained suggesting that initial tension might play a minor
role in determining the force of contraction. Consequently methods

'. were used to determine the relative importance of both initial length

and initial tension of the cardiac muscle.

377

378 ROBERT GESELL

METHODS

It.is realized that the parallel fibered muscle offers the best condition
for studying the strength of muscular contraction; that the interlacing
fibers and the reticular arrangement of the bundles of fibers in the
auricles may offer difficulties. These difficulties are largely met by
recording the pressures developed within the intact auricle rather than
the tension developed by a strip of auricular tissue. The method neces-
sarily introduces factors involved in the mechanics of the hollow sphere
and the results therefore have an additional interest in supplementing
earlier papers on the relation of cardiac volume to cardiodynamics
(1), (5), (6) and (7).

_ The methods employed are similar to those previously described (1).

The auricle suspended in saline solution in a stoppered vessel is con-
- nected with a membrane manometer and a reservoir also containing
saline solution. A simple maximum, minimum and mean valve devised
- for these experiments is interposed between the auricle and the reservoir
to permit the recording of isometric contractions with progressively
changing auricular volume (initial length). The changes in auricular
volume are recorded with a piston recorder in connection with the air
space above the saline solution in which the auricle is supsended.

In the records shown, the upper are the volume tracings, the lower
are the tension tracings. Upstrokes represent respectively an increase
in volume and increase in tension; downstrokes, the reverse.

In these experiments we are interested not only in the aingiae
of pressure developed by the entire auricular musculature which is
recorded directly with the membrane manometer but in the actual
amount of tension supported per unit of muscle as well. To determine
this we must take into account the changing auricular surface for the
same amount of musculature spreads over a changing surface all of
which is supporting the recorded pressure. Assuming the auricle to
be spherical (it is of course only roughly so) and knowing its volume,
the inner auricular surface can be computed. Multiplying this by the
recorded initial and final pressure we get the tension per unit of muscle.

' RESULTS

The data of this research were obtained from twenty-two experi-
- ments on the auricles of nine turtles (Pseudemis elegans). Tables are
published for three of these experiments accompanied in two instances
by the records from which the data were obtained. The data are tabu-

:
;
%

MYO- AND CARDIODYNAMICS 379

lated in the following way (see table 1): Column 1—number of contrac-
tion; columns 2, 3 and 4—initial pressure, final pressure and pressure
developed or the difference between initial and final pressure—all in
millimeters of water as recorded by the manometer (these are the
intra-auricular pressures sustained and developed disregarding the
changing inner auricular surface) ; columns 5, 6 and 7 represent auricular
volume, inner auricular surface and length of auricular fiber in cubic,
square and linear millimeters respectively; columns 8, 9 and 10 the
tension sustained and developed taking into account the changing
auricular surface. These are given in weight (in cubic millimeters of
water) sustained by the total inner auricular surface.

Of this data final tension and tension developed are plotted on the
ordinates against length of fiber on the abscissas, in figure 1, a, b, cand
d. An idea of the changes in initial tension sustained is obtained by
noting the vertical distance between the curves of final tension and
tension developed. In figures 4, a, b, c, d and e the curves of initial
tension are plotted as well.

THE RELATION OF INITIAL LENGTH OF FIBER TO STRENGTH OF MUSCULAR
CONTRACTION

Previous results (1) on the auricle of the turtle pointed to initial
length of fiber as the important factor determining the amount of ten-
sion developed agreeing with the conclusions of Patterson, Piper and
Starling (9) on their work on the mammalian ventricle. My conclu-
sions were reached from records similar to figure 3 of the present paper.
It will be noted in this record that the strength of contraction pro-
gressively increases as the auricle distends and also that the recorded
initial pressure, i.e., the pressure obtaining at the onset of auricular
systole, is practically constant. Initial length of fiber was apparently
the only variable at work influencing the strength of muscular contrac-
tion. Unfortunately I had overlooked the factor of increasing intra-
auricular surface accompanying the increase in initial length of auricular
fiber. We know that whereas the circumference of a sphere increases
directly as the radius, the surface of a sphere increases as the square of
the radius. Applying this fact to observations on the auricle as illus-
trated in figure 3 in which initial intra-auricular pressure remains con-
stant, it is obvious that initial length of fiber is not the only variable.
On the contrary, since the same amount of muscle is spreading over an
increasing surface all of which sustains a constant pressure, the initial

380 ROBERT GESELL —

tension of the individual fibers increases as the square of the radius
whereas the length of fiber increases only as the first power of the radius. —
Although Patterson, Piper and Starling (9) try to differentiate between -
the effects of initial tension and initial length of fiber, it appears that
they, too, had overlooked the effect which an increasing surface must
have upon the initial tension of the individual fibers.

We might in various ways argue the relative effects of initial tension
and of initial length of muscular fiber, yet it is more satisfactory to dis-

ee

sociate these factors by direct experiment. We have noted:jin some
few experiments that as the length of fiber increased, a synchronous
decrease in initial intra-auricular pressure occurred. Such results were
obtained when the auricle was undergoing oscillations of tone by allow-
ing saline to enter the auricle during the phase of tonic relaxation. Since
the intra-auricular pressure may decrease out of proportion to the
increasing surface and since tonic relaxation per se is detrimental

Fig. 1

MYO- AND CARDIODYNAMICS 381

rather than beneficial to strength of contraction, it appears that the
increasing strength of contraction invariably accompanying increasing
length of fiber is primarily due to the increasing initial length of fiber.

Having dissociated the effects of initial tension and initial length of
fiber under the conditions just described it is of interest to analyze the
quantitative data on the relation of strength of contraction to initial
length of fiber.

The results of a series of contractions with progressively increasing
length of muscle fiber are tabulated and plotted in table 1 and figure
1, a. At contraction number 8, the auricular volume is 480 cmm. and

TABLE 1 |

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) |
4 abs : ce hee % D
ol bie| aoe beeen | 4 Ge tent | Be th Bee! ia.
E BE | EBB |zeze | < ie Bz ae
¢ | 328/522 |SS8ba| B, | 32 | BB cr Ba | Ba:
B Epa | 4Da |eeZscpl 82 Ba 228 ce 42 at
S ia a” eels ag a cha %° ge Bo
mm. H2O\mm. H:0| mm. H20| cmm. | sq. mm. mm. jomm. HO emm. H:0 cmm. HO
ie ee 2.5 1.5 480 | 296 | 30.5| 296 740 | 444
6 1.4 We Gi i 835 | 426 | 36.6] 597 1622 | - 1024
9 1.7 eo; 33 1180 | 508 | 39.9] 863 2540 | 1676
13 2.4 6.0| 3.6 1644 | 672 | 46.9| 1615 4038 | 2419
19 3.1 711 4.0 | 2200] 818.0} 50.6 | 2535 5807 | 3272
25 3.7 7.7| 4.0 | 2596] 914.0] 53.6 | 3382 1038. | 3656
31 3.9 8.2| 4.3 | 2931} 990.0) 55.7] 3861 8167 |: 4257
37 5.0 9.0| 4.0 | 3161 | 1042.0) 57.2} 5210 9378 | 4168
43 5.6 9.5| 3.9 | 3335 | 1078.0) 58.2 | 6036 1024 | 4204 -
53 6.2 9.9| 3.6 | 38508 | 1117.0} 59.1 | 6961 10948 |° 3987
63 6.5. | 10.2] 3.7 | 3623 | 1140.0} 59.8] 7410 | 11628] 4218.
70 6.7 10.3| 3.6 | 3662 | 1148.0) 60.0| 7691 11824 | 4132

at contraction number 70 the volume is 662 cmm. Assuming the
auricle to be spherical the length of fiber with number 70 is 60 mm.
approximately 196 per cent greater than with number 3. The final
tension sustained at number 70 is 1627 per cent greater than the tension
sustained at number 3. The tension developed is 930 per cent greater
than that developed at number 3.

The curves of final tension and tension developed are plotted in figure
1, a. Final tension is represented by a curve with the concavity up-
wards whereas tension developed is represented by an approximately
straight line. Final tension represents two processes, sustenance of

882 ROBERT GESELL

initial tension and development of tension. Sustenance of tension
presumably is a non-energy consuming process. According to Hill,
tension developed represents the energy consuming processes of muscu-
lar contraction in which we are particularly interested.

':Three similar experiments are represented in figure 1, b, ¢ and d.
The tables are omitted. The fluctuations in curves 1, b and 1, ¢ are
due to tonus oscillations and do not concern us here. Ignoring then
these oscillations we note again that: tension developed is represented
by: a straight line. Though in some few experiments (see fig. 4) the
curve of tension developed is not as straight as in figure 1, a, b, ¢, d,
and though the computations for the curves are based on the assump-
tion that the auricle is perfectly spherical, the results on the whole
indieate that cardiac muscle behaves to extension as does striated
muscle; that in cardiac muscle tension developed is a linear function of
initial length of muscle fiber. Of particular interest as concerns the
theories of muscular contraction is the fact that this relation holds for
such great variations in initial length of muscle fiber, far greater than
those occurring in’ striated muscle.

. THE RELATION OF INITIAL TENSION TO STRENGTH OF MUSCULAR
ere i CONTRACTION

: When an auricle executes a series of isometric contractions with a
constant volume the strength of contraction remains constant. o Tf;
daring such a series of contractions, a tonus oscillation occurs, the
strength. of contraction varies even though the length of muscle fiber
must necessarily remain unchanged. It is found that final tension and
tension developed increase with the rise of initial tension synchronous
with tonic contraction. These results suggest the beneficial effect of
‘initial tension upon muscular contraction but unfortunately the change
‘4ni Initial tension is a result of a tonus oscillation which: in itself may be
aap factor influencing the clonie contraction. Roh

- Another method may be .uséd for dissociating the’ abut, of initial
bial and initial length of fiber. If a series of isometric contractions
“is obtained ‘first’ with increasing and then with decreasing length of
fiber, as in figure 2, it is seen that for any given initial length of fiber
‘the initial tension is higher in the first series of contractions in which
-the .auricular volume is increasing, than in the second series with
\decreasing volume. Other things being equal, it would follow that if
jinitial tension exerts a beneficial effect on muscular contraction the

MYO- AND CARDIODYNAMICS

ZBI

PAUALINANAIINUDUANMIOARDNOGAUSSTOOODORINSDUA ROLDAN SDDIONODAAASONDANINDRAVOORATINEODAVDRSCTUERULINVGTETOROAhAOIIIIINIIDDT

cy Ui
ie oe ase BM

6s
4) a a (9 {6 he % ts bh 44 sh i i =f ae

\NANAA

he eg
1969.59 4p gy og 55 519 bh TA HS

384 ROBERT GESELL

tension developed per given length of muscle fiber should be greater in
the series with increasing length of fiber than in the series with decreas-
ing length of fiber. Before discussing the results, attention should be
called to the method employed in obtaining such records and its influ-
ence on the interpretation of the results. In the first series with in-
creasing length of fiber, the reservoir is raised to insure an adequate
filling pressure. The valve is turned to the maximum position per-
mitting the solution to enter but not leave the auricle. Aside from the
diminution in volume permitted by capacity change of the manometer,
the contractions are isometric. In the second series with the initial
length diminishing, the reservoir is lowered to zero pressure and the
valve turned to mean position allowing a small but continuous leakage

* TRIN Wy f

Be AT gs Ks Bie
my W 41-93 46 2).
, \
SF;,

BY)

rete hl hl;

ATT TELLTALE

Fig. 3

from the heart back to the reservoir. ‘The resistance in the valve is
high enough to permit the development of large pressure changes dur-
ing contraction so that the contractions approach isometricity but not
as closely as the contractions associated with increasing length of fiber.
In consequence, a recorded tension in the second series indicates a
stronger contraction than the same recorded tension in the first series.

The data obtained from figure 2 are given in table 2 and plotted in
figure 4, a,b andc. Figure 4, a represents the curves of final tension,
4, b, curves of initial tension and 4, c, curves of tension developed. ‘The
curves of the first and second series are designated 7 and 2 respectively.
Final tensions sustained in the two series are nearly equal. The curve

MYO- AND CARDIODYNAMICS 385

of initial tension sustained is, however, markedly lower in series 2 than
in series 1. The curve of tension developed therefore is greater in
Series 2 than in series 1. Such results might suggest either that initial
tension may have a detrimental effect upon muscular contraction or that
since the curves of final tension sustained run nearly the same course
that muscle at a definite length, other things being equal, is able to
sustain a definite final tension—the actual tension developed being -
‘dependent upon the level to which the initial tension falls between
contractions.

Fig. 4

The experiment represented in figures 3 and 4, d and e, and table 3
bear on these two suggestions. Here again initial tension is higher in
the first series than in the second series. The difference in initial
tensions, however, is so slight that a marked effect upon the strength of
contraction could hardly be expected. The greater tension developed
in the second series is primarily due, in this experiment, to the higher
final tension rather than the lower initial tension.

Not knowing of the work of du Bois Reymond (10) and Osborne and
Sutherland (11), I sought for more data in the behavior of inflated and

386

ROBERT GESELL

TABLE 2

=
—_
—

CONTRACTION

~
fs
A
=-_
on
—

INTRA=-
RICULAR PRES-

AURICULAR
PRESSURE
AURICULAR
PRESSURE

AND FINAL AU-
SURE

TWEEN INITIAL
UME

FINAL INTRA-
DIFFERENCE BE-
AURICULAR VOL-

INITIAL

=
for)
~

INTRA-AURICU-
LAR SURFACE

-o
wy
—
C)
—_

RICULAR MUS-

CLE
INITIAL: TENSION -~

LENGTH OF AU-
SUSTAINED

FINAL TENSION
SUSTAINED |

(9)

as

SESSRERBSR

3
S

ee ie eee oe
aor OK CO CO W

H Or Or
aoron

OONNAWNIanokrown
=
cs

a ae eee ee

chaos in
Now

et et DO DO DD tO Ww
OnmWDwWoonoaonn

Dw Ph OO
Dm 0OOW Oo

cmm. H20

882

1986

2765
3900
5014

=
ry

—e
~~

TENSION DEVEL-~

OPED

MYO- AND CARDIODYNAMICS 387
deflated balloons. If one inflates and then deflates a balloon and plots
the pressures on the ordinates against the radius or circumference on
the abscissas, curves similar to those in figure 5 are obtained. In that
the pressures on deflation are lower per given circumference than on

inflation they are analogous to the initial pressures in the auricle with
increasing and decreasing length of fiber. In this connection it is inter-
esting to note that Langelaan (12) noted a similar hysteresis in the

TABLE 3
(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)
4 ce ee Sb z c
ee | ta | fete | Be | <3 Bp Ba ce
< a2e)#8e |eeaeeg) 6 {2 Mel az A 4 oa
B | eee | 2e2 |eeees| gf | gf | cf | s8 | 3B | Be
é pte Ese bee ab BP ee eee 2” g@ go
mm. Hg| mm. Hg| mm. Hg cmm. sq. mm mm cmm. H2O\cmm. H2O\cmm. H2O
6 0.9 2.9 1.6 640 359 33.6 323 897 574
10 pe has 3.3. 2.3 980 477 | 38.7 A477 1559 1081
15 1.0 4.1 3.1 1372 596 43.3 596 2443 1847
20 VA 4.7 3.6 1712 692 46.6 747 3252 2505
25 1.2 5.3 4.1 2028 775 49.3 930 4107 3177
30 Tio) GZ...) 4.5.1. 2320, 847 | 51.6,|. 1060. | 482%) | 3776
35 1.3 6.2 4.9 2588 912 53.5 1185 5700 4524
40 1.4 6.6 5.2 2852 972 55.3 1360 6444 5083
45 1.5 7:2 5:7 3062 1022 56.7 1533 7358 5825
50 1.5 ae" 6.1 3120.|. 1055 | -57..7>| ) 1582 8018 6435
55 1:5 8.0 6.5 3220 1055 57.7 1624 8440 6815
58 1.4 7.8 6.4 3020 1011 56.3 1415 7906 6490
60 1.3 Ye 6.4 2820 966 55.1 1255 7486 6230
65 $e | 7.0 5.9 2260 833 §1.1 916 5831 4914
70 1.0 6.2 5:2 1740 699 46.9 699 4333 3634
75 0.8 4.6 3.7 1460 623 | 44.2 529 2865 2336
80 0.7 2.0 2,2 780 409 | 35.9 306 1227 920
82 0.7 2.8 2.0 592 340 | 32.7 248 952 697
85 0.7 1.3 0.6 340 235 | 27.2 164 295 141

striated muscle of the frog. The question naturally arises, is this hys-
teresis common to muscle and rubber of significance in the processes
of muscular contraction? There is reason to believe (13) that in the
case of rubber the change in structure upon which the difference of
‘initial tension” depends is only temporary (curve / can be duplicated
after curve 2) and we might be justified in inferring that in the series of
auricular contractions with decreasing length of fiber the structure

388 ROBERT GESELL

of the muscular elements upon which the contractile processes depend —
is likewise different from the structure of the muscle during ~~ oe
in length of fiber.

There is another possible explanation also based upon the previous
history of the muscle, in this case chemical rather than physical. The
staircase phenomenon; for example, is explained on the basis of liber-
ated metabolites formed during contraction, which collect in the muscle
and influence subsequent contractions. Applying this to the auricle—
as the muscle contracts with pro-
gressively increasing length of fiber
the amount of metabolites liberated
with each contraction presumably
increases. In aseries with decreas-
ing length of fiber the reverse would
occur. But the difference between
the two series may be that in series
1 with increasing length of ‘fiber the
muscle, so to speak, never catches
up with itself in the supply of these
products, for with increasing length
of fiber, the demands on the muscle
are steadily increasing while in
the series with decreasing length of
fiber it is always ahead of itself for then the demands: on the muscle are
steadily diminishing.

These explanations of the experiments bearing upon initial tension
are admittedly speculative. The results of the experiments, however,
have a definite value in pointing to a possible detrimental effect of
initial tension upon muscular contraction rather than a beneficial effect.
The results enhance the value of the quantitative experiments on the
effect of initial length of fiber upon strength of muscular contraction.

—-

l Ae L ‘ {

Fig. 5

A NOTE ON TONUS

A note on tonus. There is an apparent analogy in the behavior of
rubber, auricular muscle and the peculiar catch muscle of the bivalve
mollusk Pecten described by Parnas (15), Uexkuell (16) and others
which may be of significance. The muscle of the Pecten though
powerful in that it can support a heavy weight is relatively weak in ©
that its lifting power is small. Recalling the curves of inflation and

MYO- AND CARDIODYNAMICS 389

deflation of the rubber balloon, the analogy is apparent. Since on de-
flation the balloon contracts and expels air and is therefore lifting,
rubber at a given length can sustain a heavier weight than it can lift.
How real this analogy is we of course do not know, yet it is very inter-
esting to perform the Pecten experiment upon the balloon in the
following manner: If the partially inflated balloon be enveloped and
supported by the hand and then compressed to 4 slightly smaller volume |
by forcing water into the manometer with which it is connected (which
is comparable to passively closing the shells of the Pecten), when the
hands are released the water fails by several centimeters to fall to the
original level. The rubber again holds more than it can lift.

To be sure the catch mechanism is developed more highly in the catch
muscle of the Pecten yet this does not necessarily spoil the analogy for
the mechanism is developed to different degrees in rubber just as it is
in muscle under varying conditions. From the description of Parnas
(15) it appears that in the weakened Pecten an appreciable interval of
time must elapse after passive closing of the valves before the muscle
will resist stretching. In that respect the analogy between rubber and
muscle may be extended, for if an inflated balloon is deflated and
promptly reinflated, the curve of reinflation is little higher than the
curve of deflation. But if, as Osborne and Sutherland (11) showed,
rubber is allowed to ‘“‘rest” the first curve of inflation may be dupli-
cated. Apparently opportunity must be given for the molecules to
rearrange themselves or approximate each other more closely if the force
of attraction is to increase. If a balloon just deflated be subjected to
a mechanical shock such as slapping sharply in the hand, the first curve
of inflation may be promptly approximated. Strong light also tends
to restore the properties of recently stretched rubber. It may be that
these differences in the properties of rubber under different conditions
are due to the formation of molecular aggregates for it has been found
(17) that shaking a rubber solution may double its viscosity through
the formation of these aggregates. It has also been noted that rubbing
of stretched rubber produces microscopic structural changes (13). It
may be that the catch mechanism of muscle in its ultimate analysis
is not comparable to the phenomenon shown by rubber, yet attention
should be called to a possible analogy between a purely chemical or
physical phenomenon and a so-called physiological phenomenon.

a ee ne

ee

"BIBLIOGRAPHY

a Geant This Journal 1916,
: (2) Burx: Skand. Arch. f. Physiol., 1895, ‘Y, 473.
(3) Evans anp Hitz: Journ. Physiol., 1914, xlix, 10.
(4) PATTERSON, PIPER AND STARLING: J¢ ourn. Physiol 1015,
(5) GuseLu: This Journal, 1911, xxix, 32. _
(6) Gesety: This Journal, 1915, -xxxviii, 404. C Pip
(7) Gesetu: This Journal, 1916, xl, 267.
(8) Bayxiss: Principles of general physiology, Tough Grek ae
(9) Srartine: On the law of the heart, Longmans Green & Co., 1
(10) pu Bois Reymonp: Festschr. f. Rosenthal, 1906, 287.
‘(11) OsBoRNE AND SUTHERLAND: Proc. Royal Soc., Series B, 19
(12) LanGetaan: Brain, 1915, xxxviii, 235. .
(13) ScuipRowitz: Journ. Soc. Chem. fadustry: 1909, xxviii
(14) SuerRineTon: Brain, 1915, xxxviii, 191.
(15) Parnas: Pfliigers Arch., 1910, exxxiv, 441.
. (16) Urxxu.u: Zeitschr. f. Biol., 1912, Iviii, 305.
‘ (17) Fou: Kolloid. Zeit., $013, ASL city ay

——- oo

THE ROLE OF THE PANCREAS IN HYPERGLYCEMIA FROM
ETHER ay

ELLISON L. ROSS anv L. H. DAVIS

From the Department of Physiology and Pharmacology, Northwestern University
Medical School

Received for publication June 25, 1920

The part played by the pancreas in producing hyperglycemia and
glycosuria in diabetics has been the subject of much study. But there
has been a tendency to forget the pancreas when dealing with glyco-
surias and hyperglycemias not directly associated with diabetes (1).
We, as well as others, have been guilty of this. In too many instances

- the attention has been centered only on liver glycogen, nerve supply to

the liver, and adrenal secretion. It is well known that the removal

_ of the pancreas will always cause a severe and uncontrollable hyper-

glycemia and glycosuria. This leaves no doubt that the pancreas
exerts an ever-present influence on the mobilization of dextrose in the
body. There is no reason to consider that the pancreatic influence may
not be subject to the same variation as the influence of any other
organs or tissues of the body. Therefore, we have been led to inves-
tigate the relation of pancreatic influence on the hyperglycemia resulting
from ether anesthesia.

EXPERIMENTAL WORK

A number of healthy dogs were depancreatized. The removal of
the organ was accomplished in two operations. At the first, the entire
pancreas was removed except the tip of the tail of the gland. This,
with its blood supply still intact, was drawn through the abdominal
wall and sutured under the skin. At the second operation, about a
week later, the remaining portion of the pancreas was removed and the
pedicle ligated.

On account of the mortality following anesthesias, we were forced to
make observations after the first operation, or on partially pancreatec-
tomized dogs, on one group of animals and observations on completely
pancreatectomized dogs of another group.

391

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO. 3

392 ELLISON L. ROSS AND L. H. DAVIS

The group of partially pancreatectomized animals were not glyco-
suric. They appeared to be in a healthy condition. The effect of
ether anesthesia on the blood sugar was determined. About 10 cc. of
blood were taken through a hypodermic needle and oxalated. Ether
then was administered by inserting the head of the animal into a cylin-
der into which air that had passed through ether was forced. After

TABLE 1

Effect of ether on glycemia of partially pancreatectomized animals

DEXTROSE IN BLOOD
ANIMAL

Before ether After 15 minutes ether Increase

per cent per cent per cent

1 0.110. 0.189 0.079

2 0.100 0.120 0.020

3 0.083 0.111 0.028

4 0.081 0.112 22 0.031
Average 0.0935 0.1330 0.0395

TABLE 2

Effect of ether on glycemia of completely pancreatectomized animals

DEXTROSE IN BLOOD
ANIMAL

Before ether After 15 minutes ether Increase

per cent per cent per cent

5 0.344 0.400 0.056

6 0.344 0.392 _ 0.048

7 0.256 0.322 0.066

8 0.285 0.327 0.042

9 0.287 0.300 0.013

10 0.377 . 0.400 0.023
Average........ 0.3155 0.3568 0.0413

fifteen minutes of surgical anesthesia, a second sample of blood was
taken in the same manner as before. The dextrose content of the
blood was determined by Benedict’s method (2). The results expressed
in percentage of dextrose in blood are given in table 1.

The second group of animals consisted of those which had had a com-
plete pancreatectomy. They all had glycosuria. They were all active,
bright and ready to eat and drink. They were given a meat diet exclu-

ea - =
‘

ROLE OF PANCREAS IN ETHER HYPERGLYCEMIA 393

sively. Two of the animals were treated exactly like those of the
preceding group. On others of this group it was thought best to measure
the changes in the output of urinary sugar as well as blood sugar.
Therefore a catheter was passed through the urethra into the bladder
and left in place throughout the test. The bladder was first emptied
and was then washed out with 10 cc. of water three times. The ani-

TABLE 3

Dextrose changes in blood and urine of pancreatectomized animals induced by ether

DEXTROSE IN BLOOD DEXTROSE IN URINE | DEXTROSE IN URINE
ANIMAL OF } HOUR OF } HOUR
Before ether ‘| After 3 hour ether Bay Oe ee be ie: anes ra
per cent per cent grams grams
7 0.256 0.327 0.411 0.062
8 0.285 0.340 0.719 0.242
9 0.287 0.303 0.336 0.227
10 0.377 0.410 1.032 0.515
TABLE 4

Dextrose changes in blood and urine of pancreatectomized animals induced by ether

INCREASE IN DEXTROSE|DECREASE IN DEXTROSE CHANGE IN BODY
ANIMAL IN BLOOD IN URINE DEXTROSE DUE TO
DUE TO 4 HOUR ETHER | DUE TO } HOUR ETHER 4 HOUR ETHER
grams grams
7 0.574 0.349 +0 .225
8 0.363 0.477 —0.114
9 0.101 0.109 —0.008
10 0.267 0.517 —0.250
First average....... 0.3262 0.3630 —0.0368
Second average..... - 0.2436 0.3676 —0.1240

Note: The first average is the average of all. Second average omits the values
for the first animal.

mal was kept on the board half an hour and at the end of that time the
urine was drawn off and the bladder washed out exactly as before.
The urine and bladder washings were combined and analyzed for dex-
trose according to Benedict’s method (3). A sample of blood was
taken and ether was given in the usual manner described above. After
fifteen minutes of anesthesia a second sample of blood was taken. At
the end of half an hour of anesthesia a third blood sample was taken
and the urine collected as before. The results expressed in percentage

394. ELLISON L. ROSS AND L. H. DAVIS

of dextrose in blood are given in table 2. The results expressed in per-
centage of dextrose in blood and grams of dextrose in the urines for
two half-hours are given in table 3. Deductions from table 3 are
given in table 4.

DISCUSSION

In table 1 is expressed the effect of fifteen minutes of ether anesthesia
on the blood sugar of animals possessing only a small fraction of pan-
creas and that in an abnormal location. The animals were undieted.
There was the usual individual variation whichmakesindividual changes
of much less significance than the average from a group of animals.
However, it is worth noting that there was a decided increase in the .
glycemia of every animal. The average blood sugar before anesthesia
was 0.0935 per cent and after anesthesia 0.133 per cent of blood dex-
trose. The average increase in these animals was 0.0395 per cent. In
a previous paper (4) the effect of fifteen minutes of ether anesthesia
on the blood sugar of seventeen normal dogs was reported. ‘The °
average blood content of dextrose before anesthesia was 0.090 per
cent and after fifteen minutes of ether anesthesia produced in our
usual way the bloods averaged 0.127 per cent. The increase was 0.037
per cent. The close agreement between the effect of ether on normal
dogs and dogs with much less than the normal pancreas, leads us to
conclude that an influence on carbohydrate metabolism equal to nor-
mal was being exerted by these small pieces of pancreas implanted
under the skin. Ether disturbed the equilibrium just the same as in
normal dogs.

In table 2 is expressed the percentage of blood sugar before and after
anesthesia of animals without a pancreas. The high glycemia is strik-
ing. It is fully three times the amount normally found. All the ani-
mals had severe glycosuria. The average dextrose content of the
blood before ether was 0.3155 per cent and after fifteen minutes of
ether anesthesia 0.3568 per cent. There was an average increase of
0.0413 per cent. It is notable that the values before and after ether
were particularly high and that the increase agrees well with the
hyperglycemia of normal animals. If there were no changes in the
rate of dextrose excretion by way of the urine, this agreement between
the increase of normal and pancreatectomized dogs would be of con-
siderable significance.

In order to determine whether there was any change in the dextrose
excretion brought about in these pancreatectomized dogs by ether anes-

ROLE OF PANCREAS IN ETHER HYPERGLYCEMIA 395

thesia, the dextrose excreted the half-hour before ether and the half-
hour during ether anesthesia was determined. From table 3 it is
noted that in the half-hour there was a decided increase in the blood
sugar percentages and also a decided decrease in the grams of urinary
dextrose. In table 4 it is possible to compare the blood increases
with the urine decreases. ‘The blood increases expressed in grams were
calculated from the percentages of dextrose, the weights of the dogs
and Howell’s blood and body weight factor (5) of 7.7 per cent.

With the exception of dog 7, there was in every case less increase
in blood sugar than there was decrease of urine sugar. The possible
cause of this exception was that during the anesthetic dog’ 7 stopped
breathing and nearly died. The asphyxia in this case was severe and
capable of causing marked disturbances of the carbohydrate regulatory
mechanism. The average which includes all the animals, shows a
decrease: in dextrose mobilization of 0.0368 gram due to ether anes-
thesia of the half-hour. The second average which does not include
dog 7 shows a reduction in dextrose mobilization of 0.124 gram. There
is no doubt that the removal of the pancreas removed at least one of
the sites of action of ether in its production of hyperglycemia.

Cannon (6) worked out the mechanism of the mobilization of dex-
trose by pain, fear and rage. These factors are capable of stimulating
the nerve endings in the adrenals, a stimulation which results in the
freeing of epinephrin into-the blood stream. This epinephrin acts
directly on the glycogen of the liver to set free dextrose. Stewart
objects to some of these findings (7).

Macleod (8) has presented data which leads him to conclude that
sympathetic nerve terminations controlling glycogenic function can be
stimulated either by excess of adrenalin in the blood or by nerve impulses
alone.

It is a well-known fact that the removal of the pancreas causes a
disappearance of glycogen from the liver and the production of an un-
controllable hyperglycemia and glycosuria. Allen (9) concludes that
derangement in the internal secretion from the pancreas is the factor
most concerned in the etiology of diabetes. The pancreatic internal
secretion must have an inhibitory effect on the liberation of glycogen,
since the removal leads to a rapid disappearance of glycogen.

Thus we have strong experimental evidence that there are three im-
portant influences being exerted on stored glycogen, i.e., adrenalin,
sympathetic nerve endings and pancreaticinternalsecretion. Thenerve
endings and adrenalin exert an influence to liberate dextrose from gly-

396 ELLISON L. ROSS AND L. H. DAVIS

cogen. Pancreatic internal secretion exerts an influence to prevent the
liberation of dextrose from glycogen. So there is for glycolysis an in-
hibitory and an acceleratory mechanism, a mechanism which is common
to many of the body functions. If this be true, no discussion or inves-
tigation of any condition which has to do with the mobilization of dex-
trose is complete unless all these factors are considered. There is no
more reason for regarding the internal secretion of the pancreas as in-
variable than for so regarding the internal secretion of the thyroid.

Our results show that ether does not bring about the mobilization of
sugar in the animal without a pancreas, as it does in a normal one.
Since the pancreas inhibits glycolysis, we conclude that ether reduces
the activity of the pancreatic internal secretion of normal dogs, and
thus increases dextrose mobilization. It may be objected that the
store of glycogen was so depleted after pancreatectomy that dextrose
could not be liberated in normal amounts. This objection will not
hold because large amounts of dextrose were being freed into the blood
and excreted by the urine hourly as was indicated by the blood and
urine content stated in the tables.

Keeton and Ross (1) presented data suggesting that the action of
ether to cause hyperglycemia was through hepatic nerve influence and
not chemical action directly on the liver cells. They did not take into
consideration the internal secretion of the pancreas. Because they did
not get hyperglycemia with ether in bi-splanchnectomized animals,
they concluded that ether could not be working on the liver directly.
The most probable cause for this result, under these conditions, was
that the cutting of the nerves to the adrenals reduced the epinephrin
output more than ether reduced the internal secretion of the pancreas.
Since the adrenals encourage glycolysis and the pancreas inhibits it,
the result would be an absence of a hyperglycemia, a result which was
obtained. :

In a previous publication (10) it was found that chloroform hyper-
glycemia was not reduced by atropin as was the case with ether hyper-
glycemia. The mechanism of the two must have certain differences.
In view of the present conclusions and since we know chloroform seri-
ously injures liver cells, it is possible to say that the results were due to
the inability of the injured nerve endings in the liver and the adrenalin
to accomplish as much change in glycogen stored in cells whose walls
had been injured and whose chemical equilibrium had been altered, as _
normally. So that possibly chloroform reduces the pancreatic internal
secretion exactly the same as ether but in addition injures the liver,

ROLE OF PANCREAS IN ETHER HYPERGLYCEMIA 397

which would result in not so great a hyperglycemia as that of ether and
also not be subject to influences of such drugs as atropin while ether is
so influenced.

Stewart and Rogoff (7) have presented much data contending that
ether hyperglycemia is not due to the liberation of excess adrenalin into
the blood stream. ‘They have cut the splanchnics on one side and
removed the adrenal gland on the other side. The cats were allowed
a number of days to recover. Then they were anesthetized with ether.
A hyperglycemia resulted. The amount of adrenalin in the blood
before and after the anesthesia was far below normal. During the
time allowed for recovery there is no doubt that there was a read-
justment between the accelerator and inhibitor glycolytic mechanisms,
as is commonly seen after a disturbance of one of the mechanisms gov-
erning heart rate. So at the end of several days the inhibitor glyco-
lytic mechanism, the internal secretion of the pancreas, had fallen to a
lower level more nearly equal to the accelerator glycolytic influence of
adrenal and hepatic nerve ending activity. So when ether was given,
according to our view the activity of the pancreas was reduced and the
accelerator glycolytic mechanism became the more powerful and
dextrose was set free into the blood stream in greater abundance.

SUMMARY AND CONCLUSIONS

The hyperglycemia induced by ether was measured on two series of
dogs. One group had partial pancreatectomies and the other group
had complete pancreatectomies. The effect on the urine dextrose of
ether anesthesia was also measured in the case of the pancreatectomized

~ dogs.

The hyperglycemia induced by ether anesthesia of partially pancrea-
tectomized dogs was the same as that of normal dogs.

The hyperglycemia induced by ether anesthesia of completely pan-
createctomized dogs was practically the same as normal. Comparing
the dextrose output in the urine half an hour before anesthesia and
half an hour during anesthesia showed a marked decrease in the elimi-
nation of dextrose. Comparing the rate mobilization of dextrose with
and without ether showed that it was markedly. decreased by the drug.

The results lead us to conclude that the chief action of ether in
causing a hyperglycemia in normal animals is to reduce the influence
of the internal secretion of the pancreas on glycolysis.

Et Pee s rss L. RC
“ prprtocRapny

(a) i ade AND Ross: This Jounal bis, aii 6. 2 rl get
(2) Benepict: Journ. Biol. Chem., 1918, xxxiv, 203. | 3.
(3) Benepicr AND OsterBeRG: Journ. Biol. Chem., 1918, xxxiv.
: (4) Ross: Journ. Pharm. Exper. Therap., 1919, xii, 377.
: (5) Howe: Textbook of physiology, Philadelphia, 1913, 155,
(6) Cannon: Bodily changes in pain, hunger, fear and uroiegt
(7) Stewart AnD Rogorr: This Journal, 1917, liv, 543. ye
(8) Mactrop: Diabetes, New York. © tht RN
(9) ALLEN: Monograph no. 11, ‘Rockefeller Institute, 1919.

PHYSIOLOGICAL STUDIES ON PLANARIA

1V. A FurtHer Stupy or OxyGEN CONSUMPTION DURING STARVATION —

LIBBIE H. HYMAN
From the Hull Zoélogical Laboratory, University of Chicago

Received for publication June 26, 1920
INTRODUCTION

In the first paper of this series (1) it was shown that in Planaria doro-
tocephala the rate of oxygen consumption rises during starvation.
This rise is readily detectable in less than three weeks after the cessa-
tion of feeding; and from this time on the oxygen consumption rises
continuously with an acceleration for at least eight weeks, when the
experiments were concluded. ‘This result agrees with the result pre-
viously obtained by the susceptibility method. The susceptibility .
method consists in observing the time of death of organisms in lethal
solutions. It is believed on adequate grounds, which need not be
reiterated here since they have been discussed in numerous papers
from this laboratory, that such time of death is in a general way a
measure or indication of the metabolic rate of organisms. By this
method it had been found many years ago by Professor Child that
the susceptibility of planarians is increased when they are starved
and that it is greater and their survival time in lethal solutions shorter —
the longer the period of starvation has endured. From this result
Child drew the conclusion that the rate of metabolism is increased by
starvation. My results on the rate of oxygen consumption confirmed
the conclusion reached by Child through the susceptibility method and
further supported Child’s general conceptions concerning the nature of
senescence and rejuvenescence (3).

Meantime, however, Lund and Allen at the University of Minnesota
sought to find evidence against Child’s viewpoint. Their point of
attack concerns the susceptibility method and they have been attempt-
ing to show that the susceptibility method is not a reliable measure of
metabolic rate. Their arguments reveal a certain amount of misunder-
standing concerning the application of the susceptibility method but

399

400 LIBBIE H. HYMAN

the matter is perhaps of little consequence now, since the main con-
clusions drawn by the use of the susceptibility method have been
verified by quantitative determinations of oxygen consumption and
carbon-dioxide production.

In a paper appearing simultaneously with mine already referred to
(1), Allen (2) sought to show that in Planaria agilis and Planaria
maculata the rate of oxygen consumption is not increased during
starvation within the periods tested by him (nine weeks). In this
paper he also made an elaborate comparison between the susceptibility
of planarians in various physiological conditions and their rate of
oxygen consumption under the same conditions and argued that since
the two methods do not (according to him) always yield the same
results, the susceptibility method is not a reliable index of the rate of
respiratory metabolism.

This comparison made by Allen would probably appear convincing
to anyone not conversant with all of the facts. But it has in reality
no adequate scientific basis since the comparison is made between the
susceptibility data obtained by us on Planaria dorotocephala and the
oxygen consumption data obtained by him on Planaria agilis and Planaria
maculata. Allen at the time of writing did not know anything about
the rate of oxygen consumption of P. dorotocephala during starvation.
How then can he maintain that it does not agree with the findings by —
the susceptibility method? As a matter of fact there is in this species
as I have shown (1) general agreement between the susceptibility
results and the rate of oxygen consumption; both show that metab-
bolism is markedly increased by starvation. On the other hand Allen
did not know or attempt to find out anything about the susceptibility
of P. agilis during starvation. How then can he maintain that in this
species there is a discrepancy between the results by the susceptibility
method and the oxygen consumption data? My own findings demon-
strate that no such discrepancy exists. It is true that the rate of
oxygen consumption in P. agilis increases very slowly during starva-
tion and the increase appears only after four to six weeks while in P.
dorotocephala it is present before the third week of starvation; but it
is also true that in P. agilis the susceptibility to potassium cyanide shows
no or little increase during this period. This fact completely demolishes
Allen’s argument. If he had taken the trouble to investigate the
susceptibility of P. agilis during starvation, he would have realized -

1 Owing to the uncertainty in the identification of the species Planaria macu-
lata, explained later, consideration of this species is left out of the discussion.

OXYGEN CONSUMPTION IN STARVATION 401

that he had no basis for his contentions. As a matter of fact, the con-
ditions in P. agilis, far from disproving the value of the susceptibility
method, furnish a remarkable demonstration of its utility; for the diff-
erences between the two species could have been discovered by means
of the susceptibility method alone.

Marked differences exist in the physiology of P. dorotocephala and
P. agilis; these differences are mostly quantitative and not qualita-
tive. P. agilis respires about two-thirds as fast as P. dorotocephala, it
consumes more food and has probably larger food reserves, it loses
weight more slowly in starvation, and requires a much longer period to
reach the same degree of rejuvenescence. Such differences must re-
ceive consideration in experimental work. To assume, as Allen did,
that because P. dorotocephala reaches a high degree of rejuvenescence
within a few weeks, other species must also, and if they do not the con-
clusions drawn from P. dorotocephala are erroneous, is an unscientific
proceeding. Each species must be thoroughly analyzed and studied as
we have studied P. dorotocephala before comparisons can be made.

The question next arises: is it true that in P. agilis and P. maculata,
as maintained by Allen, the oxygen consumption does not increase
within nine weeks of starvation? I have repeated Allen’s experiments
upon these species (cf., however, footnote 1) and find that he was mis-
taken in his conclusions. A critical examination of the data presented
by him shows that they are inadequate and unconvincing and furnish
an insufficient basis for his contentions, éspecially in view of the fact
that those contentions are counter to a large body of evidence already
at hand.

Allen has presented six experiments, three on P. agilis and three on
P. maculata. In but one of the six has the oxygen consumption been
tested from the beginning to the end of the period of starvation. This
experiment is given in his table 7. At the end of this experiment the
worms were consuming some 40 per cent more oxygen than during the
early part of the experiment. The result, therefore, in the one really
adequate experiment performed by Allen is exactly the contrary to his
general conclusion. My experiments show that the results given in
this table are essentially correct.

I wish to consider each of his experiments in a little more detail.
Tables 4, 5 and 6. deal with P. maculata. In table 4 the experiment is
begun with worms which had already been starving for ‘‘several weeks.”
We are not told just how long this period was nor do we know anything
about the rate of oxygen consumption during this part of the starva-

402 LIBBIE H. HYMAN

tion period. The oxygen consumption of these worms was then tested

during thirty-four days of starvation, during which it is said by Allen ~

to be “constant.”” This “‘constancy” means a variation of from 5.8
to 8.2 cc. of oxygen per gram per day. This variation is much greater
than that regarded in other tables as of significance and clearly indi-
cates a lack of experience with the method. Leaving this irregularity
out of consideration, it may be said that the data in the table are of no
significance since after ‘‘several weeks” of starvation a high level of
metabolism has already been attained by some species of Planaria and

' this rate may not increase further. Unless we know the complete -

history of such worms the data are of no value for the purpose. The
_reader should note, however, that according to. table 5 the average
oxygen consumption per gram per day after ‘‘several weeks” starva-
tion is 6.9 cc. Turning now.to table 5, we find that in the same species
of Planaria between the fifteenth and forty-second days of starvation
the average oxygen consumption per gram per day is about 4.4 cc.;
and again in table 6, between the fifth and twenty-ninth days of star-
vation it is 5.2. This discrepancy between the results in table 4 on
the one hand and those in tables 5 and 6 on the other was not men-
- tioned by Allen; but it is perfectly evident that it can be explained
only on the basis that the oxygen consumption is decidedly increased
after ‘‘several weeks’ starvation. We may therefore state that Allen’s
data on P. maculata are inadequate because in no case was the oxygen
consumption determined throughout a period of starvation covering a

number of weeks; and that as far as they show anything at all they —

certainly do not support Allen’s conclusion but lead in the opposite
direction. :
Allen’s tables 2, 3 and 7 deal with P. agilis. In table 2 it is shown
that the oxygen consumption in this species is less after three weeks
than after one day of starvation. This is also the case in all species of
Planaria which have been tested.. The greater rate after one day of
starvation is due to the persistence of the effect of feeding on the diges-
tive tract as explained in a previous paper (1). This table has there-
fore no direct bearing on the question of the rate of metabolism during
starvation, since the comparison must be made with worms in which
the increased oxygen consumption due to feeding has been eliminated.
In Allen’s table 3 data are given which show no difference in the rate of

oxygen consumption between worms starved one week and worms

starved nine weeks. These data are most certainly incorrect. They
are directly contradicted by the results in Allen’s table 7, in which

OXYGEN CONSUMPTION IN STARVATION 403

after nine weeks’ starvation an increase of about 40 per cent in the
rate of oxygen consumption has occurred. Perusal of the text referring
to table 3 throws some light on the origin of the error, since it is stated
that the control worms had been kept in the laboratory only a few
days preceding the test.. Since the metabolic rate of worms decreases
when they are maintained under laboratory conditions it is probable
that the control worms in this case had a higher rate of oxygen con-
sumption than they would have had if kept in the laboratory the same
length of time as the experimental worms. |

As already noted, the experiment recorded in table 7 is the only one
presented by Allen in which the metabolism of the worms was studied
from the beginning to the end of a starvation period lasting about ten
weeks. This experiment is also the most accurate, as Allen states.
Unfortunately at the critical point in the experiment, a gap of three
weeks is present. Nevertheless the results clearly show that the
oxygen consumption of P. agilis is increased by starvation, a rise of
about 40 per cent having occurred after nine weeks’ starvation. Allen
attempts to explain away this result on trivial grounds, but my own
experiments on five different lots of P. agilis, each of which was studied
throughout a period of at least twelve weeks’ starvation, prove that it
is essentially correct. The rate of oxygen consumption of P. agilis
like that of other species of Planaria is increased by starvation; but a
longer period of time is required for the increase to occur. |

It is evident that throughout his experiments Allen proceeded on a
physiologically incorrect assumption. He supposed that absolute
length of time is the important factor in starvation; whereas in fact the
primary factor is the rate at which starvation proceeds. This rate is
determined by a number of factors other than time, such as: the tem-
perature during starvation, the age (size) of the worms at the begin-
ning of the experiment, the metabolic rate of the species in question,
the rate at which weight is lost, and the amount of food reserves present
in the body. An increase in metabolism as a consequence of starva-
tion cannot be expected to occur until the animal actually begins to
use its own tissues for food and the time required for this is variable
with different species and under the different circumstances just
enumerated.

404 LIBBIE H. HYMAN

EXPERIMENTS ON PLANARIA MACULATA

1. Source and care of material. The so-called species- Planaria
maculata lives in the Chicago region in small still bodies of water. It
_ is found creeping about on the submersed vegetation. The stocks of
worms used in these experiments were collected from the lagoon in
Jackson Park in the city of Chicago. This lagoon to the east of the
Wooded Isle is filled with submersed water weeds, chiefly Elodea,
Ceratophyllum, Myriophyllum and Potamogeton. Large quantities
of these plants were brought in, packed closely in dish pans, and enough
water added to just cover the plants. Within two or three days the
plants begin to decay and as the planarians cannot endure such condi-
tions they creep up to the surface layers of plants, and after more time
has elapsed will collect at the margins of the pans near the surface.
If the surface layers of plants are removed after two or three days,
placed in pans with a large quantity of water, and violently agitated,
the worms will fall off to the bottom and begin to crawl up the sides
of the pan to the top. They can then be readily picked off and placed
in pans of clear water. Although the method is rather laborious and
time-consuming, it is possible with a little patience to accumulate a
considerable stock of such worms. They are most abundant on the
vegetation in the autumn.

Stocks so collected were kept in large pans of well water. If they
are to be maintained for any length of time, they must be fed. I at
first tried to feed them by the same method we were accustomed to
use for P. dorotocephala, namely, by placing pieces of liver in the pans.
It was at once found that this species will not take food by this method.
It was then suggested by Professor Child that this species is so sluggish
and insensitive that it does not detect food except at very short dis-
tances and that the liver should be ground and strewn over the bot-
tom of the pan. This procedure was tried but was even less successful
than the preceding plan, because not only did the worms refuse to feed

but many of them died as a consequence of the presence of the ground

liver in the pan. While giving this plan a trial, however, the correct
method of feeding was accidently discovered. In an attempt to
reduce the labor of removing the ground liver from the pans after
feeding it was decided to wash the liver, since blood and other materials

diffusing from the liver render the water so opaque that the worms ~

can no longer be seen. The liver was then ground and washed and it
was at once observed that the worms would feed voraciously on such

OXYGEN CONSUMPTION IN STARVATION 405

washed liver. Evidently some materials in the liver (bile?) are inju-
rious to this species. The feeding plan adopted was then the follow-
ing: the liver was ground in a meat grinder, poured into a strainer,
and a stream of water run through it until the water came through
clear. The washed liver fragments were then strewn over the bottom
of the pan and the worms stirred up and washed down from their
usual resting places along the sides of the pan. After three or four
hours, the worms, having fed, again accumulate on the sides of the
pan, and the liver fragments can be removed with the aid of a suction
pipette made of an atomizer bulb and a glass tube of wide bore. The
worms and the pan are then washed thoroughly two or three times to
remove all fragments of liver. By this method the worms feed readily
and grow with astonishing rapidity.

2. Taxonomy of Planaria maculata. Inspection of a stock of worms
collected as described above at once shows that two distinct types are
present. Neither of these: corresponds to the description of the true
Planaria maculata of the eastern United States. I am of the opinion
that there are at least three distinct species to which the name Planaria
maculata has been applied indiscriminately.

One of the types occurring in collections from pond weeds has the
following characteristics: the body is short and relatively broad and
not at all or but slightly narrowed behind the auricles; the pigmenta-
tion varies from chocolate to ashy brown and is decidedly arranged in
spots with conspicuous white blotches between; there is never any
white stripe down the center of the dorsal side but large specimens
show a tendency to develop a dark stripe in this region. This type is
designated in this paper as the spotted variety. It is not found sexu-
ally mature in nature but sexually mature individuals have developed
in our stocks and are at the present writing laying capsules. An inves-
tigation of the morphology of the reproductive system in this form will
settle the question of its identity or non-identity with Planaria maculata.

_ The other species present in our stocks has the following appearance:
the body is longer and more slender than in the spotted variety and
very much narrowed behind the auricles; the pigmentation is more
evenly distributed and to the naked eye not at all spotty in arrange-
ment; it is of a very dark brown, almost black color and the white
part of the eyes stands out very conspicuously in consequence; there is
always a well-marked white stripe down the center of the back. This
form is designatedin this paper as the striped variety. Although spec-
imens in our stocks have attained a considerable increase in size, no
sexually mature individuals have as yet appeared.

406 LIBBIE H. HYMAN

Both of these forms are different from the true Planaria maculata
which I have observed at Falmouth, Mass. The general proportions
of the latter are similar to those of the spotted variety but it is not
nearly so spotty in appearance and generally bears a white stripe

down the center of the back. Further, it lives in a different kind

of habitat, under stones in clear, comparatively quiet water. This
species does not live in the Chicago region as the type of habitat neces-
sary for it does not occur here. Finally its behavior in regeneration
experiments is quite different from that of our two varieties.

In view of this confusion regarding the taxonomy of this species, it
is impossible to know whether investigators working with what is
called P. maculata are really using the same species or not. This
difficulty arises in connection with Allen’s experiments. It is not
certain that the species used by Allen is the same as either of the two
varieties used by me in the present experiments. From some remarks
made by Allen to Professor Child at the 1919 meeting of the American
Society of Zodlogists, it seems probable that he was working with the
true Planaria maculata. At any rate the results which I have obtained
are quite different from his and this difference may be due in part to
the use of different species.

8. Method of procedure. From the general stocks lots of worms
were selected, each consisting of some two hundred worms of approxi-
mately the same length. Two such lots were selected of the striped
variety and three of the spotted variety. Each lot was kept in a
separate dish and its rate of oxygen consumption determined at inter-
vals, generally two-week intervals, during a period of starvation.
To prevent such worms from undergoing fission, it is only necessary
to place them in dishes already well coated with slime from other
worms.. .

The method of determining the oxygen consumption has already
been described (1). Briefly the lot of worms was placed in a 500 ce.
Erlenmeyer flask, filled air-tight with water of known oxygen content;
after an interval a sample was drawn from this flask and its oxygen
content determined. The difference between the oxygen content of

this sample and the original oxygen content of the water gives the

amount of oxygen consumed by the worms. The oxygen content was
determined by Winkler’s method. Two independent determinations

were made each time. After the experiment was completed the worms ©

were weighed. The temperature was the same each time the oxygen
consumption of the worms was tested, but between these tests the
worms remained at room temperature, which is naturally rather variable.

ei te

OXYGEN CONSUMPTION IN STARVATION 407

4. Results. The results of these tests on the oxygen consumption
of five separate lots of P. maculata at various intervals during a period
of starvation are summarized in table 1. The detailed data will be
found at the end of the paper.

5. General conclusions regarding Planaria maculata. As shown in
table 1, the oxygen consumption falls after feeding and reaches its
lowest level within three or four days. It then remains at this level
until after the second week of starvation. From this time on it rises

TABLE 1

Oxygen consumption of the spotted and the striped varieties of Planaria maculata
during starvation, showing increase in all cases. All temperatures 20+ 0.5°C.

STRIPED VARIETY SPOTTED VARIETY
Lot 1 Lot 10 Lot 3 Lot 2 Lot 4
TIME SINCE FEEDING
10-12 mm. | 8-12 mm. 8mm. 8 mm. 10-12 mm.
(Original | (Original | (Original (Original | (Original
length) length) length) length) length)
Oxygen consumed by 0.5 gram in two hours
cc. cc. ce. ce. ce.
1 day 0.18 0.25
3-4 days 0.16 0.21 0.28 0.21
1 week 0.16 0.21
2 weeks 0.15 0.22 0.22 0.28 0.21
4 weeks 0.17 0.26 0.24 0.29 0.28
6 weeks 0.22 0.35 0.29 0.36 0.30
8 weeks 0.21 0.29 0.59* 0.42
10 weeks 0 .23* 0.52* 0.61*
Per cent increase........... 53 59 147 110 190

_ ™ Means that the worms were decapitated on the day preceding the test so
that movement was eliminated. :

continuously. A marked difference was found between the two varie-
ties used. In the striped variety the oxygen consumption reached a
maximum by the end of the sixth week of starvation and exhibited no
further rise within the limits of the experiments. It is therefore not
safe to begin these tests on worms which have already starved several
weeks, as done by Allen in his experiment 4. In the spotted variety,
on the other hand, the oxygen consumption continued to increase with
an acceleration throughout the experiments. The experiment was
discontinued in each case when the size of the worms was so reduced as

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, NO, 3

408 : LIBBIE H. HYMAN

to render further determinations impracticable. In most cases the
heads of the worms were removed preceding the last test so that move-
ment is eliminated as a factor in the result. In the early stages of the
experiments, some of the stocks were not tested as this was considered
unnecessary in view of the fact that the fall in oxygen consumption
during this period is so well established that it may be assumed to

have occurred.. No tests are omitted from the table; all that were |

made are given.

EXPERIMENTS ON PLANARIA AGILIS

‘1. Source and care of material. This species is not indigenous to the .

Chicago region. About three hundred individuals were purchased
from the firm of Powers Powers, Lincoln, Nebraska. Since Allen’s
stock was also obtained from this firm, we are dealing here with the
same species. This species is stated by Mr. Powers to live in springs,
a habitat similar to that frequented by P. dorotocephala. The two
species resemble each other very much in appearance, although agilis
is more restless and attains a larger size.

The original individuals were cut into pieces in order to obtain a

sufficient number of individuals for the experiments. The experi-

ments were not begun until three or four months after this had been
done so that it is impossible that this procedure could have affected
the results. ‘The worms were fed by the same method as used for P.
maculata and ate voraciously and grew with remarkable rapidity.

2. Digestive tract of Planaria agilis. In order to make a comparison
between Planaria agilis and Planaria dorotocephala it is not sufficient,
as Allen has done, to perform a few isolated-tests. The physiology of
the species must be studied before such comparisons are valid. One of
the striking differences between these two species concerns the capacity

of the digestive tract. Planaria agilis is an extremely voracious feeder, ©

and appears to be very hungry after only three or four days’ starvation.
It will attack injured worms much more fiercely than will the other
species we have observed. After it has fed, the body is noticeably
distended so that it is quite easy to distinguish the individuals that
have fed from those that have not. This observation suggested that
the digestive tract has a larger capacity in P. agilis than in P. doroto-
cephala. Sections show that such is the case. Figure 1 gives camera
lucida outlines of the body wall and digestive tract at various levels
of section in individuals of P. agilis and P. dorotocephala of the same
length. It is readily seen that the digestive tract of P. agilis, partic-

OXYGEN CONSUMPTION IN STARVATION 409

ularly in regions through and in front of the pharynx, is more capacious
than that of P. dorotocephala. In the latter species, the anterior
part of the digestive tract has the form of a median trunk with lateral
branches, while in the former species there is a large irregular cavity
extending nearly the width of the body. By simply distending the
whole body, the capacity of this digestive tract would become very
great and as already noted such distension occurs when P. agilis feeds.

Fig. 1. A, six sections through Planaria agilis, drawn with the camera lucida,
showing outlines of the cavity of the digestive tract; most anterior section at
the top and others in order. B, sections through similar levels of Planaria

dorotocephala, showing outlines of the cavity of the digestive tract.

Both worms
15 mm. long.

Further the body is broader and thicker in P. agilis in proportion to
length than in P. dorotocephala. These facts indicate that P. agilis
can consume more food at each feeding and accumulates more food
reserves than P. dorotocephala; and therefore it must be starved for a
longer period before it will reach the same metabolic condition that the
other species reaches in a short time.

410 LIBBIE H. HYMAN

3. Loss of weight of Planaria agilis as compared with P. dorotocephala.
It was noticed that during starvation the reduction in size is much
slower in P. agilis than in P. dorotocephala. To obtain a more accu-
rate measure of this difference fifty worms of the same length of each

TABLE 2
Comparison of loss of weight of Planaria dorotocephala and Planaria agilis during
starvation. Figures are weights of fifty worms, approximately 15 mm.
long at the beginning. of the starvation period

PLANARIA DOROTOCEPHALA PLANARIA AGILIS
TIME SINCE FEEDING

Weight Total decrease Weight Total decrease
gram per cent gram per cent

1 day 0.411 0.550

1 week 0.322 21 0.503 8

2 weeks 0.284 30 0.472 13

4 weeks 0.214 47 © 0.380 30

6 weeks 0.159 61 0.289 47

8 weeks 0.107 ! 74 0.227 58

10 weeks 0.073 82 0.169 69

1 1 1

- 1
0 2 4 6 8 JO

Fig. 2. Graph showing loss of weight of Planaria agilis and Planaria doroto-
cephala in starvation. Abscissa, time in weeks; ordinate, weight in tenths of a
gram. Upper line, P. agilis; lower line, P. dorotocephala; weights for fifty worms.

species were weighed at intervals during starvation. Both lots of

worms were kept under the same conditions throughout. The data on
the loss of weight are given in table 2 and a graph constructed from
the same data presented in figure 2. Jt is evident that P. doroto-

OXYGEN CONSUMPTION IN STARVATION 411

- cephala loses weight in starvation more rapidly than P. agilis and
that the difference is greatest in the early part of the starvation period.
The loss of weight of P. agilis is practically directly proportional to
time so that the resulting graph is a straight line, as Allen has also
shown. In P. dorotocephala on the other hand the loss of weight is
more rapid at first and proceeds at a uniform rate only later in the
starvation period. The slower loss of weight in P. agilis is evidently
correlated with its greater breadth and thickness and greater supply of
food reserves. Since P. agilis loses weight more slowly than P. doro-
tocephala, a longer time will be required for it to starve to the same
degree and reach the same metabolic condition as the latter species.
This was found to be the case.

4. Oxygen consumption of P. agilis during starvation. The methods
employed were the same as those described under P. maculata. Five
lots of worms, the members of each lot of approximately the same size,
were isolated from the general stock and their oxygen consumption
determined at various intervals after the cessation of feeding. Fission
ean readily be prevented in such lots by keeping them in dishes or pans
coated with slime. A few fissions will occur and such divided worms
were removed. The results of these five experiments are tabulated in
table 3. The detailed data are given at the end of the paper.

The results tabulated in table 3 prove that in Planaria agilis as in
other species of Planaria the rate of oxygen consumption increases
during starvation. A longer period of starvation must elapse, however,
in this species before the rise occurs. This length of time required
depends, among other factors, on the original size (age) of the worms.
Thus in lot 6, where the worms were 10 to 12 mm. long at the begin-
ning of the experiment, the rise is present after four weeks of starva-
tion; in lot 7, with worms 12 to 15 mm. long, the rise begins by the sixth
week and in the other lots, with worms 15 mm. long or longer, the rise
begins by the eighth week of starvation. In all cases the rise is plainly
present by the beginning of the eighth week of starvation. This
result is therefore contradictory to the contentions of Allen, who insists
that within the length of time tested by him, namely, nine weeks, no
such rise is present. I have already pointed out that his contention
is directly contradicted by his own data given in his table 7, and that
the results in this table are correct.

After the eighth week the rise is at first rather slow or even absent
as in experiments 6 and 5 but later the increase is very rapid, and the
oxygen consumption finally attained may be more than 100 per cent

412 | LIBBIE H. HYMAN

greater than the oxygen consumption after a starvation period of one

week. After twelve or more weeks of starvation worms of this species —

are no further reduced in size than are individuals of Planaria doro-
tocephala of the same original length after six or seven weeks of star-
vation. The slower rate of reduction of P. agilis explains why the
metabolic rate increases more slowly.

TABLE 3

Oxygen tonawinpeeye of Planaria agilis during starvation, showing increase in all
cases by the eighth week

LoT 6 Lor 7 LoT 5 Lot 8 Lor 9
18°C. oC. 18°C. a0; 21°C.
lira an NT bara 10-12'mm. | 19+15’mm, | 15-18 mm. |" 15-18 mm.) 49280
(Original | (Original (Original (Original (Origingl
length) length) length) length) length)
Oxygen consumed by 0.5 gram in two hours
ce. ce. ce. cc. ce.
1 day 0.16 0.24 0.16 0.20 0.23
4 days 0.14 0.14
1 week 0.14 0.13
2 weeks 0.12 0.19 0.12 - 0.17 0.17
4 weeks 0.14 0.18 0.14 A ipe sie 0.16
6 weeks 0.15 0.19 0.12 0.16 0.16
8 weeks 0.17 0.21 0.15 0.18 0.18
10 weeks 0.17 0.23 0.17 0.20 0.19
12 weeks 0.17 0.32 0.17 0.33 0.27
16 weeks 0.31* 0.25*
Per cent increase...... 158 Vi | 109 106 65

* Means that heads were removed the day preceding the test.

5. Susceptibility of P. agilis during starvation. This matter has
already been considered in the introduction. If the susceptibility

method is reliable as a means of determining general metabolic rate,

the susceptibility of P. agilis should increase more slowly during star-
vation than is the case with Planaria dorotocephala. I have made
some tests of the susceptibility to cyanide of P. agilis during starva-
tion. These tests were not as complete as is desirable but they indi-

cated clearly enough the general situation in this species. All starving”

worms are more susceptible to cyanide than recently fed ones. Worms
starved two weeks to five weeks are slightly more susceptible than

.

a, oe

OXYGEN CONSUMPTION IN STARVATION 413

those starved one week. There is, however, no difference or very little
difference in the susceptibility of worms starved two, three, four and ‘five
weeks. «Thus between the second and sixth weeks of starvation there
is very little increase in susceptibility while during this period in P.
dorotocephala a marked increase is demonstrable. Reference to table
3 shows that during this period also the oxygen consumption of P.
agilis remains practically stationary. In later periods of starvation
the susceptibility of P. agilis increases. |

There is thus in this species a general agreement between the results
by the susceptibility method and the results from direct measurement
of the rate of oxygen consumption.

6. Comparison of starved and young (small)) individuals of P. agilis.
It is insisted by Allen in his paper that whereas small worms consume

TABLE 4
Comparison of the rates of oxygen consumption of small (young) recently fed worms,
large (old) recently fed worms, and worms reduced by starvation to a size
similar to that of the young worms. Temperature 21°C.

KIND OF WORM LENGTH TIME SINCE FEEDING | OX¥GEN CONSUMED BY

0.5 GRAM PER 2 HOURS
mm.

Youn abe pe g
Be bite Wl o's osha b's e's 6-10 5 days 0.21
fn 15-18 2 weeks ted es
Seer eee esterases ees 18-20 2 weeks 0.17
Bis 12 weeks 0.33

Starved............. oe cai 0.27 -

more oxygen per unit weight than large ones, worms reduced by star-
vation consume the same amount of oxygen as before such reduction.
This statement appears improbable on the face of it and my experi-
ments show it to be erroneous. Small worms whether their size is due
to youth or starvation consume more oxygen per unit weight per unit
time than large worms in an adequate state of nutrition. It requires
some twelve weeks of starvation to reduce P. agilis from a length of
18 to 20 mm. to a length of 6 mm. Such a reduction involves an
increase of at least 60 per cent in the rate of oxygen: consumption.
According to Allen the difference in oxygen consumption between 6
mm. and 18 mm: worms is about 60 per cent (his table 10) when
movement is not eliminated and about 40 per cent (his table 11) when

414 LIBBIE H. HYMAN

movement is eliminated. As young fed worms move about much
more than greatly starved worms, the latter figures are more correct.

In table 4 are given some data on the oxygen consumption of two
lots of young worms 6 to 10 mm. long, starved five days; the oxygen
consumption of worms 15 to 20 mm. long, from the same stock, starved
two weeks; and the oxygen consumption of the same worms after
reduction to a size similar to that of the young worms. After such
reduction the worms have a higher rate of oxygen consumption than
the young worms of similar size.

DISCUSSION

It is shown in this paper that in two varieties of Planaria maculata
(which are probably separate species) and in Planaria agilis the rate of
oxygen consumption per unit weight is increased by starvation. This
increase begins in the first named forms within four weeks from the
cessation of feeding but in Planaria agilis not until the eighth week if
the original length exceeded 15 mm. This result agrees with results
previously presented on other species (1) and disagrees with the con-
tentions of Allen (2). Planaria agilis differs from all other species
which have been adequately tested in that the oxygen consumption
remains at nearly the same level between the second and sixth weeks
of starvation, and rises only subsequently to this period. This cir-
cumstance is due to the fact that Planaria agilis starves more slowly
than other species; it has a lower metabolic rate, greater food reserves,
and a more capacious digestive tract, and consequently reduces more
slowly in starvation than other species of Planaria. The remark
of Allen that ‘‘the fact that the rate of oxidations is uniform during a
long period of starvation will serve as an important basis for the study
of respiratory metabolism in these forms’’ applies to Planaria agilis
only. There is no level of ‘‘basal’’ or “‘standard”’ metabolism in other
species, but their metabolism is continuously changing during starva-
tion. Even in Planaria agilis the rate of oxygen consumption is prob-
ably not stationary but is falling very slowly during the first half of
this period of apparent constancy and rising very slowly during the
latter half of it.

Since Allen’s paper is directed mainly against the reliability of the

susceptibility method, it remains to make some statements regarding

this method. In the first place, the susceptibility method measures
the time of death. The time of death can naturally be determined

ee

OXYGEN CONSUMPTION IN STARVATION 415

only for those parts of the organism visible to the observer, namely,
the superficial parts. Strictly speaking, therefore, survival time is a
measure of the metabolic rate of the body surface only. That we have not
in earlier publications emphasized this is due simply to the circum-
stance that the occasion for emphasizing it had not arisen. It had
always been recognized by us that the susceptibility method concerns
chiefly superficial structures and statements were made in various
publications that internal systems and organs may show different
susceptibility relations from the body surface.

It is, however, evident that when special internal conditions are elim-
inated survival time is a measure of general physiological and metabolic
conditions in the whole organism. In the lower organisms there is
only one such special internal condition which it has thus far been
necessary to consider—that is the physiological condition of the diges-
- tive tract. When this factor is eliminated by keeping it in the same
functional state in organisms which are to be compared then, as far as
our data at present go, there is never any discrepancy between the
results by the susceptibility method and the determinations of total
oxygen consumption and carbon-dioxide production. A summary of
some physiological conditions in which both susceptibility and respira-
tory rate have been determined is given in table 5.

From table 5 it will be seen that in nearly all cases where the sur-
vival time is shorter, the rate of respiratory exchange is greater, and
where the survival time is lengthened, the rate of respiratory ex-
change is decreased, the sole exceptions being those concerned with feed-
ing. That this apparent discrepancy exists was known to us for a con-
siderable time before Lund and Allen published their work. ‘Their
viewpoint, that because this discrepancy exists the whole conception is
erroneous, is certainly unscientific, to say the least. The correct pro-
cedure is to attempt to discover the cause of the discrepancy. This
has been done and it has been found that the discrepancy is due to the
behavior of the digestive tract. The respiratory rate of the digestive
tract is increased by feeding but as the surface of the body is not affected
by the feeding process, the susceptibility of the surface does not change.
On the other hand the susceptibility of the intestine is increased by
feeding, although it is somewhat difficult to observe the digestive tract
by the susceptibility method in Planaria. In more transparent forms,
like Hydra, the region of the body where food is being digested exhibits
a very striking increase in susceptibility. After feeding, the respira-
tory rate of the digestive tract falls and as the body surface is again

416 LIBBIE H. HYMAN

not involved in this process, the susceptibility of the surface is not

altered. It is therefore necessary under circumstances involving the
digestive tract to consider the body wall and digestive tract separately.
As this is continuously done in physiological experiments on higher
animals, where in order to study one physiological process it is neces-
sary to have the others-constant, we fail to see that such a procedure is
“damaging to the theory that susceptibility is a measure of rate of

TABLE 5
Summary of the results from measurements of the survival time in toxic solutions

and direct measurements of respiratory rate under different physiological
conditions in Planaria

PHYSIOLOGICAL CONDITION SURVIVAL TIME OXYGEN CONSUMPTION CO2 PRODUCTION
Age (size) Shorter the smaller | Greater the smaller | Same as for oxy-
(younger) the the worm gen consump-
worm tion
Injury Shorter in injury | Greater in injury | Same
Regeneration Shorter in regener- | Greater in regener- | Same:
ation ation
High temperature Shorter Greater | Same
Low temperature Longer Less Same
Movement Shorter than in rest | Greater than in rest | Same
In acid solution Longer | Less
Different levels Shorter in anterior | Greater in anterior | Same
levels levels
Late starvation Shorter Greater Same
Feeding No change in body | Greater Same
surface
Early starvation No’ change or| Less Same
shorter in body
surface

oxidations,”’

as Allen states.

The method of determination of total

- metabolism in which Allen places such faith also fails to give any infor-
mation about specific processes in the body unless other factors are con-

trolled and eliminated.

If one wants to measure the respiration in

muscular work one cannot at the same time give the experimental

animal a meal.

It may then be reiterated that as far as our experiments go, the

survival time in toxic solutions is in simple animals a measure in .

general of the metabolic rate, as long as internal conditions remain con-

stant.

Survival time may not be an accurate measure of total respir-

417

OXYGEN CONSUMPTION IN STARVATION

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_ OXYGEN CONSUMPTION IN STARVATION 419

atory rate because other factors than respiratory rate may be
concerned in survival time. Argument over the reliability of the
susceptibility method has now been rendered unnecessary because all of
the main conclusions drawn by the use of this method have been veri-
fied by direct determinations of oxygen consumption and carbon-dioxide
production.

SUMMARY

1. This paper is concerned with the rate of oxygen consumption
during starvation of Planaria maculata and Planaria agilis and was
undertaken in reply to a paper by Allen (1).

2. Contrary to the results of Allen it was found that in both of these
species within the time limits specified in his paper, the rate of oxygen
consumption is increased in the later periods of starvation. His data
are criticised and it is pointed out that they are with one exception
inadequate and unsatisfactory; the one exception contradicts his own
conclusion.

3. Planaria agilis has a lower metabolic rate, greater food reserves,
and loses weight more slowly in starvation than other species. For this
reason it must be starved for a longer period before its rate of oxygen
consumption increases.

4. Owing to the slow rate of starvation of Planaria agilis, there is a
period from the second to the sixth week of starvation when the oxygen
consumption is nearly constant. This is not the case in other species.

5. The susceptibility of Planaria agilis to potassium cyanide also
fails to increase during starvation as rapidly as in other species.
Contrary to the contentions of Allen, the susceptibility results in this
species are then in general agreement with the results from direct meas-
urement of the oxygen consumption.

6. Contrary to the statements of Allen, it was found that in Planaria
agilis worms reduced to a small size by starvation have as high a rate as
or a higher rate of oxygen consumption than small recently fed worms
of the same size.

7. A general discussion of the utility of the susceptibility method
as a measure of metabolic rate is given and it is pointed out that no
discrepancies are at present known to us between the results by the
susceptibility method and the results by direct determination of respira-
tory rate, except those concerned with feeding. In the case pf feeding,
it is necessary to draw a distinction between the metabolism and sus-
ceptibility of the digestive tract and those of the body wall.

increased in 'ptpahewbiorté aie ins borer ag is th at
bringing about rejuvenescence. pad sat

(1) Hyman: This Journal, 1919, xlix, 377.
(2) Pgonsi eye Journal, 1919, nei 420.

VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION
IN INTACT ANIMALS

E. G. MARTIN, A. C. FRANKLIN anp CLARENCE HIELD
From the Laboratory of Physiology, Stanford University

Received for publication June 28, 1920

Of recent years the investigation of vasomotor reflexes in mammals
other than man has been carried on chiefly on the basis of artificial
stimulation of nerve trunks, rather than by means of receptor stimula-
tion; the older physiologists, on the other hand, gave much attention
to the effects on blood pressure of excitations applied to the receptors
themselves. The latter method would appear to be the more logical if
the object sought is to determine the normal mode of functioning of
the vasomotor apparatus, although where the underlying purpose is -
the study of reflex action, with the vasomotor reflexes selected as
examples of such action, direct stimulation of sensory nerve trunks
is not only valid, but has much to recommend it, both in theory and
practice. :

One of us (M.) has been for some years interested in vasomotor
reflexes, not so much from the standpoint of their significance as regu-
lating factors of the circulation as for the light they may throw on
the reflex functioning of the nervous system itself ( (1) to (6)). In
all the experiments described in the above series of references the elici-
tation of reflex responses was by means of stimulation of nerve trunks.
Certain of the findings therein have suggested the desirability of re-
peating some of the experiments of the older investigators on the reflex
vasomotor effects of receptor stimulation, with the attention specially
directed to the bearing of such experiments on theories of nervous
action.

PROCEDURE

Our plan of study called for the elicitation of vasomotor reflexes in
conscious and in very lightly narcotized animals, as well as in decere-
brate and in deeply anesthetized individuals. It followed that the
method of recording the responses must be applicable to conscious

421

422 E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

animals. Blood pressure determinations were ruled out because of
the operative procedures involved; the choice was thus restricted to
plethysmographic methods, or to such as use temperature variations
as indices of vasomotor changes. Either of these latter have the dis-
advantage of showing only local effects, as contrasted with the general
influences revealed by blood pressure determinations. A vasomotor
response that is restricted to the splanchnic region might escape detec-
tion completely under such circumstances. This is a limitation that is
unavoidable, however, in experimentation on conscious animals. If
the site of application of the recording device is fortunately chosen it
should reveal any vasomotorchange that affects cutaneous areas. One
important desideratum is that the region selected shall be known to
have both constrictor and dilator innervation, since interpretation of
the interaction of dilator and constrictor responses is a significant
feature of the general problem (5).

Following the example of Griitzner and Heidenhain (7), our experi-
ments were made on rabbits. The varieties were those widely raised
in California for market purposes, including some common domestic

rabbits and some Belgian hares. On the whole we found the latter

less satisfactory than the smaller and hardier rabbits. We experi-

mented at some length with the nasal cavity:as a plethysmograph, —

using the method of Tschalussow (8) as modified by Mendenhall (9)
but found it unsuited for work with rabbits. We then turned to the
ear as a field of study. Since in rabbits the ears play an undoubted,
and probably considerable, part in temperature regulation (10), they
might be expected to be as sensitive to vasomotor influences as any
of the cutaneous areas. That they have dilator as well as constrictor
innervation appears from the work of Winkler (11), cited by Bayliss (10).

To record vasomotor changesin the ear weused an ordinary air plethys-
mograph, communicating with a sensitive tambour, whose lever wrote
on a slow drum. The air plethysmograph offers a double advantage
for work of this kind; in addition to being itself a sensitive recorder of
volume changes in the ear, it responds also to changes of temperature;
vasodilatation would bring about both an increased ear volume and a
higher ear temperature; the’ latter by causing expansion of the air in the
plethysmograph would add its effect to the increase of ear volume,
enlarging the tracing on the drum. Obviously sudden changes in tem-
perature due to external influences had to be guarded against, but
this was a simple matter.

VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION 423

For keeping unanesthetized rabbits in position we used a board 6
inches wide, mounted on a pedestal about a foot high. This board was
cut away at front and rear, so that when a rabbit was laid on it his
legs would hang down comfortably. A couple of short straps were
buckled around the board and over the back of the animal, and a
rest provided for his chin. With this arrangement a rabbit would
remain quiet for two or three hours, making few movements, except
when stimulated thereto in connection with our experimentation. At
first we tied the head firmly, but this proved unnecessary and since it
often seemed to impede the blood flow through the ears was abandoned.
A precisely similar arrangement was employed with anesthetized. or
decerebrate animals, except that with most of these we added a head-
clamp. The hair was regularly clipped from all four legs, and from a
considerable area of the back. :

Stimulations. Tactile, thermal and auditory stimuli were used with
success; visual stimuli, in the form of light flashed on and off before the
eyes, gave negative results so far as vasomotor manifestations ar
concerned. a

For tactile stimuli we followed the practice of Griitzner and Heiden-
hain (loc. cit.) and used blowing upon the skin, rubbing the mouth,
inside, and upon the lips, with a hard instrument, and pulling at indi-
vidual hairs with forceps, all gentle stimuli and, according to the experi-
ence of those investigators, highly effective in evoking vasomotor reac-
tions. We found the free ear the most favorable spot on which to
blow. : .

Several sorts of thermal stimuliwereused. Dipping a leg into water of
selected temperature for a measured time, thirty seconds to two min-
utes, proved moderately satisfactory. The leg, as stated above, was
clipped of hair, and was thoroughly greased with vaseline before dipping
into water. We used a container holding a liter; the temperature of the
_ relatively large volume would remain fairly steady during the period of
immersion. The position of the animal on the holder permitted the
bringing of the vessel of water up around the leg without disturbing
the experiment. The range of temperatures employed will be given
further along, in connection with the detailed account of the results of
the experiments. A second method of thermal stimulation, of which
we made much use, consisted of placing an electric light, backed by a
conical reflector of ordinary type, a short distance above the back,
from which, as noted above, the hair had been clipped. To insure
that the rays from the light should not strike upon the plethysmograph

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 3

424 E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

directly, and so warm the air within it, we interposed a double screen;
this consisted, first, of a large sheet of cardboard placed upright be-
tween the tabbit’s back and head, and cutting off from the whole head
and its surroundings any direct rays from the light; and second, of a
cylinder of sheet asbestos, about double the diameter of the plethysmo-
graph, placed about it. A sensitive thermometer inserted between the
asbestos cylinder and the plethysmograph showed that no change of
temperature occurred upon turning on the light. Another sensitive
thermometer was laid along the animal’s back, with the bulb in close
contact with the skin, to enable some idea to be formed of the rate of
warming of the back, as well as the maximum temperature reached.

Since it was probable that the temperature of the thermometer bulb ©

would rise faster than that of the skin, where the bulb was freely
exposed to the light, we protected the bulb, in some of the experi-
ments, with a small bit of asbestos. This precaution was not wholly
satisfactory, since it was now probable that the skin in contact with the
thermometer bulb, being protected with asbestos, as well as by the bulb
itself, would warm up more slowly than areas fully exposed to the
light, and so introduce an error in the opposite direction. To cool the
skin of the back we placed upon it a cloth bag of cracked ice, or obtained
the desired effect by pouring a small quantity of ether upon it.

Various auditory stimuli were tried, but the only really satisfactory

one consisted of a shrill whistle, blown near the ear of the rabbit, but .

with precaution not to allow the blast of air to strike the animal.

VASOMOTOR RESPONSES IN NORMAL RABBITS

_ For experiments on conscious animals we were obviously limited to
stimuli which would be endured without struggling, since quiet was es-
sential to the securing of a convincing record. This meant the confining
of the stimulation, in general, to the levels below the threshold of pain,
or the selection of stimuli which have no marked painful quality, even
when intense. The investigation became, in a sense, a comparison of
the thresholds of vasomotor and skeletal muscle reflexes, since our

practice was to push the stimulation, whenever possible, until the ani-

mal began to struggle, observing meanwhile such changes in ear
volume as occurred.

Tactile stimuli. Confirming the findings of roaster and Heiden-
hain (7) that gentle stimulation is effective in arousing vasoconstriction,
we obtained repeated shrinkage of ear volume by blowing with the

oo: GPC Sie >:

VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION 425.

breath on the exposed ear, or the back (clipped of hair) or the hind
leg, or the nose. The onset was prompt; the maximum effect was
reached within fifteen seconds, and the return to former volume was
completed within thirty to forty-five seconds. Contact of an ice pack
with the back caused, on several occasions, an ear shrinkage that was
obviously due to the contact and not to any lowering of temperature,
since the onset was as prompt as in the blowing experiments, and re-
_ covery occurred as quickly. Localized irritation, as by pulling at
small group of hairs with forceps, did not produce any demonstrable
change in ear volume.

A feature of these responses which we wish to stress is that they
were definitely more pronounced early in the course of the experiments
than later. There appeared to be a habituation or fatigue which
operated to cut down their magnitude. To illustrate: a large black
male rabbit, which had been experimented on on several previous
occasions, gave, within the first five minutes of the experiment of Feb-
ruary 19, 1920, typical responses to blowing upon the ear. Ten min-
utes later the response was still evocable, but much reduced; a half-
hour later the same sort of stimulation failed to elicit any response.
Throughout the period stimuli of one sort or another were applied every
minute ortwo. The animal was then removed from the apparatus and
allowed to run about the floor for ten minutes. It was then replaced
in position for recording ear volume changes and similar stimuli applied.
Shrinkage of ear volume occurred, comparable with that obtained at
the very beginning of the experiment; again, however, there was a
diminution of effect, so that at the end of fifteen minutes the response
was no longer obtainable.

Auditory stimuli. Shrill whistling, prolonged for five or six seconds,
brought about ear shrinkage very much like that obtained from tactile
stimulation. The latency was short, and the duration about equal to
‘that of the responses to blowing on the ear or back. To assure our-
selves that the effect on the plethysmograph was actually due to a
change of ear volume, and not to a movement of the tympanic mem-
brane, we stuffed the external auditory meatus with vaseline; this
procedure did not change the response. These observations are in
accord with those of Dogiel (12). We observed a habituation to audi-
tory stimulation ‘similar to that reported above for tactile. The vaso-
constriction from whistling was frequently greater at first than the
maximum from blowing on the back or ear, but quickly became less,

426 E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

and complete failure occurred earlier. A short rest outside the appa-
ratus restored the effectiveness of the stimulation momentarily. ,
Thermal stimuli. We attempted to lower the temperature of the
skin of the back by laying.an ice pack on it and by pouring ether on
it. In spite of repeated attempts the first method failed to yield
demonstrable vasoconstriction in conscious rabbits. The animal would
endure the presence of the ice pack for about two minutes, but would
then begin to struggle so violently that the pack had to be removed.
No change in ear volume occurred during the two minutes in which
the pack was in contact with the back, except for the transitory vaso-
constriction due to contact described above. The chief interest of this
observation lies in the apparent demonstration that cooling of the back
becomes sufficiently uncomfortable to arouse the conscious animal to
definite efforts at escape before the stimulation of cold reaches the
point of evoking demonstrable vasoconstriction. We were uniformly
successful in obtaining vasoconstriction when ether was poured on the
back. The effect developed promptly, reached a maximum within a
half-minute, and usually passed off within two or three minutes. A
small volume. of liquid, roughly 1 cc., gave the best results. On one
occasion we tried pouring about 5 cc. on the back. Vasoconstriction
began to develop in the usual way, ‘but before it had attained maximal
extent the animal displayed signs of annoyance at the odor, and pres-
ently began to struggle. We attribute the constrictor effect induced
by ether to the cooling brought about by the rapid evaporation, and
we have evidence from the thermometer placed against the back that
there was prompt and marked cooling; on two occasions the back tem-
perature fell eight degrees in two minutes. The possibility that the
ether may have had an irritating effect apart from the cooling is not
excluded, although no marked irritation is experienced from contact of
ether with the human skin. It is possible that our superior success with

ether as compared with the ice pack was due to a degree of fright induced

by the strange odor, which kept the animal quiet except in the case
where an excessive dose was used, when the desire to escape became
paramount.
Vasodilatation (swelling of ear) was readily obtained in conscious rab-
bits by warming the back with an electric light in the manner described
above. The animals usually endured the warming for several minutes
without showing signs of discomfort. In every trial but one of our series
vasodilatation began to show itself before any skeletal muscle move-

VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION 427

ments were made. In the one exceptional case there were signs of rest-
lessness before vasodilatation became perceptible, but the movements
were not extensive enough to necessitate discontinuance of the experi-
ment, and increase in ear volume began to show itself before struggling
became pronounced. There was considerable variation in the latency
of the vasodilatation; in some cases the ear began to swell within a
few seconds after turning on the light; in others two or three minutes
elapsed before visible increase in ear volume appeared. In the excep-
tional case mentioned above, in which struggling preceded ear swelling,
the warming continued for seven minutes before the bodily movements
began; the swelling of the ear did not become demonstrable until
eleven minutes after applying the electric light. As stated in a former
paragraph, we do not consider the recorded changes in back tempera-
ture very significant because of the large probable error, inherent in the
use of a mercury thermometer laid in contact with the skin. It is,
however, perhaps worth noting that on four occasions-the rise in skin
temperature preceding the beginning of struggling on the part of the
animal coincided at 6°.

VASOMOTOR REFLEXES IN DECEREBRATED RABBITS

Our method for preparing the rabbits was that usually employed in
experiments involving the procedure of decerebration. Light ether
anesthesia was maintained throughout the operation up to the com-
pleting of the cut across the brain stem. Clamps were applied to the
carotids, and the vertebral arteries were occluded by pressure of the
‘fingers and thumbs of an assistant at the sides of the vertebral column
immediately behind the skull. In rabbits stoppage of the circulation
through the vertebrals is easily accomplished in this manner. As
soon as possible after completion of decerebration the pressure on the
vertebrals was released and the clamps on the carotids removed.

Tactile stimuli. The only suggestions of vasoconstrictor reflexes
following tactile stimulation were obtained within the first fifteen min-
utes after decerebration. On several occasions we noted what looked
like extremely slight positive responses, but only once was there a
reaction that at all approached in magnitude our usual responses to
similar stimulation in normal rabbits. On this occasion the reaction
followed laying the hand gently on the rabbit’s head. About a min-
ute later repetition of the stimulus was followed by vigorous galloping
movements on the part of the animal. Perhaps it is worth noting that

428 E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

whenever our procedures aroused decerebrate rabbits to violent move-
ments they took the form of galloping; this contrasts sharply with the
struggling of conscious rabbits, whose effort was invariably to push
themselves backward off the board on which they lay; an effort expli-
cable when one recalls that the chief unaccustomed factor in the situa-
tion was the plethysmograph tube on the ear. In no case did tactile
stimuli applied during the later stages of an experiment give positive

vasomotor results. That this failure was not due to diminished sensi-

tivity of the animals was shown by the presence during the same stage
of the experiment of definite skeletal muscle responses to slight stimuli.

Auditory stimuli. We were unable to obtain a definite vasomotor
reaction to shrill whistling in any instance, although skeletal muscle
_ responses were readily elicitable in nearly all our trials. In one case
the galloping activity, noted above as characteristic of violent effort
in decerebrate rabbits, followed an auditory stimulus, although in most
of our trials only slight movements of ear or head were noted. We wish
to stress the point that two sorts of gentle stimulation, tactile and
auditory, which in normal animals typically bring about well-marked
vasomotor reflexes, fail almost completely in this regard in decere-
brates, although skeletal muscle reflexes are obtained about as well in
one form as in the other. :

Thermal stimuli. We obtained typical vasoconstriction from pour-
ing ether on the back of decerebrated rabbits; in appearance this
corresponded fully with the response of normal animals to similar stim-
ulation. Warming the-back by means of an electric light gave vasodila-
tation (ear swelling) in all our trials except two. There appeared to be
less tendency than in normal rabbits toward struggling; at least the
animal would usually remain quiet for a longer time after the light
was turned on than would the normals. On two occasions no ear
swelling was manifest; on one of these there appeared to be a slight ear
shrinkage. We carried out an extensive series of observations on the
results of immersing a leg in water. As stated above, the leg was thor-
oughly greased with vaseline before immersion. A hind leg was used in
most of our experiments. The front leg was tried, but in no case with
positive results. Water at 1°C. (ice water) was used repeatedly but
neither with decerebrates nor with rabbits in any other state did we
ever obtain a perceptible vasomotor change therewith. For the tests

with warm water temperature intervals of 5°C. were used, beginning —

with 35°. No changes in ear volume were observed at temperatures
below 50°. At 50° vasodilatation occurred regularly; at 55° vasodilata-

|
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VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION 429

tion occurred in approximately half the trials and vasoconstriction in
the other half; at 60° and 65° only shrinkage in ear volume was seen,
except that on one occasion a slight increase in ear volume preceded the
constriction. These observations suggest that there is a temperature
point at which reversal of vasomotor effect occurs. .

VASOMOTOR REFLEXES IN RABBITS UNDER LIGHT ETWER ANESTHESIA

In our experiments in which ether was used as the anesthetic pains
were taken to keep the narcosis as shallow as possible; some variation
in depth of anesthesia was inevitable with the method of administra-
tion, which was by an ordinary nose cone. Tactile stimuli appeared to
be less effective in eliciting vasomotor responses than in unanesthetized
rabbits; at least such as we tried were ineffective, although they were
pushed to the point of inducing struggling. Shrill whistling was fol-
lowed by transient shrinkage of the ear; very definitely in the early
stages of the experiment; less so in the lites stages. It was our impres-
sion thai there was a habituation to the stimulus, similar to that sug-
gested by the experiments on conscious animals, but the possibility
that increasing depth of narcosis might account for the lessened effect
is not excluded.

Warming the back by means of an electric light resulted in vaso-
dilatation; with very light narcosis the animal would endure the warm-
ing without struggling for only a few minutes, so that extended expos-
ure to the warming could not be studied. Immersing a hind leg in
water at 45°C. was followed in one instance by definite ear swelling,
although shortly afterward temperatures of 49°, 50° and 51° gave
negative results. Water at 52° brought about commencing vasodila-
tation, but almost coincident with the beginning of the change in ear
volume the animal became restless and the hot water had to be with-
drawn. When the leg was immersed in water at 55° the animal struggled

so promptly and violently that no vasomotor record could be obtained.

VASOMOTOR REFLEXES IN RABBITS PARTIALLY ANESTHETIZED WITH
URETHANE

We were much interested in the possible effects of urethane on-the
responses to receptor stimulation, since Martin and Lacey °(1): had
shown that the threshold of vasomotor reflexes from nerve-trunk stimu-
lation is not very much raised by urethane. We performed our’ ex-

430 E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

periments with urethane in two series; in the first only half the stand-

ard dosage was used (1 gram per kilo body weight); in the second we

used standard full dosage. The appearance of rabbits that have re-—

ceived only half the usual dose of the narcotic differs characteristically
from that presented by fully anesthetized animals. They show a de-
gree of prostration, but chiefly in the rear half of the body. The hind
legs are frequently quite useless, but some control is retained over the
front legs, and the neck muscles appear to be only slightly affected so
that the head is held in about the usual position. This is in sharp
contrast to the complete muscular relaxation of rabbits under the
influence of-full dosage.

Tactile stimuli. The same kinds of tactile stimuli were used as under
the other experimental conditions employed in the investigation,
namely, blowing on or pinching the ear, blowing on the back or leg,
rubbing the inside of the mouth with a blunt instrument. In about
half the trials ear volume showed slight decrease, indicating some vaso-
constriction; in the others no change was seen. On two occasions

pinching the ear brought about struggling, showing that skeletal

muscle reflexes were rather readily evocable.

Auditory stimuli. In only one out of nine trials did whistling fail
to elicit vasoconstriction; one of the results recorded as positive was so
slight as 10 be questionable; all the others were definite.

Thermal stimuli. Cooling the back by pouring ether on it did not
give as satisfactory results as in normal or decerebrated rabbits. We
did not obtain in any instance as good a record of ear shrinkage from

this procedure as appeared regularly in those series. The partially —

urethanized animals spoiled the record by struggling much more fre-
quently, but in the few cases in which struggling did not occur clear-
cut vasoconstriction was not manifested, although the record indicated
its presence to a very slight degree.

Warming the back with an electric light was followed regularly by
well-marked vasodilatation. The warming was ordinarily endured for

only about four minutes; at the end of that time struggling would be-

gin and the light have to be removed. There would have been time,
meanwhile, for the ear swelling to show itself. Immersing a hind leg
in warm water gave vasodilatation at 45°, 50°, and 52°; at the latter
temperature the effect was very slight; possibly there was a habituation

from the previous immersions at the lower temperatures. At 54° the

animal struggled thirty seconds after applying the warm water; no
swelling of the ear had developed at that time.

VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION 431

Observations on the late effects of urethane. We were interested in the
late effects of urethane on the relative sensitiveness of the vasomotor
and skeletal muscle reflex paths; accordingly we reéxamined our ani-
mals at the end of twenty-four hours, and in one case of a half-dose
animal again after forty-eight, and after seventy-two hours. Our
rabbits that had received only half the usual urethane dosage were in
general appearance nearly over the effects of the drug on the day fol-
’ lowing administration. They ordinarily showed some lack of coér-
dination in the movements of the hind legs, but in other respects behaved
much like normal rabbits. Such skeletal muscle reflexes as attended
our receptor stimulations were definitely more easily elicited than on the
preceding day. In contrast to this near recovery of skeletal muscle
control is the interesting fact that vasomotor reflexes were nearly
inelicitable. Vasodilatation resulted from warming the back with an
- electric light, but all our other procedures were negative, except that
in a single instance rubbing the inside of the mouth was followed by a
minute curve in the tracing which may have been an indication of very
slight vasoconstriction.

After forty-eight hours the single rabbit that was observed extensively
presented, in all respects, the appearance of complete recovery. It re-
_ sponded to mechanical and auditory stimulation by vasoconstriction in
precisely the fashion of normal rabbits. The only difference that we
observed was that no ear shrinkage followed the pouring of ether on the
back. To determine whether this response would be reéstablished we
repeated the tests on the following day, namely, seventy-two hours
after narcotization. In addition to positive results from all our other
tests we now obtained definite vasoconstriction when ether was poured
on the back.

VASOMOTOR REFLEXES IN RABBITS FULLY ANESTHETIZED WITH URETHANE

As stated above, rabbits under full urethane narcosis show complete
muscular relaxation. In our experience this comes on quite promptly;
it is accompanied by a pronounced slowing of the breathing. We
made a number of counts of the respiratory rate before and after admin-
istering urethane, and obtained slowing from a preliminary rate rang-
ing between 140 and 180 a minute to a rate of 60 to 70 a minute.

Tactile stimuli. We obtained definite changes in ear volume follow-
ing tactile stimulation in about 70 per cent of our trials; since we ex-
perimented with various means of stimulation the percentage of nega-

432 E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

tive results is probably not unduly large. Contrary to our experience
with rabbits in other states as regards narcotization, in all of which
mechanical stimulation gave only vasoconstriction, we observed in
nearly a third of our positive trials in this series swelling of the ear
(vasodilatation) ; in the remaining two-thirds the usual vasoconstriction
was seen. In an attempt to account for the occurrence of contrary
responses from similar stimulations we classified the tactile stimuli

employed as gentle or harsh. To the first group were assigned strok- ~

ing the skin, handling the ear gently, and blowing on the ear or back;
in the second were included pinching the skin hard with fingers or for-
ceps and rubbing the inside of the mouth with a blunt instrument; the
free ear was the area most frequently selected for pinching. All our
vasodilator responses but one resulted from the application of stimuli
classified as gentle; 70 per cent of the constrictor responses, on the other
hand, followed harsh stimulation.

Notwithstanding the complete muscular relaxation characteristic of
fully urethanized rabbits skeletal muscle reflexes were not~-wholly
absent. On three occasions mechanical stimulations were followed by
struggling. Two of these were after harsh stimuli; the third occurred
after blowing on the skin, a stimulus classified by us as gentle.

Auditory stimuli. Shrill whistling was followed by ear shrinkage

(vasoconstriction) regularly in the early stages of the experiments. In

only three early trials did we fail to get the response. In all these
experiments stimulation was begun as soon as possible after signs of
complete narcotization had developed. In only one case did we observe
a positive effect as much as forty minutes after the beginning of ex-
perimentation. In general urethanized rabbits seemed to respond
positively to auditory stimulation only during the very early stages
of narcosis. In all our experiments of this series good responses to tac-
tile stimuli were elicitable a half-hour or more after auditory stimula-
tion had ceased to be effective. In two instances transient ear swelling
followed the whistling; these were the only cases in our entire experience
in which any other positive response than vasoconstriction followed
auditory stimulation.

Thermal stimuli. Both our methods of lowering skin temperature,
laying an ice pack on the back, and pouring ether thereon, brought

about vasoconstriction repeatedly. We failed to get this result only

twice, both times during the very late stages of the experiments. Rais-
ing the back temperature by means of an electric light was followed

Se

VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION 433

just as regularly by swelling of the ear (vasodilatation). In several
instances the onset of the dilator reaction was considerably delayed.
During this latent period we observed several times, but not invari-
ably, a slight but perfectly definite ear shrinkage, which suggests the
possibility of a local vasodilatation in the warmed area of the back
which would cause an increased flow of blood through that region at
the expense of Other parts of the body. With the development of gen-
eral reflex vasodilatation active swelling would succeed this passive
shrinkage in such areas as the ear. On only two occasions did the
warmth on the back lead to struggling. This contrasts sharply with
the invariable occurrence of this reaction in normal rabbits.

Immersing one hind leg in warm water was followed by swelling of
the ear (vasodilatation) in just half our trials; all the others were nega-
tive except two, in which there appeared to be a slight shrinkage of the
ear. Positive vasodilatations were obtained at temperatures of 50°
and upward; none were seen at 45°, although that temperature was
tried repeatedly. We obtained in two cases ear swelling at 65°; subse-
quent tests at 70° and 75° were negative. We have reason to suspect
that injury to the receptors occurred at 65°, since the leg that had been
immersed in this very hot water was much swollen and blistered on
the following day. For comparison it might be well to note that
water at 50° to 52° is extremely painful to the human hand if i immersion
is maintained for as much as a minute, as we demonstrated upon our-
selves. The observations which we interpreted as showing vasocon-
striction occurred at 53° and 58°. There was struggling, sometimes
violent, at nearly all temperatures above 53°. The latency was suffi-
cient to allow the change in ear volume to show itself while the ani-
mal was still quiet. . In one case the skeletal-muscle response to immer-
sion of a hind leg in hot water took the form of definite running
movements.

Late effects of full urethane narcotization. In an earlier section we
described observations on the recovery from half doses of urethane.
Similar observations were made on the late effects of full doses. At the
end of twenty-four hours we found substantially the same situation
with respect to vasomotor responsiveness that is recorded above for
partially urethanized animals, namely, an almost complete insensitive-
ness. We have one observation of ear swelling following warming the
back with an electric light, but with by far the longest latent period
(thirteen minutes) seen in the investigation. We have also one record
of vasoconstriction resulting from pouring ether on the back. All our

434 E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

other numerous tests, including all the kinds described in this paper,
were negative. These rabbits showed much less progress toward re-
covery of skeletal muscle control than did those under half-urethane
after a similar interval. They did, however, respond regularly by
struggling to most forms of mechanical stimulation, to ether on the
back, and to immersion of a hind leg in water at temperatures above
51°C. The respiratory rate was that of complete urethane narcosis,
namely, 60 to 70 a minute.

A single rabbit that was examined at intervals until completely recov-
ered gave the following history: at thirty hours after administration of
urethane the breathing was still slow, 72 in the minute; there were no
well-marked ear volume changes as the result of stimulation, although.
very small curves appeared in the tracings in connection with the
application of some of the stimuli. An interesting feature of such as
did appear was that with one exception they were of opposite sign
to the usual responses; thus, both tactile and auditory stimuli gave
what would have been interpreted as vasodilatation if the curves had

been more pronounced, whereas the ordinary effect of such stimuli

is definite vasoconstriction. One tactile stimulus did give apparent

ear shrinkage, this being the single exception mentioned above. After .
forty-six hours the breathing in this rabbit had returned to the normal

rate, 170 in the minute; there was still considerable muscular incoérdi-
nation. The vasomotor responses to stimulation were more marked
than after thirty hours, but again both auditory and tactile stimuli
were followed by dilatation instead of by the usual constriction.
Warming the back with an electric light gave dilatation; immersing a
hind foot in warm water (45°-55°) was without effect on ear volume;
at 55° the animal struggled. Fifty-four hours after anesthetization
both tactile and auditory stimuli yielded vasoconstrictor reactions,
although in five out of eight trials there was a slight preliminary dila-
tation preceding the constriction. Pouring ether on the back resulted
at this time in the usual constrictor response. At the end of seventy-
six hours the vasomotor reactions were in every respect similar to those
previously obtained by us from unanesthetized rabbits.

DISCUSSION °

The transient vasoconstrictor response obtained by us under mild

tactile stimulation appears to be identical with that described by Griitz-
ner and Heidenhain (7) for a similar situation. The investigators just

a

VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION . 435

mentioned observed a marked heightening of the response under curare
poisoning, and were so impressed therewith that their further study
concerned itself with the reflex as exhibited by rabbits in curare paraly-
sis, to the exclusion of its consideration in unpoisoned animals. In
addition to their finding that the vasoconstriction was heightened in
curare paralysis they showed that very severe stimulation of the
cutaneous endings, instead of affording even more pronounced results,
failed to induce any response whatever. Before discussing the features
in which our observations agree with those of Griitzner and Heiden-
hain a brief consideration of these curare effects seems desirable. The
point about them which was chiefly stressed by the authors was that
they appeared only at a certain dosage of curare, a fact which probably
explains the failure of recent workers in the same field (Sollmann and
Pilcher (13), Martin and Stiles (3)) to report the phenomena. To our
mind this confinement of the effect to a certain stage of curare poison-
ing signifies an acute disturbance of nervous equilibrium due to the
drug, and negatives the application of the observations to the interpre-
tation of normal nervous functioning. We are not willing to accept,
for this reason, the view of Griitzner and Heidenhain that the marked
rise of blood pressure seen in their curare experiments is a mere accen-
tuation of the slighter rise obtained from similar stimulation in normal
animals. They consider the possibility that the action on the vasocon-
strictor may be a secondary result of primary psychic excitation, and
reject it on the ground of the finding that the curare rise of pressure per-
sists after cutting across the brain-stem. Dogiel (cited by Luciani,
12) found that normal animals showed vasoconstriction in response to
auditory stimulation, and that the response disappeared under curare.
We confirm the finding so far as normals are concerned. In the sec-
tion of this paper dealing with reflexes in decerebrate animals we have
reported our virtual failure to obtain vasoconstriction from either tac-
tile or auditory stimulation in uncurarized decerebrate rabbits. We
incline, therefore, to the view that both in our experiments and in
those of Griitzner and Heidenhain and of Dogiel the transient vasocon-
striction seen in normal animals was due to a secondary influence on the
vasomotor mechanism following psychic excitation. This interpreta-
tion places the reaction in precisely the same category with the familiar
plethysmographic findings in man, wherein mental alertness is accom-
panied by arm shrinkage. In our opinion this view is strengthened by
our observation that there is an apparent habituation to the stimulus.
This-is just what one would expect should follow repetition of a stimulus

436. +E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

which depends for its effect on psychic excitation, particularly when the
stimulation itself is of so mild a character as was used in this connec-
tion. The confinement of our positive results to the very early stages
of urethane and ether anesthesia agrees also with this conception.
By way of further confirmation we have the finding of Jacobson (14)
that localized skin stimulation in human beings is unproductive of
vasoconstriction through the operation of a direct reflex, although, as is
well known, definite vasoconstriction occurs in man whenever mental
alertness is elicited, however slight the exciting agent. The discussion
up to this point summarizes into the generalization that mild tactile
or auditory stimulation is productive of vasoconstriction only when it
operates to bring about psychic excitation, which latter is the immediate
cause of the vasomotor activity. ,

In addition to the transient vasoconstriction just discussed we ob-
tained a similar, but more prolonged, reaction when an area of skin was
chilled. The significant combination of influences here appears to be a
physiologically intense stimulus (lowering the back temperature 8
degrees in two minutes) and a widespread area over which the stimula-
tion is applied. That this combination is highly disturbing is shown
by the prompt objection made by unanesthetized animals to an ice
pack on the back. The cooling by pouring ether on the back was
endured, but only, in our opinion, on account of fright induced by the
odor of the ether. Deeply anesthetized and decerebrated animals gave
the reaction regularly, showing that it is not dependent on- psychic
excitation.

Our most prominent exhibitions of vasodilatation occurred as the
result of applying warmth to the skin, and of the two methods em-
ployed the more striking as well as the more constant was that which
more nearly approached the normal conditions of the environment,
namely, the gradual warming of a large area of the back by means of an
electric light. There is nothing in our experiments to show whether this
dilatation is reflex or is due to a direct effect of warming the blood,
but the experiments of Winkler (cited by Bayliss, 10) gave evidence that
a similar manifestation was definitely of reflex origin. A feature we
wish to emphasize in connection with this reaction-is that it occurs in
response to a very gentle type of stimulation. If we may judge by the
behavior of conscious animals, no disturbing element was contained in

the back warming until it had been in operation for a number of min- ©

utes. Our other method of eliciting vasodilatation, by dipping a leg
into water, gave much less uniform results. The sensory area in-

VASOMOTOR REFLEXES FROM RECEPTOR STIMULATION 437

’ volved was relatively limited, and the range of stimulation-strength
which would evoke the reflex was narrow; cold water and water of
moderate warmth gave negative results; water too hot aroused skeletal-
muscle activities which nullified the observations. In several cases
the response was reversed; this occurred usually, but not invariably,
at the highest temperatures that could be used satisfactorily. The
suggestion is that the attainment of water temperatures of definitely
painful character tends toward the vasomotor response usually associat-
ed with pain, namely, vasoconstriction, while the gentler stimulation of
warmth tends rather to induce vasodilatation. In none of our experi-
ments with thermal stimuli was there any indication that psychic exci-
tation is concerned in the reaction. The experiments of this group,.
while harmonizing on the whole with the accepted view that the specific
effect of warmth stimulation is vasodilatation, and of cold vasocon-
striction, seem to us also susceptible of explanation in terms of the
volume of nervous discharge set up. That is, that gentle or restricted
stimulation, evoking only a moderate discharge, induces typically
vasodilatation, while intense or widespread stimulation, which sets up
a considerable volume of nervous impulses, tends to arouse vasocon-
striction. It will be seen that this is an attempt to avoid relating
particular vasomotor reactions to the stimulation of specific sense organs,
and to assign them rather to certain volumes of nervous discharge. In
a subsequent communication additional evidence to this effect will be
adduced, and a more detailed consideration of the bearing of this con-
ception on the whole problem of reflex conduction presented.

SUMMARY

Experiments on the effects on ear volume of tactile, auditory and
thermal stimulation in normal, decerebrate and narcotized rabbits
lead to the conclusion that when vasoconstriction follows mild stimula-
tion it is to be interpreted as a secondary effect, following primary
psychic excitation. Gentle or limited stimulation not arousing mental
alertness typically induces vasodilatation. Reflex vasoconstriction,
not dependent on psychic excitation, requires intense or widespread
stimulation for its elicitation. é

438 E. G. MARTIN, A. C. FRANKLIN AND CLARENCE HIELD

BIBLIOGRAPHY

(1) Martin AND Lacgy: This Journal, 1914, xxxiii, 212.
(2) Martin AND Stiuzs: Ibid., 1914, xxxiv, 106.
(3) MARTIN AND Stites: Ibid., 1914, xxxiv, 220.
. (4) Stites anpD Martin: Ibid., 1915, xxxvii, 94.
(5) MarTIN AND MENDENHALL: Ibid., 1915, xxxviii, 98.
(6) MARTIN AND StTiLHs: Ibid., 1916, xl, 194.
(7) GritTzNER AND HeIpENHAIN: Arch. f. d. gesammt. Physiol., 1878, xvi, 47.
(8) TscHaLussow: Ibid., 1918, cli, 524.
(9) MENDENHALL: This Journal, 1914, xxxvi, 58.
(10) Bayxiss: Ergebn. d. Physiol., 1906, v, 330.
au) WINKLER: Sitzungsb. d. Kais. Akad. d. Wissensch. zu Wien, Math.-naturw.
Klasse, 1902, exi, ili.
(12) DoaiEL: Cited by Luciani, Human physiology, English transl., London,
1911, i, 360.
(13) SOLLMANN AND PILCHER: This Journal, 1910, xxvi, 2238.
(14) Jacospson: Virchow’s Arch., 1876, Ixvii.

STUDIES IN PLACENTAL PERMEABILITY

ee Fem DIFFERENTIAL RESISTANCE TO CERTAIN SOLUTIONS OFFERED
BY THE PLACENTA IN THE Cat

R. 8. CUNNINGHAM
From the Anatomical Laboratory of the Johns Hopkins University

Received for publication July 1, 1920

The great development of chemistry in its relation to vital phenom-
ena during the past twenty years has made imperative a more accurate
and comprehensive examination of the various problems concerned
with the permeability of the placenta. The many forces controlling

the passage of substances into cells and through cellular membranes
- have become much better understood, and since the placenta can be
considered as a cellular membrane separating the maternal circulation
from the fetal, these advanced conceptions are available for applica-
tion in the study of its permeability, so long recognized as of great
importance in the physiology of the fetus. The greatest proportion
of all workers in this field have approached the study of placental
transmission by administering substances which might be qualitatively
recognized in the fetus, and have merely stated whether or not they
passed. Some have indicated the duration of the experiment, while
others seemed to have considered the recognition of the substances as
sufficient. Much of this work will, however, prove of permanent
value because the large number of substances that have been used will
permit of various classifications, and thus indicate further lines of
investigation. 2

Nicloux (11) has made use of this opportunity, and has divided the
substances into two groups, as follows: a, Substances soluble in water
and diffusible, crystalloids; these traverse the placenta. 6, Sub-
stances insoluble in water and not diffusible, colloids; these do not
traverse the placenta. He has also (12) studied very accurately
the transmission of ethyl alcohol administered to pregnant dogs and
guinea pigs, and has found that in about one hour and thirty minutes
after the introduction an equilibrium has been reached between the

439

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOI.. 53, NO. 3

440 R. S. CUNNINGHAM

alcoholic content of the maternal and fetal bloods. Combining the
results of his own experiments with his analysis of the work of others,
he concludes that while many other factors should be considered simple
dialysis is the most important.

As one considers the question of approach to a further study of this
most extensive and complex subject, another classification seems advis-
able, in order to indicate the possible methods which may be utilized.
All substances coming into contact with the placental membrane on
the maternal side must fall into one of two great classes, those normally
present in the circulation and those which reach it accidentally. It is
_ obvious that the former will meet with an established mechanism which
is adapted to furnish constantly some given type of activity, varying
-with the substance under consideration. On the other hand the large
number of foreign substances which have been studied in one way or
another, and the still larger number which we can easily conceive of
obtaining adventitious entrance to the maternal circulation, musi of
necessity meet a very different condition because of the lack of any
specific adaptation on the part of the membrane. Any study of foreign
substances must be secondary to the search after the primordial reac-
tions of the placenta to normal constituents of the maternal blood, but
nevertheless these reactions may be often studied to advantage by
methods which utilize such adventitious materials.

From the standpoint of the physical laws which substances obey in
their relations to membranes they obviously fall into the following
groups: gases, liquids, crystalloids and colloids. Nicloux (13), in
- reporting elaborate observations on carbon monoxide, concludes that
this gas, as well as oxygen and carbon dioxide, follows exactly the laws
of gaseous diffusion in traversing the placenta. His work on alcohol
has been referred to; his studies on chloroform and ether are of equal
interest and importance. In the study of crystalloids there has been
no work comparable to Nicloux’s on gases and liquids. Among the
better contributions on the relation of colloids to the placenta are those
of ‘Wertheimer and Delezenne (19) on peptones, Goldman (8) on acid-
azo-dyes, Ascoli (1) on albumens, and Wertheimer and Meyer (20)
on methemaglobin,

: It is evident from this brief and incomplete outline of the work that
has-been done that the point of optimum attack for further investiga-
tions must be in the study of fluid and salt interchange between the
mother and fetus. The information obtained from a careful investiga-
tion of this mechanism, with any method, must be of great importance

STUDIES IN PLACENTAL PERMEABILITY 441

both directly and in the study of the more complex activities of the
placenta in relation to the metabolism of proteins, fats and carbohy-
drates. The most fruitful method in the study of the normal path-
ways of fluid drainage is that proposed by Weed (17), and used by him
to demonstrate the route followed by the cerebrospinal fluid in drain-
ing from the subarachnoidal spaces into’ the great sinuses. He in-
. jected a solution containing 0.5 per cent each of potassium ferrocyanide
and iron ammonium citrate into the subarachnoidal spaces, and pre-
cipitated the Prussian blue in the course of its drainage through the
arachnoidal villi. Later Shipley and Cunningham (16) demonstrated
the passage of fluid into the veins and capillaries of the omentum by
withdrawing that structure from the peritoneal cavity through a
midline incision, and immersing it in a similar solution. -~Many other
investigators have made use of the Prussian blue reaction, but have
not used the balanced solutions for the study of fluid pathways. It is
essential that these solutions be practically balanced to avoid the for-

-. mation of soluble Prussian blue during the process of fixation. Both

these salts pass readily through blood vessel walls, through arachnoidal
villi, through pectinate villi and through the kidney. In each of these
cases it is probable, if not certain, that they follow the normal direction
of fluid passage. ’

When projected, the study of these salts in their relation to placental
permeability was intended to analyze, if possible, any differential
adaptation of parts of that structure to the passage of water and salts.
In the case of these salts we are dealing with substances which
are foreign to the organism, but which offer the advantage of being
easily followed, not only to their ultimate location, but in the route
traversed. A study of the transmission of salts normally present
in the maternal organism would be of more value if they could only be
followed in their course through the placenta. If by this method any
‘information can be gained regarding salt and fluid interchange, this
may be applied later to considerations concerning the normal salt
constituents of the blood. Starting with the assumption that fluids
pass easily from mother to fetus it was hoped that the cellular media-
tion could be analyzed in the same way as in the cerebrospinal system,
the eye and in the peritoneum. It was a priori thought that both
sodium ferrocyanide and iron ammonium citrate would easily traverse
the placenta, because comparable salts had been shown to do so with
ease by previous workers. Bar (2) described experiments in which
potassium ferrocyanide was injected into the uterine veins of rabbits,

442 R. S. CUNNINGHAM

and was found after thirty minutes in the amniotic fluid. If then the
potassium salt passes with such ease, it seemed probable that the
resistance would not be greater to the less toxic sodium ferrocyanide.
No such comparison was possible in the case of the iron ammonium
citrate. However, certain organic salts have been found to traverse
the placenta; Porak (14) showed that potassium acetate passed in
experiments on rabbits, Zweifel (21) used sodium salicylate, admin-
istered to women in labor, Fehling (7) used sodium salicylate on rab-
bits, Launois and Briau (10) used this same substance on guinea pigs
and rabbits, and Savory (15) used strychnine acetate on cats, dogs and
rabbits. It is only by observing the effects of many different salts
that we may determine any of the laws governing the actual interrela-
tion of salts and tissues. In the placenta it is well known that the
acid-azo-dyes and colloidal metals, as well as many other colloidal
sols will not pass from the maternal to the fetal circulations. From
this it is clear that there is a definite resistance offered to a group of
substances whose molecules are large but which have, as far as we know
at present, no other characteristic in common. These substances will
all pass through capillary walls, through the lining of the peritoneum
and through the kidney; and they will diffuse into certain cells in the
body while other cells remain impermeable to them.. On the other
hand, capillary endothelium appears to be relatively impermeable to
proteins, sometimes completely so, while easily permeable to the sub-
stances named above. Relative permeability is then a certainty both
in tissue cells and in those cells functioning as membranes separating
body fluids.

The experimental animals used in the past for these investigations on
placental permeability have been confined almost entirely to rats, ©
guinea pigs and rabbits: A very few workers have used dogs, cats
and sheep; while many have performed experiments on women in
labor, but few comparative studies have been made using the same sub-
stance in different species of animals. Bremer (5) calls attention to
this in discussing anatomical characteristics of various placentae, and
suggests that many of the conflicting statements in the literature can
be explained by the variations in the animals used, and that more prog-
ress could be made by using a greater variety of species in the study of
any given substance. He has studied the placentae in a large group of
animals, in relation to the problem of fetal excretion, and therefore in
relation to other organs which may assume all, or at least part, of the
excretory function: the pronephros, the Wolffian body and the kidney.
He says: .

STUDIES IN PLACENTAL PERMEABILITY 443

Mammalian embryos may be divided into two classes; those which retain func-

tional Wolffian bodies until the kidneys are sufficiently developed to excrete
urine, as is the case in birds and reptiles, and those in which the Wolffian bodies
degenerate before the kidneys reach functional ability. The first class includes
the pig, sheep and cat; the second the rabbit, guinea pig, man and rat.
In those animals without the possibility of a continuous urinary excretion within
the embryo, i.e., with an early degeneration of the Wolffian body, the placenta is
provided with an apparatus similar to that found in the glomeruli of the Wolffian
body or the kidney, thin plates of epithelium overlying the fetal capillaries.
These appear in the placenta at about the time when the Wolffian body commences
to degenerate, or in the case of the rat, which never develops mesonephric glo-
meruli, at about the time of the normal development of the glomeruli in other
embryos. These plates continue and increase in number till term. They are
apparently of greater extent in animals whose embryos are provided with large
Wolffian bodies. In the placentae of those animals with a continuous embryonic
urinary excretion, similar plates are not found, whether the placentae be of the
opposed or conjoined type. From these facts it appears that embryonic and
fetal urinary excretion takes place wholly through the placenta in the rat, at
first through the Wolffian body and later through the placenta in the rabbit,
guinea pig and man, but never through the placenta in the pig, sheep or cat.
A knowledge of these differences should lead to more intelligent experiments
on the permeability of the placenta.

Bremer does not discuss the question of differential permeability, but
his observations are extremely interesting and important from a phys-
iological point of view, and particularly emphasize the absolute neces-
sity of studying all problems of embryological physiology from a com-
parative viewpoint, as variation in species may be sufficient to produce
very conflicting results. Bremer’s theory, founded entirely upon
anatomical observations, has received but very little real physiological
examination; it is, however, supported by the previous results obtained
by Krunkenberg (9) who found that substances which did not pass
through the placenta in dogs and cats, but were retained by the fetus,
found their way easily into the maternal circulation in the case of
guinea pigs and rabbits. Savory (15), on the other hand, described
experiments which are contradictory. He found that strychnine
acetate when introduced into canine fetuses caused convulsions in the
mother in nine minutes and death in twenty-eight. His experi-
ments on cats and rabbits gave the same results. Bremer considers:
“ , . . that these modified plates have apparently become inactive
membranes, through which a purely physical osmosis may take place,
but which themselves may be supposed to be physiologically inert.”
While he applies his observations to excretion only, there seems to be
no reason to preclude that the permeability is equal in both directions

444 R. S. CUNNINGHAM

for any membrane which is physiologically inert. Therefore experi-
ments dealing with maternal to fetal transmission in animals having
plates must be considered equivalent to a similar experiment in the
opposite direction.

Goldman (8) has shown that trypan blue will not pass the placental
barrier in the rat, but is easily excreted by the maternal kidneys. In
some unpublished experiments I have found that trypan blue intro-
duced into the circulation of rabbit fetuses in a late stage of gestation,
was excreted by the fetal kidneys, but did not pass through the pla-
centa into the maternal circulation. These experiments have also
been performed on the fetuses of cats with the same results. It is
obvious at once that the plates of the renal glomerulus must be diff-
erent in their permeability from those in the placenta, when measured
by this one class of colloidal sols. This does not in any way invalidate
Bremer’s conclusions that these placental plates indicate the route of
fetal excretion and are physiologically inert, but prevents accepting
the plates found in the placenta as an exact equivalent of those in the
kidney. baa

Admitting Bremer’s hypothesis to be substantiated, only one phase
of the general problem will be settled because even if certain species of
animals have specialized membranes which permit the passage of
substances excreted by the fetus, still there is the essential necessity of
the fetus receiving salts, water and foodstuffs from the mother, and
these must pass the placenta irrespective of any variation in species.
The problems still unanswered regarding the permeability of the pla-
centa are innumerable. They are of vital interest both as regards their
local and direct significance and in their bearing on phenomena else-
where in the organism. They range from the general problem of cell
permeability and the resistance of membranes, to special ability of the
placenta as an organ to select, change and even synthesize substances
needed in the fetal metabolism. The placenta is a single structure
which probably exercises the combined functions of many types of
cells, and therefore any laws which may be found governing its activi-
ties will undoubtedly be applicable in part to other cells. But the
placenta differs so essentially in function from any other single tissue
that even those laws having the most widespread application. will
probably have special significance here. As far as is possible all the —
laws developed for single cells and simple membranes should be applied
to the special case of this complex and more primitive structure.

STUDIES IN PLACENTAL PERMEABILITY Y45

The present communication is the first: of a series projected,::and
partly under way, having as their object the analysis of some: of the
more fundamental problems relating to placental permeability...» .

The plan has been to first analyze, by whatever methods are avail-
able, the normal fluid and salt interchange and to determine if possible
avhat forces are most active in these processes. Then to determine: as
far as possible the nature of the placenta as'a membrane and by what
means certain groups of substances are excluded by it, while others.are
allowed to pass; and to ascertain whether this reaction is comparable
to others in the body or peculiar to the placenta alone. Finally-it is
desired to determine what réle the placenta plays in the fetal meta-
bolism, and to establish the nature of its relation to each type of -sub-
stance that must be recognized as essential for the fetal nutritions.s:::

MATERIALS AND METHODS Tike 988)

. In this attempt to analyze the fluid interchange cats were sused
exclusively, and those in a late period of gestation were selected. :'The
entire series consisted of animals that had passed the fortieth day :of
pregnancy. The youngest fetus in the series measured 78: mm., and
the oldest 121 mm., crown rump diameter. - The technique has been
uniform throughout except in certain experiments designed expressly
to test some specific point arising in the course of the work. | -All.ani-
mals received balanced solutions of sodium ferrocyanide and iron am-
monium citrate, the usual strength being 14 per cent.of each salt. ‘The
percentage strength was varied in some cases; these will be mentioned
in the appropriate place. In all operations the solution was admin-
istered as an injection by means of a burette attached to a glass.can-
nula introduced into the vein of the forearm. Intratracheal. ether
anesthesia was used in the majority of these experiments, and ,.was
found entirely satisfactory. It is well known that the cat maintains
almost normal respiratory and circulatory functions under ether
anesthesia. After establishing results which were entirely constant
for ether, controls were decerebrated following Weed’s (17) technique,
and certain crucial experiments repeated: The results obtained: were
in all cases exactly the same as found in similar experiments under
anesthesia. By this the possibility of any effect by the anesthetic.on
the placenta was eliminated, and this was essential since we. knew
from Nicloux’s (11) work that ether passes the placenta and it might
In consequence render the placental membrane either more or. less

446 R. S. CUNNINGHAM

permeable to the substances used in the experiment. The salts were
weighed out and dissolved immediately before the experiment. They
were not mixed until the cannula had been inserted, when they were
poured together, and then directly into a burette and the injection
begun. Both sodium ferrocyanide and iron ammonium citrate are
eliminated very rapidly by the kidneys, so that some method had to be
devised in order to keep the concentration of the salts constant in the
blood stream. The method finally decided upon was the preliminary
interruption of the renal excretion by ligation of the renal vessels.
After this the desired amount of the experimental solution was run in
slowly, and the animals kept undisturbed until sacrificed. In order
to avoid the possibility of this procedure causing changes inblood
pressure, experiments were performed to test the effect of administer-
ing these salts after ligation of renal vessels. Amounts more than double
those usually given caused only a transient rise of pressure, so that the
hydrostatic element may be ruled out. This method proved so satis-
factory that it was used entirely, except for a few animals in which a

continuous injection was maintained throughout the entire experiment —

of just enough solution of the salts to keep them present in the blood
stream. Quantitative determinations were not made, but precipitation

tests of Prussian blue in the maternal blood plasma were carried out in |

order to indicate the presence of both salts in approximately the same
amounts, as in experiments done with the preliminary renal ligation.
These experiments were considered necessary to exclude the possibility
of the lack of excretion from the kidneys having an effect upon the
placenta, but the results with the kidneys functioning and when ligated
were the same. Cats were exposed to varying doses for periods of
time ranging from ten minutes to ten hours, and were then killed.
‘The uterus was opened so that the amniotic sac bulged out, the fluid
carefully withdrawn with syringe and needle to avoid contamination,
and then examined chemically. The fetuses were removed and their
abdominal cavities opened; whenever the bladder was distended it was
treated in precisely the same manner as the amniotic sac. Small blocks
of fetal kidney and placenta were fixed directly in Bouin’s fluid, the
acetic acid in this mixture being sufficient to precipitate the Prussian
blue. In the analysis of the fluids under examination, solutions of ferric
chloride and ferric sulphate were used to determine the presence of

sodium ferrocyanide, while a ferrocyanide was used to test for the ferric -

iron. It was found that there is sufficient iron present normally in fetal
urine and amniotic fluid to yield a Prussian blue reaction with sodium

*

STUDIES IN PLACENTAL PERMEABILITY 447

ferrocyanide and hydrochloric acid when allowed to stand; the test for
ferric iron was therefore read immediately. The exact details of all
tests will be given when the reports of the histological findings are
made, and need not be considered further here. The results of the
introduction of these salts into the blood stream of the living fetus are
from a series of experiments, still quite incomplete, concerning the
general methods of fetal excretion, and which will be reported later.
Such materials as bear upon the present problem will be referred to
here. In regard to the question of the validity of the tests given above
the following experiment will indicate both the delicacy of the test
and the certainty of finding our reagents in the fetal urine or amniotic
- fluid if they had reached the fetal circulation.

Cat P. P. 28. Intratracheal anesthesia. Midline abdominal incision with
exposure of uterus.1 One cubic centimeter of a solution containing 1 per cent
each of sodium ferrocyanide and iron ammonium citrate was introduced into the
umbilical vein of one of the fetuses, which was removed after two and a half
minutes, and the bladder exposed. A drop of acid added to the fetal urine gave
a brilliant blue color. Another fetus was exposed and the same procedure fol-
lowed except that the time interval in this case was twenty minutes. The blad-
der was found to be contracted, test of the amniotic fluid yielded Prussian blue.
The fetuses measured 80 mm.

It is quite evident that the fetal kidney will very quickly eliminate |
both salts, and that if the bladder has contracted after this elimination
has begun the amniotic fluid will show their presence. We found, in
many of our experiments on the fetus, that the bladders were empty.
From this it seems probable that the increased flow of urine had stimu-
lated them to contract. It is evident that the tests used were suffi-
ciently delicate to indicate any exchange of these salts between mother
and fetus even when relatively small doses were given to the mother
intravenously.

EXPERIMENTAL RESULTS

The results of many experiments indicated that the placenta of the
cat can be functionally divided into three parts in regard to its reaction
to the salts here used. Duval (6), in studying the histology of the
placenta, has described three layers in the labyrinth of the cat during
later states of gestation: the maternal endothelium which remains
intact and is included within the invading trophoblast of the ovum;

1 The technique of these operations will be reported with the other details on
embryonic excretion.

448 R. S. CUNNINGHAM

the ectodermal layer, which is syncitial in character; and the embry-
onic endothelium which lines the embryonic vessels even where they
penetrate deeply into the syncytial ectodermal layer. The maternal
endothelium is everywhere continuous, the cells being very promi-
nent in contrast to the thin, delicate cells of the fetal capillaries. In
every experiment of the entire series the reaction of the maternal
endothelium was precisely the same. No variation in this reaction
could be discovered in any part of the placenta, despite the fact that
certain areas showed more blue than others on examination with the
low power of. the microscope. This was probably attributable to a
variation in the blood supply due to mechanical causes. Everywhere
on histological examination the characteristic blue granules could be
seen both in the bodies of the endothelial cells and lying between them
and the ectodermal layer. In exactly the same manner the fetal
endothelium is easily permeable to both salts. -This passage takes
place very rapidly because in experiments of ten minutes duration,
the blue granules are found in, and beyond, the endothelial cells. It
is impossible to deny that the salts may pass between the endothelial -
cells as well as through them, although there are no granules pre-
cipitated in a manner which would indicate this, as they seem to be
equally distributed throughout the entire extent of the cytoplasm.
That they both pass through the maternal endothelium rapidly is
evident and that they pass only through, and never between these
cells is probable, but can not be stated as absolutely proved. This
difficulty is obviously met with in any membrane, and has been com-
mented upon by Weed (17) in regard to the arachnoidal villi; this will
be referred to later. It is probable then that the maternal endothelium
present in the placenta has the same degree of permeability in regard
to these salts as endothelium elsewhere in the body. At least it can be
asserted that they pass here in the same manner, that is, with the same
histological distribution as was shown in the veins and capillaries of
the omentum by Shipley and Cunningham (16). The maternal endo-
thelium is easily permeable to both of these salts, the fetal endothelium
is equally so, but the fetal ectoderm reacis in an entirely different
manner. The behavior of the ectodermal layer permits the division of
the experiments into two groups, relative both in terms of permeability
and duration of time. In the first group are included experiments of
duration varying from ten minutes to four hours and a half. In not
one of these has any trace of either sodium ferrocyanide or iron am-
monium citrate been demonstrated in amniotic fluid, fetal urine, or

STUDIES IN PLACENTAL PERMEABILITY 449

tissue extract. And in every case the bladders of the fetuses were
found on removal to be full of urine. In the second are included those
from five to ten hours in duration, the latter being the maximum of
the series. In all the experiments in this group traces of sodium
ferrocyanide were found in the fetal urine or amniotic fluid, but in none
of them could even the slightest trace of the iron ammonium citrate be
demonstrated. The amount of ferrocyanide which had passed the
placenta was in no case very large. In experiments of from five to
six hours only a trace was found, this was increased somewhat in those
of longer duration, the maximum being approximately: that amount
which would be excreted by a fetus in twenty minutes if 3 cc. of a 1
per cent solution had been injected intravenously. In iWakieeH con-
trast to the fetuses of the first group, the bladders were found in most
cases to be contracted and entirely emptied of their urine. And in
every one of these the ferrocyanide could be demonstrated in the
amniotic fluid. ‘This interesting reaction of the fetal bladder is s illus-
trated i in the following experiments:

Cat P. P. 25. 9:00 a.m. Intratracheal ether.

Each kidney delivered retroperitoneally, and the vessels ligated.

Cannula inserted into vein of left forearm of mother, connected to burette
containing 50 cc. of solution of 13 per cent each of sodium ferrocyanide and iron
ammonium citrate.

9:20 a.m. Intravenous injection begun.
9:30a.m. 5 ce. in.
9:40 a.m. 10 ce. in.
9:50 a.m. 15 ce. in.

~ 10:00 a.m. 20 cc. in.

10310 a.m. 25 cc. in. Injection stopped.

' Animal in excellent condition. No change after injection began except in-
creased salivation. The animal remained in excellent condition until sacri-
ficed at3p.m. Total duration of experiment, five hours and forty minutes. The
uterus was immediately removed and found to contain three fetuses in the left
horn and two in the right. The uterine wall was incised over the caudal end of
each fetus in such a manner that the amniotic sac bulged out freely; this was
punctured with a needle and the fluid withdrawn. Each fetus was removed, all
were noted to be alive. The abdominal walls were incised and the urine removed
when present.

Right horn of uterus: Both fetuses had empty bladders, the amniotic fluids
contained a trace of ferrocyanide, but no iron ammonium citrate. They meas-
ured 89 mm. and 92 mm.

Left horn of uterus: Fetus 1. Bladder full, but not distended. Urine nega-
tive for ferrocyanide and iron ammonium citrate. Measured 91 mm.

Fetus 2. Bladder full, and distended. Traces of ferrocyanide in urine, no
iron ammonium citrate. Measured 90 mm.

450 R. 8. CUNNINGHAM

Fetus 3. Bladder empty, trace of ferrocyanide in amniotic fluid; no iron ammo-
nium citrate. Measured 94 mm.

Cat P. P. 28. 9:30 a.m. Intratracheal ether.

Each kidney delivered retroperitoneally and the vessels ligated.

Cannula inserted into vein of left forearm of mother, and connected to burette
containing 50 ce. of solution of 2 per cent each sodium ferrocyanide and iron
ammonium citrate.

9:50 a.m. Intravenous yy jection begun.

10:00 a.m. 5 ce. in.

10:10 a.m. 10 ce. in.

10:20 a.m. 15 ce. in.

10:30 a.m. 20 cc. in. .

10:40 a.m, 25 cc. in. Injection stopped.

_ The only immediate effect of injection was increased salivation. Heart rate
remained about same throughout.

6:20 p.m. 10 cc. of blood withdrawn from maternal ian centrifuged imme-
diately, a few drops of acid added, blue color developed.

6:20 p.m. Ether to death.

Total duration of experiment, eight hours and thirty-five minutes. The
uterus was removed and found to contain four fetuses, two in each horn. The
uterine wall of each fetus was incised carefully and the amniotic fluid removed
and tested. The fetuses removed and their bladders examined. In three, the
bladders were found to be entirely empty, and the amniotic fluid was positive for
ferrocyanide and negative for iron ammonium citrate. In the fourth fetus the
bladder contained a single drop of urine which gave a positive test for ferro-
cyanide with ferric chloride and the amniotic fluid reacted as in the other three
fetuses. Tests for iron ammonium citrate were negative. The fetuses were all
alive and measured 80, 82, 84 and 86 mm. respectively.

On histological examination of the placentae of animals subjected
to experiments of this type, the reaction of the ectoderm is most re-
markable. In the shortest experiments, of duration less than an hour,
the Prussian blue is found throughout the maternal endothelium and
in a fine blue line between this layer and the syncytial ectoderm.
There are no granules to be seen anywhere within the ectoderm, but
they are closely adherent to the outer border both of the syncytium
and the inter-capillary giant cells. Indeed these present a most
remarkable picture of large round cells everywhere surrounded by a
fine blue border, but nowhere can any granules be seen within their
cytoplasm. In experiments of longer duration, about two hours, blue
granules are to be seen within the ectodermal layer; these have not
penetrated far into the cytoplasm but tend to form lines relatively |
close to the maternal poles of the ectodermal nuclei. This arrange-
ment is very irregular, frequently the granules collect in small masses
instead of lines and filaments. In experiments of six, eight and ten

STUDIES IN PLACENTAL PERMEABILITY 451

hours duration there is very little change in the histological findings.
The blue granules are found deeper in the cytoplasm of the ectoderm,
in some places they are scattered almost throughout the entire struc-
ture. None can be seen in the fetal endothelium, or between the
endothelial cells and ectoderm. Also the inter-capillary giant cells
have absorbed a small amount of the two salts so that a few fine blue
granules are seen in the peripheral third of the cytoplasm, while the
rest of the cell is entirely clear.

The principal advantage of the method used in these experiments is
that, by the precipitation of Prussian blue in situ, it is possible to
interrelate the results obtained by examination of fetal excretion with
the histological location of the point at which these results must have
been determined. These placentae are sufficienty interesting to merit
a more detailed histological description than would be advisable here;
this will be given later when comparative results on other species are
available.

DISCUSSION

The principal facts demonstrated by these experiments are the
differences in the permeability of the endothelial and ectodermal layers
of the placenta, and the differences in the resistance offered to the
two experimental salts by the fetal ectoderm. It is evident that the
placenta may be considered as two protoplasmic membranes which
are directly applied to each other in such a way that they appear ~
anatomically as a single membrane separating the two bloods, but have
in reality entirely different physiological properties. .

In considering a given living membrane we have to deal with three
phases of activity, the entrance of a substance into the membrane, its
_ passage through the membrane, and its exit from the membrane.
The factors involved in each of these may be entirely different, and
those relating to one membrane may differ entirely from those relative
to another. The simplest membrane in the organism is such a one as
that described by Weed (17) in the arachnoidal villus where he found
the exchange through the membrane to be effected by a simple hydro-
static filtration; osmotic pressure having little or no participation.
That the passage of sodium ferrocyanide through the entire placenta
is not a simple filtration dependent upon hydrostatic pressure alone, is
evident from several experiments carried out expressly to determine
this point. After animals had been exposed in the usual manner and
to the usual dose of this salt, the blood pressure was raised to a max-

452 R. S. CUNNINGHAM

imal degree by the continued introduction of physiological saline under
pressure of about 500 mm. of water. Despite this procedure no
variation in the passage of this salt as previously determined in animals —
with normal blood pressure was noted. Simple filtration by hydro-
static pressure can therefore be considered as a negligible factor.

It is evident that all materials necessary for the nutrition of cells
and all products of cell metabolism that either come from the blood
stream or are eliminated by it, must pass through the capillary wall. |
Therefore the endothelial membrane must be permeable to water,
salts and even to some colloids, since some of the substances included
above are in that state. This remarkable degree of permeability shown
_by capillary endothelium has been explained in various ways. It has
been suggested that the endothelial cells are capable of separating
from each other and allowing large molecules, as well as small, to pass
through by simple filtration. Others have thought that the inter-
cellular cement substance is physiologically inert and contains large
pores through which diffusion of colloids may take place. The other
possibility is, that the endothelial cells are constructed in such away
that their cytoplasm is permeable to large molecules. The nature of
the mechanism by which permeability is regulated still remains un-—
settled; Bayliss (4) after reviewing many observations at variarice with.
each other concludes that:

Different views are held as to that property of a membrane which makes it
permeable to some solutes and impermeable to others. Reasons are given in
the text for accepting, with some modifications, the original sieve theory of
Traube, according to which the passage of a solute through a particular mem-
brane, depends on the size of the pores in the membrane in relation to the molecu-
lar, or particulate, dimensions of the solute. The hydration of solutes must be
taken into account. In a few cases, the question of solubility in the substance
of the membrane appears to play a part.

That capillary endothelium is permeable to both iron ammonium.
citrate and sodium ferrocyanide has been shown in the case of the
omentum by Shipley and Cunningham (16). The forces involved in.
the passage of these salts through the maternal endothelium of the
placenta are probably no different from those operative in the case of
any other capillary wall. That osmosis and diffusion are the most
important is assured, but that other factors are involved is shown by
the observations that substances can be absorbed from the peritoneal -
cavity against osmotic pressure and concentration gradient, and like-
wise substances can be secreted by the kidney under similar circum-

STUDIES IN PLACENTAL PERMEABILITY 453

stance. While these factors are not yet entirely understood they are
explained in terms of negative osmose, which in turn is explainedin
terms of electro-endosmose, or the pull of a charged particle through
a membrane pore by an opposite charge on the membrane (Bartell
and Hocker (3)). The experiments in this series do not assist in analys-
ing the factors pertaining to the permeability of the maternal endo-
thelium, and therefore of endothelium in general, except that they
show conclusively the passage of these salts through the cytoplasm of
the cells. , |

We have seen that both sodium ferrocyanide and iron ammonium
citrate penetrate the ectodermal layer in about two hours, while the
majority of cells in the mammalian organism appear to be semi-per-
meable as regards these salts, even when exposed to them for much
greater periods of iime. So that the limiting membrane of the fetal
ectoderm must differ in its normal permeability from the limiting mem-
brane of cells in general. The differences in structure or composition
between membranes permeable to certain crystalloids and others semi-
permeable to them, are as yet unknown. Whether other factors than
osmosis and diffusion are concerned in the penetration of the ecto-
dermal membrane can not now be stated. When we consider the
- passage of these substances through the cytoplasm and the exit of one
while the other is. retained, other factors must be considered. After
about five hours the sodium ferrocyanide passes through the cell
membrane adjacent to the fetal capillaries and can be detected very
quickly in the fetal urine. At this time tests fail to reveal any citrate
as having passed through, nor do experiments of several hours longer
duration yield different results. It is evident, therefore, if thesé tests
are sufficiently accurate, that no citrate has been able to pass out of
the fetal ectoderm despite the fact that it has unquestionably entered
it. The rate at which sodium ferrocyanide passes through the placenta
is extremely slow in comparison with its passage through other living
membranes which have been studied. While this doesnot prove, it
certainly suggests thai other factors than osmotic pressure and diffu-
sion are involved. It is possible however, to conceive that the ecto-
derm is so difficultly permeable to sodium ferrocyanide that we are
actually measuring the normal time element of its diffusion, in other
words, the ratio of osmotic pressure to the resistance of membrane.
The determination of the osmotic pressure of the maternal and fetal
bloods would be of great assistance in indicating how much salt ex-
change depends on this factor. Such determinations have not been

454 R. S. CUNNINGHAM

done. It is evident that if an osmotic balance is maintained this
exchange would be easier than if the presence of metabolic products:
in the fetus should have raised the pressure on that side; but it is
possible to conceive that even if the osmotic pressure in the fetal blood
is equal to, or lower than in the maternal blood, the retardation of the
passage of sodium ferrocyanide might be caused by an unstable com-
bination with intracellular compounds; on the other hand, if the fetal
pressure be proved to be higher, some activity comparable to that of
secretion must be postulated.

We can conclude much more surely that other factors than osmosis
and diffusion are operative in regard to the peculiar action of the ecto- -
dermal membrane toward the iron ammonium citrate, because both
the ferrocyanide and citrate are known to diffuse through many dif-
ferent membranes at approximately equal rates. Of course it must be
admitted that we have not excluded the possibility of the citrate pass-
ing through the ectodermal layer if exposed for a longer period of time.
In this case we would be forced to conclude either that some intra-
cellular activity had delayed the citrate longer than the ferrocyanide,
or that the fetal surface of the ectoderm was much more easily per-
meable to ferrocyanide than to the citrate, and consequently different,
not only from many. other living membranes, but also from the mater-
nal side of the ectoderm. On the other hand, if longer experiments
substantiate those of six, eight and ten hours duration, we are forced
to the conclusion of some even more radical change taking place within
the fetal ectodermal layer. Two possibilities may be. mentioned,
although it is evident that both are entirely without experimental
evidence. First, it is conceivable that some substance present in the
cell prevents the further passage of the citrate by a specific adsorption.
Second, as many organic acids and salts of organic acids are changed
and broken down in the metabolism of the animal body, it is therefore
quite possible that the ectodermal syncytium is capable of breaking
down iron ammonium citrate into substances no longer detectable by
our tests, which were after all only capable of demonstrating the pres-
ence of ferric iron. It should certainly prove feasible to devise meth-
ods for determining whether this salt, and perhaps many other salts of
organic acids, can be decomposed in the fetal ectoderm. We have con-
sidered so far, in endeavoring to explain the results of our experiments,
the effects that the tissues involved may have had upon the salts intro-
duced; but it is necessary to consider also what effect the salts may-
have had upon the tissues. While the death of the cell after exposure

STUDIES IN PLACENTAL PERMEABILITY 455

to these salts can be easily determined by the brilliant blue staining of
the nucleus, minor injuries do not produce any change which can be
recognized in histological preparations. In general it is known that
permeability of cells is increased by injury; if then the ectoderm has
been changed by these salts we must conclude that it is normally
semi-permeable to them. But it is extremely difficult to understand
how one of these salts can be retained and the other pass out into the
fetal blood, if they can not penetrate the ectoderm until they have
injured it in some degree. Admitting that it is injured, the results
which have been obtained can only be explained in one of twoways:
either the injury is insufficient to destroy the activity which prevents
the passage of the citrate, or else this function is a chemical reaction
developed as the result of the injury. These explanations are both
rather improbable, so that it seems more likely that the degree of injury
is slight. Whether or not longer exposure to these salts, and particu-
larly to the citrate, would depress the cell activities enough to allow
the citrate to pass through is entirely problematical and need only be
stated here as a possibility. Finally, it is worth while to consider
what bearing these experiments have upon the theory advanced by
Bremer and referred to before. According to his theory excretory
products of fetal metabolism in the cat do not pass through the pla-
centa, but are excreted by the fetal pronephros, Wolffian body or kid-
ney, as the case may be. Since the fetal as well as the adult kidney
excrete both of these salts very rapidly, i.e., within two or three min-
utes, and since it requires five hours for one of them to pass the pla-
centa, it seems strong evidence that his interpretation of the absence
of placental excretion in the cat is correct. Although still quite incom-
plete, the work on the excretion of the embryo, referred to above,
also substantiates Bremer’s view as far as the cat is concerned.

BIBLIOGRAPHY

(1) Ascout: Zeitschr. f. physiol. Chem., 1902, xxxvi. 498.

(2) Bar: Thesis, Paris, 1881.

(3) Barrett anp Hocker: Journ. Amer. Chem. Soc., 1916, xxxviii, 1029.
(4) Bayuiss: Principles of general physiology, London, 1918.
(5) Bremer: Amer. Journ. Anat., 1916, xix, 179.

(6) Duvau: Journ. d. l’Anat. et d. 1. Physiol., 1894, xxx, 231.
(7) Feuurna: Arch. f. Gynaek., 1876, ix, 523.

(8) Gotpman: Die Vitale Farbung, Leipzig, 1912.

(9) Kruxensere: Arch. f. Gynaek., 1885, xxvi.
(10) Lavnors AND Briav: Lyon. Med., 1898, Ixxxvii, 323.
(11) Nictovux: Obstetrique, 1909, no. 11, 840.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 3

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(16) SuipLey anp CUNNINGHAM: This Jc
(17) Weep: Journ. Med. Research, 19 ins,
(18) WEEp: Journ. Physiol., 1914, | -
(19) WERTHEIMER AND DELEZENNE:
(20) WerrHemer anp Muyer: “Arch. d.y phys.
en) were: Areh. i Gynaek., 187, xii, 235.

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THE PRODUCTION OF INTRACELLULAR ACIDITY BY
NEUTRAL AND ALKALINE SOLUTIONS CONTAINING
CARBON DIOXIDE

M. H. JACOBS

From the Laboratory of Zodlogy, University of Pennsylvania

Received for publication July 1, 1920

It has recently been pointed out by the author (1) that the appar-

- ently contradictory views as to the mode of action of CO. on the mam-

malian respiratory center expressed on the one hand by Winterstein
(2), Hasselbalch and Lundsgaard (3), (4) and others, and on the other
by Laqueur and Verzdr (5), Hooker, Wilson and Connet (6) and Scott
(7) are not necessarily in conflict. Carbonic acid may conceivably,
like any other acid, act primarily through its hydrogen ions, as the first
group of workers have held, and yet differ so greatly from other acids
in certain peculiarities connected with its powers of penetrating living
cells as to have practically as specific an action as that postulated by the :

second group.

One of the most interesting of these peculiarities is the relatively
small importance of the hydrogen ion concentration of a solution con-
taining carbon dioxide in determining the degree of intracellular acidity
produced in a cell exposed to it. The author has already called .atten-
tion to the fact that a neutral or even a slightly alkaline CO.-bicar-
bonate mixture is practically as toxic to tadpoles as a pure CO» golution
of the same concentration, and that such a solution-has a sour taste.
He has interpreted these results as being due to the fact that the
H.CO;, whose dissociation is held in check by the bicarbonate, can
freely enter cells (perhaps as CO), while the bicarbonate cannot, the
carbonic acid dissociating when within the cells to the extent per-
mitted by the new conditions of equilibrium prevailing there, which
may easily result in a hydrogen ion concentration higher than that of
the surrounding medium.

While the observations already recorded have probably been cor-
rectly interpreted, it has seemed desirable to supplement them with a
case where the rise in intracellular acidity under the conditions in ques-

457

458 M. H. JACOBS

tion is actually visible to the eye. Such a case might be furnished by a
cell containing a sufficiently sensitive natural indicator whose color
changes would give visible evidence of any increase in the hydrogen
ion concentration within the cell. After a rather lengthy search, ma- |
terial of this character has been found in the colored flowers of Symphy-
tum peregrinum, a cultivated plant belonging to the family Boraginaceae.
These flowers, like those of many other members of the family, are
pink in the bud, later becoming blue; the former color is associated with
a higher, the latter with a lower hydrogen ion concentration. The exact
pH of the turning point has not been determined, but is wie the
range of carbonic acid solutions.

The use of colored flowers to study cell penetration by acids, though
Haas (8) obtained good results with this method, has not been very
much favored in the past because of the fact that such cells are generally
cuticularized and are not readily wetted by aqueous solutions. In the
present case, however, it was found that if the end of the tubular corolla
were snipped off with a pair of scissors and the whole flower were
then slipped over the tapering end of a glass rod which could be dropped
into a test-tube containing the solution to be studied, the change in
color appeared quickly and regularly and could easily be followed,
especially along the “‘ribs’” correspondng to the attachment of the
stamens, and at and above the depressions which are found at the
points of attachment of the ‘‘corolla scales.”” To observe the change .
in color, a hand lens was used, though even with the naked eye the
results were easily visible. For example, the difference between a
flower exposed to a saturated solution of CO, and one kept in distilled
water was usually apparent in five minutes at a distance of five or six
feet. The change in color with weak solutions of acids always began
on the ‘‘ribs’’ and above the depressions mentioned, and gradually
spread from them to other parts of the flower.

As a considerable number of similar experiments with this material
all gave essentially the same results, it will be sufficient to describe a
typical one. Four flowers from the same plant, as nearly alike in
color as possible, were selected. The first was exposed to a satu-
rated solution of COs, in distilled water (pH approximately 3.8); the
second to pure distilled water of pH between 5.0 and 6.0 (the slight
acidity being due to CO: absorbed from the air); the third to an M/2
solution of NaHCO; saturated with CO. at atmospheric pressure and
having a pH of ca. 7.4, and the fourth to an M/2 solution of NaHCO;

PERMEABILITY OF CELL WALL TO CARBON DIOXIDE 459
considerably more alkaline than pH 8.0. The course of the experi-
ment may be indicated most clearly in tabular form as shown in
table 1.

It will be observed that the increase in acidity of the cells was not

‘proportional to the pH of the external medium, since those exposed to

Color changes in flowers

TABLE 1

of Symphytum exposed to various solutions _ eee
tobe 5

TIME | jertttep ele £002 | OW BTILLED | (3) m/2 NaHCOs + COs. | (4) m/2 NaHCO:
11:20*| Blue Blue Blue Blue
11:21 | Pink color appear- | No change | Pink color appear- | No change
ing on lower ing on _ lower
“Tibs’’ and above “ribs”? and above
depressions depressions
11:23 | Upper “‘ribs’’ be-| No change | Upper “ribs’’ be-| No change
coming pink coming pink
11:25 | Pink spreading lat- | No change | Pink spreading lat- |} No change
erally from lower erally from lower
“ens” “ribs”
11:27 | Lower portion of | No change | Lower portion of} No change
flower mostly vio- flower mostly vio-
let with pink let with pink
“Tibs”’ “‘tibs”’
11:31 | Upper portion of | No change | Upper portion of | No change
flower shows con- flower shows less
siderable pink and violet than (1) but
violet outside of “ribs” are, if any-
“Tibs”’ thing, pinker
11:38 | About the same No change | About the same No change
11:45 | Same No change | Same Becoming |
slightly bluer
than (2)
1:05 | Same No change | Slightly bluer than | Beginning
before but still! turn greenish
pinker than (2) !
2:30 | Same No change | About the same as | Decidedly
; (2) greenish
* Started.

’

the slightly akaline CO,.-bicarbonate mixture became distinctly acid,
while those in the distilled water with a hydrogen ion concentration
perhaps one hundred times as great did not. There was also com-
paratively little difference (up to the time when the bicarbonate finally

460 M. H. JACOBS

began to penetrate) between the two solutions saturated with COs,
though the concentration of hydrogen ions in the one was approximately
four thousand times that in the other. Evidently, therefore> the neu-

trality or slight alkalinity of a solution does not rule out the possibility —

of decided effects of hydrogen ions where CO, is one of the substances —
concerned, and the stimulation of the mammalian respiratory center in
Scott’s (7). experiments by blood of pH 7.6 would not necessarily be in
conflict with the orthodox view of the réle of hydrogen ions in this
process.

It may perhaps be of interest to show that the effects of COs on liy-
ing cells here described may be imitated by a simple model whose
- construction depends on the fact that CO, is freely soluble in xylene
(as well as in other lipoid solvents and lipoids) while NaHCO; is not.
The “cell” is made as follows. A small “‘shell vial,’ 10 by 35 mm.,
is filled to within 4 mm. of the top with a solution of phenolsulpho-
nephthalein made very slightly alkaline by the addition of a trace of
- NaHCOs. It is next filled level full with xylene (from which the COs,
if necessary, has been removed by allowing it to stand in contact
with an aqueous Ba(OH). solution, and the escape of the xylene is
then prevented by the following simple expedient. A test tube is
dipped into a fairly thick celloidin solution and the film which closes
its mouth on removal is immediately pressed over the opening of the
vial to which it adheres as a thin transparent covering, freely perme-
able to salts, acids, etec., but preventing the loss of the xylene when the
cell” is immersed in water. The vials as thus prepared are kept
until needed in an upright position in water. In some cases cotton-
seed oil was used in place of xylene with essentially similar results,
though it is less convenient to work with, and results are obtained

more slowly with it than with xylene.

A typical experiment with ‘artificial cells’? was the following. The
solutions used were the same as those studied with the Symphytum
flowers, namely, distilled water saturated with COk, distilled water,
M/2 NaHCO; saturated with COs, and M/2 NaHCO;. With the dis-
tilled water and NaHCOs, there was no change in the color of the indi-
cator; with the two solutions containing COs, though, as before, the
actual hydrogen ion concentration of the one was perhaps four thou-
sand times as great as that of the other, there was an approximately
equal rate of change in color of the red indicator solution to a bright
yellow. In both cases, the change began to be visible within two

PERMEABILITY OF CELL WALL TO CARBON DIOXIDE 461

minutes and was complete in about an hour and a half. The line of
demarcation between the red and the yellow portions of the solution
was very sharp, especially at first.

The result of one alkaline solution turning acid when placed in
another alkaline solution is a rather striking one. In this case it evi-
dently depends on the relative solubilities of CO. (and perhaps H.COs),
on the one hand, and of NaHCO; on the other, in lipoids and lipoid
solvents. In living cells there is frequently a close correlation between
the lipoid solubility of substances and their powers of penetration.
Without at all postulating a lipoid membrane in Overton’s original
sense, it is nevertheless possible that the relative solubilities of the
substances concerned in the lipoid and aqueous phases encountered in
the structure of a typical cell may have much to do with the physio-
logical behavior of CO.-bicarbonate mixtures. |

In connection with the points already mentioned, it is of interest
to compare the penetrating powers of CO, with those of other acids,
particularly those which, from the results of previous workers, appear to
enter living cells with the greatest readiness. Such a comparison was
in progress at the time when a severe storm put a temporary end to the
available supply of Symphytum flowers. Reserving, therefore, for a
subsequent paper the details of the work already done and certain
points requiring further elucidation, it may be said briefly that of all
of the acids studied (carbonic, benzoic, salicylic, valeric, butyric, acetic,
sulphuric and hydrochloric), carbonic is by far the most effective when
pure solutions of eyual pH are compared, in causing a visible change in
intracellular acidity. For example, with a solution saturated with
CO, at ordinary temperatures, a visible change in color usually begins
in one or two minutes, while with the acids next in the order of their
effectiveness (benzoic and valeric) fifteen to thirty minutes are required,
with butyric, acetic and salicylic acids following in the order named.
The mineral acids are only very slightly effective.

These results, as far as the acids other than carbonic (which appar-
ently has not yet been studied in this manner) are concerned, agree fairly
well with the findings of Haas (8) and of Collett (9) and also with the
observation of the author (1) that tadpoles are fatally injured in satu-
rated CO, solutions in two or three minutes, while in solutions of other
acids of the same pH, from one to several hours are required to produce
the same result. | | epee 7

If carbonic acid be compared with other acids of the same normality
instead of the same pH, it appears far down the list, as would be ex-

462 M. H. JACOBS

pected, not because it does not enter cells readily—for there is much
evidence that it does—but because, on account of its weakness (i.e., its
low degree of dissociation), it is necessary for so much more of it to enter
the cell before a change in the color of the indicator can occur than in
the case of the more strongly dissociated acids. 7

One further point may be noted. If a solution, e.g., N/10, of NaOH
have various acids added to it (in as concentrated a form as possible to
avoid dilution) until the point of neutrality, as shown by phenolsul-
phonephthalein is reached, there should at this point, in the case of
fairly strong acids, be practically no free acid present, while in the case
of a weak acid like H2CO;, there should be a considerable amount.
This difference may easily be made visible in the case of carbonic and
other lipoid-soluble acids, such as benzoic, salicylic and butyric, by
using the “‘artificial cells’’ already described. In one such experiment —
where the solutions were all neutral and N/10 with respect to total base,
the indicator solution within the vial began to change color in three
minutes with carbonic acid, while with butyric, benzoic and salicylic
acids (all of which are highly effective with the “artificial cells’ in the
free form on account of their solubility in xylene) no change had occurred
in three hours. Had the water used been so free from COs as to make
possible the use of an indicator solution with a lower buffer value, the
effect of the smaller amounts of the other acids could probably have
been detected as well, but as the experiment was not intended to be
accurately quantitative, but merely to show that a very considerable
difference exists between carbonic and the other acids, it was not con-
sidered necessary to take this precaution.

As the final conclusion to be drawn from the various experiments
described in this paper, which have been made partly on: living plant
cells and partly on a simple “artificial cell,’’ it may be stated that the
physiological behavior of COz probably depends to a considerable ex-
tent on two of its chemical and physical peculiarities: a, the weakness
of H.CO; as an acid, permitting the existence of a relatively large
amount of the free but undissociated acid (as well as dissolved COz)
in the equilibrium that exists at neutrality or slight alkalinity; and },
the readiness with which the undissociated acid, or its anhydride, COs,
enters living cells, perhaps in virtue of its lipoid solubility. The com-
bination of these two peculiarities is responsible for certain of the .
remarkable, and in some respects, unique, physiological properties of
carbon dioxide.

PERMEABILITY OF CELL WALL TO CARBON DIOXIDE 463

SUMMARY

1. It has been found that the flowers of Symphytum peregrinum
contain a natural indicator sensitive to carbonic acid, which may be
used to study cell penetration by COs.

2. Using this material, it appears that a condition of intracellular
acidity can be produced by a slightly alkaline solution of CO, in M/2
NaHCo0O; almost as effectively as by a solution of CO, in distilled water,
though the hydrogen ion concentration of the latter solution is approxi-
mately four thousand times as great as that of the former.

3. A similar result may be obtained by the use of an “artificial cell”’
whose construction is described.

4. From pure aqueous solutions of acids of the same pH, carbonic
acid changes the color of Symphytum flowers more quickly than do
any of the other acids studied. _

5. The ability of neutral and slightly alkaline solutions containing
CO, to produce intracellular acidity is probably due to at least two
factors: a, the weakness of HsCO; as an acid and 8), the lipoid solu-
bility of CO. or HCO; or both, and the lack of such solubility in the
case of bicarbonates.

BIBLIOGRAPHY

(1) Jacoss: This Journal, 1920, li, 321.

(2) WintTEeRSTEIN: Pfliiger’s Arch., 1911, cxxxviii, 167.

(3) HasseLBaLcH AND LUNDSGAARD: Biochem. Zeitschr., 1912, xxxviii, 77.
(4) HassetBpatcu: Biochem. Zeitschr., 1912, xlvi, 403.

(5) LaquEuR AND VERzAR: Pfliiger’s Arch., 1912, exliii, 395.

(6) Hooker, W1Lson AND Connet: This Journal, 1917, xliii, 351.

(7) Scorr: This Journal, 1918, xlvii, 43.

(8) Haas: Journ. Biol. Chem., 1916, xxvii, 225.

(9) CottettT: Journ. Exper. Zo6l., 1919, xxix, 448.

THE EFFECT OF SALT INGESTION ON CEREBRO-SPINAL
FLUID PRESSURE AND BRAIN VOLUME

FREDERIC E. B. FOLEY anp TRACY JACKSON PUTNAM
From the Laboratory for Surgical Research, Harvard Medical School
Received for publication July 6, 1920

Recently Weed and McKibben (1), (2) demonstrated the important
physiological fact that it is possible to reduce the cerebro-spinal fluid
pressure and diminish the bulk of the brain by the simple procedure of
injecting a hypertonic solution into the blood stream. Dr. Harvey
Cushing, appreciating the clinical possibilities of this discovery, was led
to suggest the present study on the following speculations:

It is common knowledge that people who suffer from headaches suggesting
‘tension headaches’? may get relief by a thorough intestinal evacuation, par-
ticularly when this is accomplished by salines. This has led to the view that
constipation in itself is provocative of such discomforts. It would be of profit
to repeat the observations of Weed and McKibben and then attempt to accom-
plish the same results by giving the hypertonic saline by mouth instead of intra-
venously. It seems possible that the withdrawal of fluid from the blood may _
increase its salt content sufficiently to cause the same result but without
actually introducing salt into the blood stream. The fact established by Weed
and McKibben might be adaptable to certain cranial operations, making them
easier by lowering tension and diminishing brain volume. ,

INTRODUCTORY

In this laboratory some years ago, L. H. Weed began a series of
experiments in an effort to answer certain questions relating to the
cerebro-spinal fluid circulation which had been suggested by Doctor
Cushing (3). His studies related largely to the circulation of the -
fluid and to the methods of its escape from the cerebral chamber (4).
It was demonstrated that the normal escapes for fluid were by way
of the arachnoid villi directly into the large dural sinuses and along
the perineural spaces about the cranial and spinal nerves. He
subsequently showed how the arachnoid spaces pad dural sinuses
developed (5).

On.the side of pure physiology we are not so secure. It has recently
been claimed that a solution of the primary problem of fluid produc-

464

CEREBRO-SPINAL FLUID PRESSURE 465

tion has not been accomplished. ‘There-is much presumptive evidence
of an experimental and clinical nature that the choroid plexus behaves
as a secreting gland in this connection. Such has been the generally
accepted belief. Later students of the subject, and in particular
Becht (6) and Becht and Matill (7), have objected to some of these
generally accepted broad facts. They have recently published the
results of a very critical reinvestigation of the subject and conclude
that there is no indisputable evidence that the fluid is a secretory
product of the choroid. Dixon and Halliburton (8), 1913, published
an elaborate investigation of the action of certain drugs and tissue
extracts on the ‘‘secretion.’”’ Their results are somewhat discounted
by the work of Becht and Matill who point out that the results obtained
may have been due to intracranial vascular (pressure and volume)
changes caused by the injected substances and recorded as fluid secre-
tion changes through the inadequacy of the methods employed. ‘These
authors applied the same criticism to that part of the work of Cushing
and Weed (9) in which they claimed to demonstrate an increased
secretion of fluid following the administration of hypophysis extract.
The criticism does not seem warranted since in some of their experi-
ments Cushing and Weed measured the increased flow of fluid from a
catheter within the ventricle. The catheter was sealed off from the
subarachnoid space by its contact with the walls of the aqueduct.
Under such circumstances the changes in capacity of the subarachnoid
space, incident to vascular changes, could not impart a significant
alteration to the flow from the ventricular catheter. |

Aside from the primary broad facts of anatomy, secretion and ab-
sorption channels, the work of Weed and McKibben brings out the
most definite and significant fact established in regard to the detailed
physiology and pharmacology of the cerebro-spinal fluid. The demon-
- stration that intravenous injections of hypertonic solutions reduce
cerebro-spinal fluid pressure and brain bulk offers a new point of
departure into the problems of the physiology and pathology of the
cerebro-spinal fluid.

A brief summary of the work of Weed and McKibben (loc. cit.) is
essential to a presentation of the present study. In experiments on
cats it was shown that hypertonic solutions of electrolytes injected into
the venous circulation caused a marked fall of the cerebro-spinal fluid
pressure. Hypertonic solutions of other crystalloids (glucose) caused
a similar fall of pressure though not so great in extent. Saturated
solutions of sodium chloride, sodium bicarbonate and sodium sulphate

466 F. E. B. FOLEY AND T. J. PUTNAM

were employed. Conversely, it was found that distilled water (a
hypotonic solution) had the opposite effect and caused a rise of pres-
sure. It was later observed that the hypertonic solutions caused the
brain to diminish in bulk while the hypotonic solution had the oppo-
site effect, the brain increased in bulk. It was suggested that these
changes were mediated through changes in the osmotic value of the
blood.

EXPERIMENTAL

In experiments of this sort the methods employed are important toa
proper interpretation of results. For this reason the method employed
is set forth in considerable detail.

Animals. Most of the experiments were made on cats. The animals
were taken from stock, usually without any preparation. In a few
instances castor oil was given twenty-four hours before experiment.
Some of the animals were allowed no food or water for this period.
These measures did not appear to affect markedly the results and were
given up. The animals were securely fixed in the prone position, the
neck flexed and the head held tightly in place. For the recording of
accurate pressure changes the fixation and position of the animal are
most important for slight movements greatly affect the pressure.

Anesthetics. We have used the following anesthetics: Ether a, by .
cone and b, by the intratracheal method; c, morphine-atropine-urethane ;
d, luminal and e, morphine-chloretone. In our experience morphine-
atropine-urethane is the most satisfactory anesthetic for the experi-
ments on cats. The anesthesia requires no attention, it is uniform
throughout the experiment and appears to have little effect on the
fluid pressure. Chloretone does almost as well. With ether anes-
thesia the animals lose heat so rapidly that even with them placed on
electric pads and under the warmth of several electric bulbs we have
found it impossible to keep them in good condition for long periods of
observation (8 to 10 hours). Changes in the depth of ether anesthesia
markedly affect the intracranial fluid-blood pressure relationship
causing changes quite apart from the purpose of experiment. The
animals eventually fail when morphine-atropine-urethane is used, but
in our experience it gives the most satisfactory period for observation.

Manometer and connections. The fluid pressures were read from a
U-tube manometer connected to a needle in the subarchnoid space.
The method of establishing the manometer connection is of impor-
tance. The whole system was filled with normal saline, the column of

CEREBRO-SPINAL FLUID PRESSURE 467

fluid being set at 100 mm., somewhat below the initial pressure usually
to be anticipated. A wire obturator extended through a water-tight
puncture in the tubing on into the needle. With the apparatus so
arranged the puncture through the occipito-atlantoid ligament was
made. The obturator was withdrawn from the needle but its end was
still left in the tubing to occlude the puncture through which it was
inserted. By this method we were able to avoid the great loss of
fluid and artificial fall of pressure incident to beginning with an empty
system into which the fluid must rise to fill the manometer. The
bore of the manometer was such that 100 mm. pressure change required
a displacement of 0.26 cc. of fluid.

The importance of the displaced fluid in experiments of this sort has
been emphasized. It will vary directly with the bore of the manom-.
eter. The possible bearing of this fact on the results was realized
and so a number of control experiments with a larger bore manometer
were conducted in which 100 mm. pressure change required a dis-
placement of 1.06 cc. of fluid.

_ Arterial and venous pressures. These were recorded in some of the
first experiments. It soon became apparent that they were not essen-
tial to an interpretation of the enormous fluid pressure changes that
occurred and so their recording was given up.

Routes of administration: Intravenous doses were given from a buret
through a cannula in the femoral vein. It was possible to control
accurately the rate of flow. Gastro-intestinal doses were given through
a stomach tube. Duodenal doses were given by a syringe and needle
into a loop of duodenum exposed through a small mid-line incision.
Colonic doses were given by a rectal tube held in place by a purse string
suture in the anus.

Normal pressures. We wish first to record the values we have ob-
tained for normal pressures. In addition to the present series Weed
and McKibben (loc. cit.) and Becht (loc. cit.) furnish the most compre-
hensive data on the subject. In a series of one hundred experiments
on cats under the experimental conditions described, the average of
normal pressures of the cerebro-spinal fluid was 133 mm. normal saline.
This is 14 mm. higher than the value of 119 mm. given by Weed and
McKibben and 21 mm. higher than the value of 112 mm. given by
Becht. The former authors cited used an empty manometer into
which the fluid had to rise. It is not clear whether or not Becht began
with a filled system in the experiments on which this figure is based.
At any rate, this factor in our experiments probably,.in part, explains

468 F. E, B. FOLEY AND T. J. PUTNAM

the higher value of our normal pressures. When an appreciable amount
of fluid has been displaced it takes a considerable period of time for a
new equilibrium to be established. Under these circumstances readings
taken early in an experiment only represent a point in the transition
toward equilibrium. ‘The average normal with ether anesthesia was
127; with urethane 155 and with chloretone 127. The higher value of
our normal readings may be connected with the use of urethane. The
highest initial pressure we recorded was 255 mm. The lowest positive
pressure was 65 mm. In three animals the pressure at the beginning
of experiment was negative, viz., —10 mm., —20 mm., —25 mm.
These values existed after the 100 mm. column of fluid in the manom-
eter (0.26 cc.) had run into the subarachnoid space. The experi-
mental details were carefully checked up in these cases so that we are
sure the figures represent true values.

INTRAVENOUS DOSES

A number of experiments in which the solutions were given intra-
venously were carried out. They abundantly confirmed the work of
Weed and McKibben (loc. cit.). In addition they furnished a basis
for comparison between intravenous and gastro-intestinal doses of the
solutions.

Ringer’s solution regularly caused a marked though unsustained
rise of the cerebro-spinal fluid pressure. For example an intravenous
dose of 50 cc. was followed by a rise of the cerebro-spinal fluid pressure
of 100 mm. Within an hour the pressure had fallen to approximately a
normal value again. On the other hand, the same amount of water -
given intravenously caused a marked and sustained rise of the cerebro-
spinal fluid pressure (100 mm.) which, an hour after the injection, was
still at its height. These changes were not accompanied by significant
changes in the blood pressure. Figure 1 illustrates the effect on cere-
bro-spinal fluid pressure and blood pressure of an intravenous injection
of 25 ec. 30 per cent sodium chloride solution. The cerebro-spinal
fluid pressure promptly fell from 175 mm. to —78 mm. The fall is
sustained, for two hours and fifteen minutes after injection the pressure
remains 197 mm. below its level at the beginning of experiment.

These experiments are typical of our results with intravenous injec-
tions. The changes are greater than those recorded by Weed and
McKibben but qualitatively they are identical with their experiments.
In longer continued experiments we have found the lowered cerebro-

CEREBRO-SPINAL FLUID PRESSURE

spinal fluid pressure persisting for long periods.

469

In one experiment,

for example (10 cc.¥30 per cent NaCl intravenously), it remained
110 mm. below its initial level five hours and twenty minutes after

injection. In this experiment
a fall of 238 mm. took place in
‘fifty-four minutes or at the rate
of approximately 4.4 mm. per
minute. The return toward

normal occurred more slowly, |

at the rate of approximately
0.7 mm. per minute. :

Unless the rate of injection
of the hypertonic sodium chlo-
ride is carefully controlled the
cardiac and respiratory changes
may prove fatal. Death from

these causes occurred in several ©

experiments.

GASTRO-INTESTINAL DOSES

General effect. By a single
experiment our main anticipa-
tion in regard to salt ingestion
was shown to be well warranted.
To an anesthetized dog a mas-
sive dose (180 cc.) of 30 per cent
sodium chloride was given by
stomach tube. The cerebro-
spinal fluid pressure promptly
fell from 150 mm. (normal
saline) to zero. This was
accompanied by a rising arterial

wl.

‘\
1 \ aN
r ft rd Sas
igs V mete oP]
60 \ A
phe Vel
20 \
0 \

3 T]he. Thr. [30 2 [hr 2hr|30
sd \ ee
60 Se re
7

80

Fig. 1. Intravenous injection of 25 ce.
30 per cent sodium chloride solution. Dog
weight6.0kgm. Etherbycone. Cerebro-

spinal fluid pressure (mm. saline) and blood
pressure (mm. Hg.) curves. During the .
injection there was marked cardiac and
respiratory disturbance—the respirations
became rapid and shallow, almost ceasing,
the heart rate rose and the beats became
feeble. Note the 70 mm. fall of arterial
pressure during this period. A prompt re-
covery occurred with return to a higher
value than previously. An abrupt: fall,
253 mm. of cerebro-spinal fluid pressure
occurred. Two hours and fifteen minutes
after injection this pressure remains at
—22 mm. or 197 mm. below the level at the
beginning of experiment. The change is
independent of blood pressure.

This is the essential fact. The

pressure during the whole experiment.
subsidiary facts—size of dose, response to different substances, vascular
changes, duration of the effect and so on—will be presented in the
following experiments. | |

Size of dose. Figure 2 illustrates the effect on cerebro-spinal fluid
pressure and blood pressure of injecting 35 cc. 10 per cent sodium
chloride solution into the rectum. Five minutes after the injection

470 F. E. B. FOLEY AND T. J. PUTNAM

was begun the cerebro-spinal fluid pressure began to fall. In one hour
and ten minutes it had fallen from 112 mm. to —44 mm., a drop of
156 mm. This was at the rate of 2.2 mm. per minute. The change is
quite independent of the blood pressure which, as may be seen, remains
quite constant. The change here is not so extensive nor rapid as in
the case of intravenous doses—compare figure 1 with figure 2.

That the extent and rate of change obtained in this experiment are
not the maximal ones to be obtained by intestinal doses was soon
apparent. Figure 3 illustrates this fact. In this experiment 50 ce. of

| ——s ie 0 °
* \
= ;
e \ 140 Me
20 . i N
' —3o Thr a Thr 30 2 [hr toe \
Sul fads " (
60 Whe a \
Fig. 2. Thirty-five cubic centime- is Seca
ters 10 per cent sodium chloride solu- Saf re

tion by rectal tube. Cat weight 2.5

kgm. Intratrachealether. Cerebro-
spinal fluid pressure (mm. saline)
and blood pressure (mm. Hg.). Soon
after the injection is begun the cere-
bro-spinal fluid pressure begins to

fall—_156 mm. in 1. hour and 10 .

minutes. Rate 2.2 mm. per minute.

The arterial pressure rises a little

during the injection but shows no
significant change.

Fig. 3. Fifty cubic centimeters
30 per cent sodium chloride by
rectal tube. Cat weight3.2kgm.
Intratracheal ether. A fall of
270 mm. occurred at the rate of
4.2mm. per minute. The curve
closely resembles that obtained
with intravenous doses, an
example of which is shown in
figure 1.

30 per cent sodium chloride solution were put into the rectum. The
fall of pressure and thé rate at which it occurred were here equal to
intravenous doses, a fall of 270 mm. at the rate of 4:2 mm. per minute,
compare figure 1 with figure 3.

Changes of this extent are maximal and larger doses add nothing to
the effect. Much smaller doses, 20, 10, 5 cc. of 30 per cent salt solu- |
tion still cause very marked falls of pressure. An injection of 5 ce. of
30 per cent sodium chloride solution into the duodenum caused a fall

CEREBRO-SPINAL FLUID PRESSURE 471

of 104 mm. in the cerebro-spinal fluid pressure. Comparing this with
the injection of a similar amount of the same solution intravenously,
little difference between the effects produced is found. The intra-
venous dose causes a slightly more rapid fall. /

Route of administration. As between rectal, duodenal and gastric
doses little difference in the effect is to be noted when large doses are
employed. Smaller doses, however, are most efficient in the duodenum.

Concentration of solution. Solutions of much lower concentrations
than those mentioned so far are capable of producing changes of the
same sort. Here, however, the rate of change is much slower and the
fall of pressure is decidedly less in extent. In a number of experiments
the average maximal effect to be obtained with large doses (40, 50,
150 ec.) of 2 per cent sodium chloride per duodenum was a fall of 97
mm. pressure at the average rate of 2.5 mm. per minute. Figure 4

: £3

30 thr. the. 30 2hr. Chr 30 Shr.

Fig. 4. Injection of 150 cc. 2 per cent sodium chloride solution, containing 2.7
per cent magnesium sulphate into the duodenum. Cat weight 3kgm. Urethane
anesthesia. Curve of the cerebro-spinal fluid pressure changes (mm. saline).

represents an experiment of this sort. The average maximal effect
obtained with the large doses (20 to 35 cc.) of 30 per cent sodium: chlo-
ride on the other hand was a fall of 258 mm. pressure at the average
rate of 3 mm. per minute.

The large volume of solution in the case of the doses of lower con-
centration probably brought the solution into contact with a larger
area of intestinal mucosa than was the case with the smaller volume of
‘solution when 30 per cent salt was used. Were it not for this factor,
the lower concentrations would have very likely affected the change ©
at a still slower rate.

Comparison with intravenous doses. These large doses of concen-
trated salt solution in the gastro-intestinal tract cause maximal effects

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 58, NO. 3

472 F. E. B. FOLEY AND T. J. PUTNAM

as regards the extent of the fall of pressure. Comparing the results of
such experiments with those in which the intravenous route was used
the extent of fall in the two cases is approximately equal. The intra-
venous doses produce the effect at a slightly more rapid rate.
Duration of effect. Some of the experiments were continued for long
periods. It was found that the lowered pressures endured for a con-
siderable period. The curves are too long for reproduction but the
following tabulation of four experiments (table 1) illustrates the point.

TABLE 1

Cerebro-spinal fluid pressures at hour intervals following salt ingestion

HOUR INTERVALS
INITIAL

ria ts loa | 8: 1.6 42 foe

10 cc. 380 per cent NaCl.| 160 |—78|/—35|) +4) +32) +45
20 ce. 30 per cent NaCl.} 240 |—40/—50} 0| +60/+110/+140/+130/+150/+145
20 cc. 30 per cent NaCl.| 120 |—70|/—85|—40} —2) +20) +388) +55
10 cc. 30 per cent NaCl.| 230 | 0/+40/+84/+138/+182

TABLE 2

Cerebro-spinal fluid pressures at long intervals after salt ingestion

PRESSURE
punc. | NORMAL | "ENoe
TURED r
20 ec. 30 per cent NaCl 48 hours before puncture 100 133 33
20 cc. 30 per ‘cent NaCl 24 hours before puncture} ©" "
15 cc. 30 per cent NaCl 22 hours before puncture 75 133 58
15 cc. 30 per cent NaCl 18 hours before puncture} *"’
35 cc. 30 per cent NaCl 17 hours before puncture..... 55 133 78
35 cc. 30 per cent NaCl 22 hours before puncture..... 123 133 10
PCO ALR «0.08.9 - tye cane sea g:a-hcw pow ae DIS See nO a 55 4 88+ 133 45—

We felt that possibly under the experimental conditions, anesthesia
etc., it was impossible for the pressures to recover. For this reason
other animals were given doses of 30 per cent sodium chloride or satu-
rated Na SO, the day previous to experiment. The following day ~
their pressures were determined. As we did not know their pressure
before the ingestion of salt, the results are not of great value. The
pressures found the next day can only be compared with our average
normal pressures for animals under similar experimental conditions.
The data bearing on this subject are given in table 2.

CEREBRO-SPINAL FLUID PRESSURE 473

In these four experiments the average of pressures found many hours
after salt ingestion is 88 mm., i.e., 45 mm. lower than our average of
133 mm. in the normal animal.

Unabsorbable salts. Experiments in which the unabsorbable salt,
sodium sulphate, was used were also carried out. Changes qualita-
tively like those following sodium chloride occurred. The extent of
change, however, and the rate at which it occurred were somewhat
smaller in the case of the sulphate. Figure 5 is plotted from a sulphate
ingestion experiment, a fall of 202 mm. occurred at the rate of 2.2 mm.
per minute. Compare with figure
3, an experiment in which a large
dose of sodium chloride was given.
The average extent of fall after .
large doses of sulphate was 122 mm.

at the average rate of 24mm. per 4 \
minute. Smaller doses caused « \
roughly proportionate falls of pres- 2 x
sure. Asin the case of chloride in- +5 m.
‘ ; the} 30 Che
gestion, the low pressuresendure for 2
a considerable period. 40 mat
This ingestion of salt does not Mie
60

appear to make the animals refrac-
tory to a second dose at a later
time. This was demonstrated by
administering the salt on two succes-
sive days. Following the second
administration the typical pressure
changes occurred.

Glucose solutions. Concentrated
solutions of glucose given into the
gastro-intestinal tract are capable

Fig. 5. Injection of 35 cc. satu-
rated (120° F.) sodium sulphate solu-
tion into the duodenum. Cat weight
3.5 kgm. Chloretone anesthesia. A
prompt fall of cerebro-spinal fluid pres-
sure occurred—202 mm. at the rate of
2.2 mm. per minute. This rate is
somewhat slower than in the case of
similar doses of sodium chloride.

of producing qualitatively these same results, though quantitatively
smaller in extent. Thus following the administration of 35 cc. of
concentrated glucose solution into the duodenum the cerebro-spinal
fluid pressure fell 140 mm. This is the largest fall we have obtained
with dextrose solutions. The results are not as constant as with the
salts.

Hypotonic solutions. The effects produced with hypotonic solutions
are not nearly so striking. Several experiments were conducted in
which water was injected into the duodenum. Thirty-five cubic centi-

474 F, E. B. FOLEY AND T. J. PUTNAM

meters caused rises of pressure averaging 45mm, This rise is not very —
well sustained for within an hour practically normal pressure values
obtained. Water seems somewhat more effective when it follows by
several hours the ingestion of a hypertonic solution.

Alteration of brain bulk. A number of observations ‘were made on
the change of brain bulk following the administration of 30 per. cent
sodium. chloride solution. The method employed was like that de-
scribed by Weed and McKibben. The temporal muscles were dis-
sected back and the dura with the underlying brain was exposed,
through two trephine openings. The sodium chloride solution was
injected into the duodenum. Within a short time following the admin-
istration of the solution the brain began to recede from the margins of
the skull. Previously it had been bulging under the tense dura. The
changes were very obvious, for within twenty to forty minutes the
bulging had completely disappeared and the surface was concave. On
incising the dura at this stage the sulci were found to be quite broad
while the convolutions were contracted and narrow.

Proof of secretion and absorption pressure changes. The question
naturally arises as to whether or not, in experiments like those re-
corded, the changes in the height of the manometer column may not
be due simply to the dislocation of fluid from the manometer into the
cerebrospinal fluid spaces coincident with the diminution in the size
of the brain. That this factor plays some part in the pressure changes
recorded, there can be no doubt for obviously as the subarachnoid
spaces enlarge with a diminishing brain volume, fluid must run in to
occupy them. On the other hand, there is very good evidence that
the ingestion of these solutions causes alterations in the ratio between
secretion and absorption of the fluid, producing pressure changes quite
independent of brain volume changes. This fact was demonstrated by
a number of experiments. With a certain dose of hypertonic solution
the pressure changes of the cerebro-spinal fluid were recorded in a
manometer of such bore that 100 mm. pressure change required a dis-
placement of 0.26 cc. of fluid. The pressure changes following this
same dose were then recorded in another animal using a larger bore
manometer, in which 100 mm. of pressure change-required a displace-
ment of 1.06 cc. of fluid. Figure 6 shows the two curves obtained. If
the whole process were merely one of change in brain volume, the sec-
ond curve (large bore manometer, broken line) should illustrate a fall -
of pressure at a very much slower rate and very much less in extent,
for the fluid displaced into the subarachnoid spaces would change the

CEREBRO-SPINAL FLUID PRESSURE A475
height of the column in the small bore manometer a great deal more
than in the large bore manometer. Such is not the case, for the new
ratio established between secretion and absorption is capable of main-
taining the pressure at a new level, independently of the volume of the
cerebro-spinal fluid spaces or the size of the manometer used. A later
report will be made of the remarkable changes which these solutions
bring about in the absorption mechanism and in the currents of the
fluid channels. They appear to be capable of reversing the flow in the
perivascular spaces and the ventricular system.

The clinical significance of these
problemsismost apparent. They |
concern the states commonly
referred to as ‘‘pressure symp-
toms.” Up to the present the
facts established by research have
been more anatomical than physi-

ological and it is significant that ss)
the clinical advances have been AG ~
mostly applications of these facts 2 Dy Phir
and have little to do with physi- “° MS A ea Ra Te Bo
ology. ig Pena Gr
” ~
Fig. 6. Curves from two separate experi-
SUMMARY AND CONCLUSIONS - ments. The broken line describes the

cerebro-spinal fluid pressure as recorded in
a large bore manometer, the continuous

_ 1. Weed and McKibben dem-

onstrated that it is possible to
reduce the cerebro-spinal fluid
pressure and diminish the bulk
of the brain by injecting hyper-
tonic solutions into the blood

line describes the same pressure changes in
another animal, but recorded with a small
bore manometer. In each casethe animal
(cat) was given 10 cc. of 30 per cent sodium
chloride solution intravenously.

stream. Conversely, they showed that the pressure and the bulk of
the brain could be increased by the injection of hypotonic solutions.
Their work has been repeated and their general conclusions confirmed.

2. We have shown that the introduction of hypertonic salt solutions
into the gastro-intestinal tract has a similar effect. This route of
administration is more convenient, and by its use the disturbances of
circulation and respiration common with intravenous infusions are
avoided. |

3. Twenty to thirty cubic centimeters of a 30 per cent sodium chlo-
ride solution introduced into the duodenum or rectum of an average

476 F. E. B. FOLEY AND T. J. PUTNAM

sized cat produced a maximal fall of cerebro-spinal fluid pressure.
Following such doses, the average fall of pressure in a large series of
experiments was 258 mm. of water. Larger doses added nothing to
the extent of the fall. A dose of 5 ce. produced a fall of 104 mm. of
water, and intermediate doses gave roughly proportionate results.
Following this fall in pressure there is a gradual rise. Thus, seventeen
to forty-eight hours after such injections, four animals showed pres-
sures averaging 45 mm. less than the average normal. |

4, Sodium chloride solutions in only slightly hypertonic concentra-
tion are also effective in causing a fall in cerebro-spinal fluid pressure,
although to. produce appreciable changes, large doses are required.
Doses of 40 to 150 cc. of 2 per cent sodium chloride solution caused
falls of pressure averaging 97 mm. of water, in cats.

5. Saturated solutions of sodium sulphate, which is not absorbed
from the gastro-intestinal tract, produced qualitatively similar results,
but less in extent and at a slower rate. With concentrated dextrose
solutions the fall is still less and its rate still slower.

6. Water injected into the duodenum produces a small rise of pres-
sure in the normal animal, but it disappears more rapidly than the fall
incident to salt ingestion. If the animal has been given a concentrated
saline solution the day before, the rise in pressure following the admin-
istration of water is more marked and of longer duration.

7. Such changes in cerebro-spinal fluid pressure were shown to be
independent of changes in arterial or venous blood pressure.

8. These changes of fluid pressure are accompanied by a decrease in
the size of the brain.

9. The manometer readings (pressure values) obtained after salt
ingestion are not due solely to changes in brain volume and capacity
of the cerebro-spinal fluid spaces, but primarily represent new ratios
between secretion and absorption of cerebro-spinal fluid.

BIBLIOGRAPHY

(1) Weep anp McKisseEn: This Journal, 1919, xlviii, 512.

(2) Weep anp McKrssen: This Journal, 1919, xlviii, 531.

(3) Cusuine: Journ. Med. Research, 1914, xxxi, 1.

(4) Wrxp: Journ. Med. Research, 1914, xxxi, 51.

(5) Weep: Contrib. Embryology, no. 14, 1917, Carnegie Inst. of Washington.
(6) Becut: This Journal, 1920, li, 1.

(7) Becut anp Matix: This Journal, 1920, li, 126.

(8) Drxon anp HaturpurTon: Journ. Physiol., 1913, xlvii, 215.

(9) Weep anp CusuHine: This Journal, 1915, xxxvi, 77.

ANTAGONISM OF DEPRESSOR ACTION OF SMALL DOSES
OF ADRENALIN BY TISSUE EXTRACTS

J. B. COLLIP

From the Department of Biochemistry and Physiology, the University of Alberta,
Edmonton, Alberta, Canada

Received -for publication July 6, 1920

In a previous communication the writer (1) has shown that extracts
made from heart, spleen, pancreas, testes, anterior and posterior lobe
of the pituitary body, and the thymus, thyroid and parathyroid glands,
in addition to stimulating the isolated uterus of the rat, guinea pig,
virgin dog and cat, antagonized the inhibitory action of adrenalin on
this tissue. It was also shown that the same extracts depress the car-
diac vagus of the terrapin. It was demonstrated also that the pressor
effect following the intravenous injection of a definite dose of adrenalin
was augmented and longer maintained when splenic extract was pre-
viously injected. It was suggested that this latter result might be due
to the depression or paralysis of some part of the vasodilator mechanism
by the splenic extract.

Further investigation has confirmed the writer in this opinion.

Methods. The extracts used were made by boiling with distilled
water in amount sufficient to give a practically isotonic solution, desic-
cated glandular products supplied by Armour & Company, Chicago; or
else by extracting fresh tissue with distilled water on the water-bath,
filtering and concentrating the filtrate to an aliquot volume of the
original tissue. ;

The animals used for testing out the extracts were the dog and rab-
bit. The experimental animal was placed under ether anesthesia and
a cannula was inserted into the left carotid artery. This was con-
nected with the mercury manometer. Injections were made into the
left external jugular vein by means of a “‘ Record” syringe. If the fluid
injected were small in amount the vein was at once flushed with Ringer-
Locke’s solution to insure that all the fluid should enter the general
circulation.

477

478 J. B. COLLIP

Results. It was found that extracts of such widely divergent tissues
as those of the spleen, skeletal muscle and parathyroid gland caused the
pressor response of a definite dose of adrenalin administered by the in-
travenous route to be augmented and longer maintained. Thus it was
found that 3 cc. of adrenalin, 1:50,000, injected into a dog weighing 14
kilos, produced a rise of 32 mm. in blood pressure with a return to nor-
mal in 40 seconds, whereas the same amount of adrenalin injected 5
minutes after the administration of 25 cc. of extract of ox spleen pro-
duced a rise of 52 mm. in blood pressure with a return to normal in 4
minutes. The injection of 0.5 cc. of 1:50,000 adrenalin into a rabbit
weighing 2 kilos produced a rise of 54 mm. in blood pressure with a
return to normal in 80 seconds while the same amount given after the
injection of 5 cc. of extract of ox muscle produced a rise of 62 mm. in
blood pressure with a return to normal in 34 minutes.

In another instance a dog weighing 22 kilos with both vagi cut re-
sponded to 4 cc. of adrenalin 1:50,000 by a rise of 80 mm. in blood pres-
sure with a return to normal in 1 minute 50 seconds; 20 cc. of extract
of ox spleen were then injected and when the depressor action of this —
had passed off 4 cc. of adrenalin 1:50,000 were again injected. This
produced a rise of 110 mm. in the blood pressure with a return to normal
in 3 minutes.

An animal in which the depressor effect of small doses of adrenalin is
readily elicited is most suitable to demonstrate the antagonism of this
latter action by tissue extract. Figure 1 illustrates the antagonism of
the vasodilator action of small doses of adrenalin by extracts of various
tissues. The experimental animal in this instance was a male dog 25
kilos in weight. Shortly after ether anesthesia had been induced, the
injection of 2 cc. of 1:50,000 adrenalin produced a fall of 22 mm. of
mercury in the blood pressure (fig. 1, 1). Both vagi were cut and the
injection of 3 cc. of 1:50,000 adrenalin produced a fall of 32 mm. in the
blood pressure. One cubic centimeter of 1:1,000 adrenalin was next
injected and the blood pressure rose 86 mm. The injection of 3 ce. of
1:50,000 adrenalin then produced a slight rise in the blood pressure
which was followed at once by a slight fall in the same with an immedi-
ate return to normal. This latter effect stands in sharp contrast with
the effect of 3 cc. of 1:50,000 adrenalin prior to the injection of the
stronger extract (fig.1, A). After 10 minutes had elapsed the injection
-of 3 ce. of 1:50,000 adrenalin produced an effect similar to that following |
the first injection of 3 cc. Figure 1, B, shows the conversion of a fall of
16 mm. in blood pressure following the intravenous injection of 3 ce.

ANTAGONISM BETWEEN ADRENALIN AND TISSUE EXTRACTS 479

of 1:50,000 adrenalin. into a rise of 20. mm. after the injection of 10 cc.
of extract. of thyroid gland.

Figure 1 (C, D, E, F, G and #) illustrates the same type of phenom-
enon induced by extracts of pancreas, thymus gland, corpora lutea,
-anterior lobe of pituitary, testes and parathyroid glands respectively.

This antagonism of the depressor action of small doses of adrenalin
by extracts of various tissues was not of long duration. The primary
effect of adrenalin in small doses given intravenously returned in from
10 to 30 minutes after the injection of the tissue extract (fig. 1, D and G).

Discussion. The antagonism of thedepressor action of small doses of
adrenalin and the augmentation and prolongation of the pressor re-
sponse to the larger doses would appear to be phenomena of the same
order. As Hartman (2) and others have shown, there is a definite vaso-
dilator mechanism which is activated by adrenalin. Different opinions
have been expressed as to the exact causation of adrenalin dilatation.
Dale (3) was of opinion that small amounts of adrenalin stimulate the
vasodilator endings. Cannon and Lyman (4) attributed the two ef-
fects, vasodilatation and vasoconstriction, to opposite actions according
to the state of the muscle,—relaxation when tonically shortened, con-
traction when relaxed. Gruber (5) suggested that the central nervous
system was involved in the dilatation from adrenalin. Hartman, Kil-
born and Fraser (6) obtained vasodilatation in the hind limb and intes-
tine by the central action of adrenalin. They concluded that adrenalin
stimulates vasodilator cells situated in the sympathetic and posterior
spinal root ganglia.

It would appear that the tissue extracts studied depress iz in some man-

ner some part of the vasodilator mechanism. The effect of a small dose
of adrenalin which normally produces a fall in blood pressure is thus
neutralized in whole or in part by the tissue extract as far as the dilator
action is concerned, and constriction only or else a lesser degree of dila-
tation takes place. When a larger dose of adrenalin is given, one such as
causes a rise in blood pressure, the administration of tissue extract de-
presses the vasodilator apparatus and the vasoconstrictor effect is thus
augmented and prolonged. Whether the point of action of this peculiar
- constituent of tissue extract is the nerve ending, nerve fiber or nerve cell
of the vasodilator apparatus is undetermined. The writer inclines to
the view that the action is peripheral since the antagonism of the inhib-
itory action of adrenalin on the uterus and the depression of the cardiac
vagus of the terrapin produced by similar extracts was shown to be due
to peripheral action (1). It is however possible that the vasoconstrictor

J. B. COLLIP

14 Om
/30

Fig. 1. Dog 0; 25 kilos. Ether anesthesia

A: a, 3 ce. of adrenalin 1:50,000 injected into left external jugular vein.
b, 1 ec. of adrenalin 1: 1000 intravenous.
c, 8 ce. of adrenalin 1: 50,000 intravenous.

ANTAGONISM BETWEEN ADRENALIN AND TISSUE EXTRACTS 481

mechanism may be rendered more sensitive to adrenalin by tissue
extract.

_ The short duration of the antagonistic action of tissue extract, given
“s intravenous injection, upon adrenalin dilatation indicates that the
active principle is either neutralized or destroyed fairly rapidly when
present in the general circulation.

SUMMARY

1. The fall in blood pressure produced by a small dose of adrenalin is
antagonized by various tissue extracts.

2. The rise in blood pressure produced by a definite dose of adrenalin
is augmented and prolonged by administration of tissue extract.

3. It is held that both these types of effects are of the same order.

B: a, 3 ce. of adrenalin 1:50,000 intravenous.
b, 10 ce. extract of thyroid gland intravenous.
¢, 3 ec. of adrenalin 1:50,000 intravenous.

C: a, 3 ce. of adrenalin 1:50,000 intravenous. ’
b, 20 ce. extract of pancreas intravenous.
¢, 3 ce. adrenalin 1:50,000 intravenous.

D: a, b and c, 3 ce. of adrenalin 1:50,000 intravenous.
Between 1 and 2, 20 ce. of extract of thymus gland injected into left
external jugular vein.
8, five minutes after 2. z

E: a, 3 ce. adrenalin 1: 50,000 intravenous.
b, 20 ec. of extract of corpora lutea intravenous.
c, 10 ce. of extract of corpora lutea intravenous.
d, 3 ce. of adrenalin 1: 50,000 intravenous.
F: a, 3 cc. of adrenalin 1: 50,000 intravenous.
b, 10 ce. of extract of anterior lobe pituitary intravenous:
c, 3 ce. of adrenalin 1:50,000 intravenous.
2, taken eight minutes after /.
G: a, b and c, 3 ce. of adrenalin 1: 50,000 intravenous.
Between i and 2, 40 cc. of extract of testes injected into left external
jugular vein.
3, taken ten minutes after 2.
H: a and b, 3 cc. of adrenalin 1 : 50,000 intravenous.
Between / and 2, 10 cc. of extract of parathyroid glands injected into
left external jugular vein.

I: a, 2 ce. of adrenalin 1: 50,000 intravenous. Vagi intact.

Note: In all other instances vagi were cut.

acs ae ie: antagonism of
extract is of short duration. — fe

OBSERVATIONS ON A SEX DIFFERENCE IN THE PRESENCE
OF NATURAL HEMOLYSIN IN THE RAT

YOSHIO SUZUKI
From the Wistar Institute of Anatomy and Biology

Received for publication July 6, 1920

It has been often found that without previous treatment the serum
of one animal can hemolyse the red blood corpuscles from an animal of
another species. Such hemolysin is called ‘‘natural hemolysin.’”’ The
instances of the presence or absence of natural hemolysins against dif-
ferent species of animals have been noted among the various animals
which are commonly used in the laboratories. However, so far as I
am aware, no systematic observations have been made with the cor-
puscles or serum of the rat in regard to the hemolytic activities of these
fractions against those from other animals, and for this reason we have
carried out the following investigation.

The technique used is as follows:—The rat was lightly etherized and
the blood was collected from the carotid artery. The serum was diluted
to ten times its volume and in each case 0.2 cc. was mixed with 0.5 ce.
of 10 per cent washed blood corpuscles of the pig. To this mixture 1.3
ec. of normal salt solution was added, thus making up the total amount
to 2 cc. This mixture was placed for half an hour in an incubator at
37°C. Sometimes a much greater dilution of serum was also tried, but
unless otherwise stated the results refer to the ten-times dilution.

It was found in the first instance that the rat serum did not hemolyse
the red blood cells of the pig; in other words the rat serum did not pos-
sess the anti-pig natural hemolysin. This result was obtained from the
mixed sera of a dozen adult rats of both sexes combined.

However during further experimentation it was accidentally noted
in one case that the serum of the rat used when mixed with the pig
corpuscles hemolysed the latter readily. This particular rat happened
to be a female immediately after parturition. Since we had previously
found, as we thought, that the rat serum lacks the anti-pig hemolysin,
but yet*in this particular instance a highly active anti-pig hemolysin
was found, we decided at once to repeat the observations. Table 1
gives the results of the further observations.

483

484 YOSHIO SUZUKI

We find from table 1 that during the first five to six days after par- -
turition the anti-pig hemolysin is present, but is less frequently present
after this period. This interesting result induced us to study further.
As a first step we tested for the presence or absence of anti-pig natural

TABLE 1
After parturition. Anti-pig natural hemolysins in female rat
par aren | sore | POPE | law =| eekourome | aa
grams mm, days
1 wea ote Normal
2 (2) ape Normal |
2 150 - Normal
3 171 90 sb Normal
3 300 o Normal
5 180 90 +++ Normal
5 (8) Normal
6 168 ee ha a Slightly infected
9 (2) _ Normal
10 115 — Normal
(hie 171 184 oe ote Normal
16 131 179 -- Normal
25 165 183 — Normal
TABLE 2

Natural anti-pig hemolysin in the rat serum under various conditions
Total number of rats examined = 211

FEMALES MALES
PRESENCE OF ANTI-PIG NATURAL HEMOLYSIS
IN RAT SERUM

Absent Present Present Absent

per cent per cent per cent per cent
PROGHADCY. oc 64%, cunied ae 4.590 09 oes 35.8 64.2
1 to 7 days after parturition............ 27.3 72.7
7 to 25 days after parturition........... 83.3 16.7
Normal adultes 23000-0607. 5) Pua 57.7 42.3 17.6 82.4
Normal young—under 50 days.......... 87.5 12.5 14.3 85.7
Infected, lunge. 6:0 sv ep icone ei yee 59.3 40.7 50.0 50.0

hemolysin of rats under various conditions, and obtained the results
which are shown in table 2

1. The number of cases in which the serum possesses the anti-pig
hemolysin is greater among the females in pregnancy, during the first ©
week after parturition, and in males in infection of the lungs.

SEX VARIATION IN NATURAL HEMOLYSIN IN RAT

485

2. The hemolysin is usually absent in rats of both sexes whose ages
are less than fifty days.

3. Among the adult females the hemolysin is found in almost half
the number of rats examined. Whether or not this high frequency was
due to the use of rats at an early period of pregnancy cannot be deter-
mined. These females however had been with the males.

TABLE 3

Anti-pig natural hemolysin. Observations on the same animal at two or more ages

AGE AT THE TIME OF EXAMINATION

REMARKS

30 days 70 days
30 — —_
3 - +
Q ~ sop
9 _ +-+* * Emaciated
Q - (+)
9 es t+
75 days 96 days
J an ae
Q _ +% * Pregnant
3 Q —
106 days 119 days 132 days 166 days
rot _ _ + (sick) +* * Sick, killed
g +t et Sick, killed
g + + Died after operation
9 oe Sia =
9 _ + Found dead
9 +++ |Notexam.| +++ | +++
216 days 232 days
~f i PL Sick, killed
3 - +
@o |tt+t] +++
29 — Wate
°) on +++ Sick, killed

4, Among normal males we find a comparatively small number of

cases in which the hemolysin is present.

5. Among males with infected lungs, 50 per cent of the cases possessed
the hemolysin.

486 YOSHIO SUZUKI

We may conclude from these data that anti-pig natural hemolysin |

is far more frequently present in the female than in the male.

In order to verify the statistical findings, and also further to inyesti-
gate whether or not the natural hemolysin appears suddenly associated
with both pregnancy, as well as with lung infection, and also with the
idea of determining whether or not the degree of titration increases

with the advance of pregnancy or of infection, the following experiment —

was undertaken.

The rats at various ages were examined individually for the presence
or absence of the anti-pig natural hemolysin. The same. rats were
again examined after a lapse of several weeks. In each instance the
- blood was obtained by puncture of the heart. As table 3 shows, this
puncture was made in two instances at four different ages. ‘ae

It was intended to carry such an examination throughout the entire
span of life. However disturbances in the rat colony from ecto-para-
sites unfortunately prevented the completion of this work. Neverthe-
less the results obtained, though imperfect, show several interesting
points, and these are presented in table 3.

From this table 3 we see that in many instances the hemolysin was
not present at the earlier age but appeared later. In every instance
where the hemolysin was found at the earlier age it was found later also.

DISCUSSION

Recently Kolmer and Casselman (1) examined a large number of
human sera for natural hemolysins against the erythrocytes ofthe
sheep, dog, ox, goat, hog, rat, chicken, horse, rabbit and guinea pig.
They found a considerable variation in the activity of the human sera
to these various blood cells. Kolmer and Casselman did not however
mention any variation associated with either sex, disease or pregnancy.

While the present investigation was nearing completion, I received

from Doctor Obata a paper (in Japanese) on comparative immunologi- — |

cal studies on the fetal and adult sera in man. Doctor Obata found in
man practically all that I have found in the rat; that is the anti-sheep
natural hemolysin occurs more frequently in women than in men, and
furthermore the hemolysin is not present in the fetal blood. Doctor
Obata found however that the anti-sheep natural hemolysin is equally
distributed among non-pregnant women and pregnant women, while
my own observation on the rat shows a much greater frequency in both
pregnancy and the first week after parturition than in the non a
adult female.

SEX VARIATION IN NATURAL HEMOLYSIN IN RAT 487

From the foregoing observations I conclude that

1. Anti-pig natural hemolysin is not usually present in the serum of
younger albino rats under thirty to fifty days of age.

2. In older rats this hemolysin may be present in the serum of both
sexes but more frequently in that of the female than in that of the male
rat.
3. During pregnancy, as well as during the first week after parturition,
the anti-pig hemolysin is not only more active but the proportion of

cases is far greater than among normal non-breeding females.

4, The number of cases in which the anti-pig hemolysin is present
tends in males to increase with the appearance of lung infection.

BIBLIOGRAPHY

_ (1) Kotmer anp Cassetman: Journ. Infec. Dis., 1915, xvi.
(2) OpaTa: Chugwai Izi Shinpo, 1914, no. 866.

/
THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 53, No. 3

STUDIES ON ABSORPTION FROM SEROUS CAVITIES
Ill. Tuer Thiriscsh OF DEXTROSE UPON THE PERITONEAL MESOTHELIUM

R. S. CUNNINGHAM
From.the Anatomical Laboratory of the Johns Hopkins University

Received for publication July 15, 1920

It has long been known that absorption from the peritoneal cavity
takes place rapidly and effectively. The absorption of solutions and
particulate matter has been studied by many investigators, but very
little effort has been made to use the peritoneal cavity as a route
by which therapeutic agents might be administered. The basis of
practical intraperitoneal therapeusis must be founded upon three
general factors: a, the physiological and toxicological effects on the
organism of substances administered by this route; b, the variations
upon tissue resistance caused by the administration of any substance;
and c, the direct effect upon the mesothelium. The determination of
the effect of any substance upon the organism must be carefully con-
trolled for each method of administration, as it is well known that there
is a great variation in the effect of the same substance given by different
routes. Such methods need not be discussed here, since they are in
constant and practical use.’ In the peritoneal cavity the effect upon the
resistance of tissues which are directly exposed to the first shock of
the administration should be considered with very great care.

Blackfan and Maxcy (1). have reported the use of the peritoneal
route for the administration of physiological saline to patientsrequir-
ing additional fluids, with much success and no apparent ill effects. In
their report they also give animal experiments in which they found
rapid absorption ofthe saline; the peritoneum remaining smooth and
glistening, no adhesions forming, and no abrasions of the viscera taking
place. They have also shown that the introduction of salt solution is

attended with no risk of infection if reasonable surgical precautions are .

taken, but they have not controlled their experiments by a study of the

comparative resistance of normal animals, with animals which have

been subjected to the intraperitoneal injection of repeated doses of
488

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ooh eae ws — a

(oe -
A Fe eerie

9 = bse oe ne i ae ee aay Sete Tot Stewarts
SER REIS RE Se

“pet

ABSORPTION FROM SEROUS CAVITIES 489

physiological saline. This is obviously easy to determine with regard
to any given substance, and any given exposure to that substance.
There has heretofore been no method for an early and certain diag-
nosis of injuries, or at least changes in the mesothelium. The forma-
tion of adhesions and an aseptic inflammation could of course be con-
sidered as definite criteria, but these are at best crude methods, and
only indicate comparatively late stages of change in the mesothelium.
It is now possible to estimate the effect of any given substance since
it has been determined that definite morphological changes in the meso-
thelium can be produced by the repeated administration of certain solu-
tions and suspensions. These changes have been produced by the
introduction of particulate matter, laked blood and soluble starch. A
preliminary note on these experiments has been published (Cunningham
(2)); the detailed report has not been published but is in preparation.
These findings seem to offer a possible criterion for the comparison of the

effect which any substance will have upon the peritoneal mesothelium.

Dextrose was selected for study in this connection because, if it could
be administered via the peritoneum without injurious effects, selection
of that route would eliminate many difficulties incident to adminis-
tration in other ways. The purpose of this contribution is not di-
rected to the settlement of the action and fate of dextrose in the circu-
lation, or to the reaction of the organism to this substance, but simply
to the effect which it might have upon the mesothelium of the peri-
toneal cavity.

Technique and material. Rats were used for these experiments. The
animals were anesthetised with ether, the abdomen shaved and washed
with alcohol, and then a drop of tincture of iodine applied... The dex-
trose solution was introduced by means of a 10 cc. record syringe, a
fresh needle being used for each animal. The syringe and needles were
sterilized by boiling. The dextrose, Merck’s “pure,’’ was prepared by

dissolving in freshly distilled water and sterilizing in the Arnold for

one hour on each of two successive days. The solutions were never
sterilized under pressure, because this usually caused the breaking down
of the dextrose molecule. Cultures of these solutions made at the time ©
of administration were uniformly sterile.

Two series of animals are to be reported at this time. The first
consisted of twelve rats, each of which received 10 cc. of a 10 per cent
solution of dextrose; seven of these were killed at two, four, six, eight,
ten, twelve and fourteen hours respectively after the injection. The
other five were killed at intervals between ten and fourteen hours; this

490 R. S. CUNNINGHAM

was done in order to determine more exactly the time at which the fluid
had been entirely absorbed. At autopsy ‘cultures were taken, and the
fluid in the peritoneal cavity was removed with a pipette; by this means

practically all the fluid present could be recovered. Quantitative deter- —

minations of the sugar content were not done, because the actual rate
of absorption of the sugar was not required for the present purpose.
This will be referred to later. Tissue was fixed in Bouin’s fluid, and
prepared for section in the usual manner.

The second series consisted of twelve rats which received 10 ce. of a
10 per cent solution of dextrose every twenty-four hours for fourteen
days. Six of these were killed at two, four, six, eight, ten and twelve
hours respectiyely after the last injection. Three were killed at short
_ intervals after twelve hours, in order to determine the end point of
absorption as in the first series. The other three were kept as controls,
and will be referred to later.

Experimental results. Figure 1 represents the amount of fluid found
in the peritoneal cavities of the animals in the first series, while figure 2
is a similar curve for the second series. From these. curves it is evident

that the absorption of dextrose from the peritoneal cavity is not im--

paired by repeated doses of this solution, but the differences between
the two curves are not great enough to establish conclusively that the
exposure increases the ability of the peritoneum to absorb. |
Autopsies on animals of series I revealed no changes in the peritoneal
cavity, while examination of those in series II showed a slight brownish
tint in the taches laiteuses of the omenta; otherwise they were entirely
normal. No adhesions were found in any of the animals and the folds
of the omenta were normal and free in every case. Usually three or
four points of injection were visible as red areas about 1 mm. in diam-

eter on the anterior abdominal wall; those from earlier punctures were —

almost entirely healed. Cultures taken at autopsy were sterile.

On histological examination of tissue from all the animals in the first ©

series, the serous mesothelium appeared everywhere entirely normal and
no changes were found in the taches laiteuses. On the other hand in
the animals in the second series, which had received repeated doses of
the dextrose solution, the mesothelium of the diaphragm, the spleen,
the omentum, and in one case even the body wall, showed quite decided
changes. The diaphragm and spleen always showed the most marked
changes, which varied very little in the animals of the group, but the
changes in the mesothelium of the omentum were much more variable,
even in the same animal, while the mesothelial cells of the body wall

Gs,
eT

i

re Bee no Sa re Ve
‘

ABSORPTION FROM SEROUS CAVITIES 491

showed slight increase in size in only one animal. This distribution
of changes corresponded very closely to that found after the use of
laked blood, and was most interesting in that the diaphragm and spleen
were always most markedly affected, the omentum somewhat, while

_ the intestines, the liver, and in most cases the body wall, showed little

or no change.

-The mesothelial cells of the diaphragm and spleen showed everywhere
a definite increase in size, the nuclei having become rounded and vesic-
ular, and the cytoplasm increasing considerably in thickness. ‘The
borders of the cells were often withdrawn from the neighboring cells,
so that a small area of the subjacent connective tissue seemed to be |
uncovered. Here and there, especially over the diaphragm, a few cells

: cana
ed 1

lin 3 a

4 10K
se \ +.
sie Le" N

2 \ “|:

é 4 z

a Time in| HOURS \ < Time iw | HOURS. q

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Fig. 1. Curve showing amount of Fig. 2. Curve showing amount of

fluid in peritoneal cavities of normal fluid in peritoneal cavities of rats which
rats after introduction of 10 cc. of a 10 had received fourteen injections of 10 cc.

‘per cent solution of dextrose. of a 10 per cent solution of dextrose.

had become almost separated from the underlying structures and were
attached by only a small pedicle. Areas scattered over the diaphragm,
which represent the space normally occupied by two or three cells, were
bare; the cells evidently having desquamated. In the splenic meso-
thelium desquamation had not taken place to any great extent, although
an occasional cell area was bare. Perhaps the most remarkable fact
noticed was that, in the mesothelium of the diaphragm especially, but
also in a less noticeable degree in the case of the spleen, many of the
cells which had rounded up and increased in size were undergoing divi-
sion. Mitotic figures could often be seen in several stages: In one
section three figures were found in the area covered by the oil immer-
sion lens. The cytoplasm of the mesothelial cells became much more
basophilic as they rounded up and increased in size, but in no case could
any granules in the cytoplasm be detected. Pycnotic nuclei were

492 R. 8S. CUNNINGHAM

never seen in any of these enlarged cells. One other interesting obser- —

vation must be recorded; these large cells when carefully studied under
very high magnification aveidAbie presented a surface which was coy-
ered with fine projections simulating in appearance the cilia of normally
ciliated cells. They differed from true cilia in that they varied consid-
erably in length and width, and were somewhat irregular.

_ After examination of sections had demonstrated that dextrose pro-
duced such definite changes, other experiments were performed in order
to determine how rapidly these changes occurred. Rats were given 10
ce. of dextrose solution and killed after one, two, three, four and five
days respectively. After twenty-four hours no change was noticed.
After forty-eight hours the only changes were a very slight thickening
of the cell-bodies and a delicate irregularity of the peritoneal surface
of the mesothelium, which suggested that the cells were beginning to
form the cilia-like projections described above. After three or four
days the cells had increased in size, and had become somewhat cuboidal
in shape; while here and there some of them had begun to round up and
the projections had increased. After the fifth injection the cells had
rounded up and the free surfaces were covered with the characteristic
cilia-like formations, but no evidence of any separation or deat
tion was apparent. .

_. Sections of the omenta of animals in the second { series showed that
the brown coloration mentioned earlier was due to a few fine granules
of brown material in the cells of the taches laiteuses. These granules
were very few in number and were scattered quite widely. There
was no apparent change on careful comparison of sections of normal
omentum with those of the rats in series II except for these few fine
granules. Sections of anterior mediastinal lymph glands were examined
to determine whether or not there was sufficient granular material in
the sugar solution to have produced the changes noted in the meso-
thelium; but only an occasional: granule could be found in any of the
cells, which are usually loaded with them, when particulate matter has
been absorbed from the peritoneal cavity.

In order to determine the effect of rest after exposure to dextrose the
three rats, saved as controls from the series of animals which had re-
ceived fourteen doses, were killed six, eight and ten days after the last
dose was given. In all of these, sections demonstrated that the meso-
thelium was everywhere absolutely normal in appearance. From this
it was evident that recovery had taken place completely in six days.

aa ei ote ee

eT ee a

ABSORPTION FROM SEROUS CAVITIES 493

DISCUSSION

These experiments do not settle the question of the feasibility of
using the peritoneal cavity as a route for the administration of dex-
trose; but they furnish certain evidence which is of value, both in regard
to this practical application and to the general reactions of the peri-
toneal mesothelium. The experimental results which are favorable for
the use of this method are: the general effect upon the animals, the
nature of the curve of absorption, the rapid recovery of the mesothelium,
and the absence of adhesions. But the changes in the mesothelial
cells can not be considered as directly favorable, and may even prove a
definite contraindication.

The animals suffered no ill effects as far as could be judged from
their activity, general appearance and weight. While the rats were
not weighed every day, their weights were taken before the first injec-
tion, and occasionally thereafter; none were found to have lost weight,
while several actually gained a little. In studying the two curves it
is evident that the length of time required for the complete absorption
of the dextrose solution was about the same in both series of animals;
but the increase in fluid, soon after the injection, was considerably
greater in the first than in the second. From these observations it is
evident that the continued exposure of the surfaces of the peritoneum
did not produce sufficient injury or change to decrease their ability to
absorb this particular solution.

In addition to this conclusion these curves suggest the discussion of
certain general features of absorption, concerning which we have very
little conclusive evidence. Immediately after the injection of the sugar
solution the total amount of fluid in the peritoneal cavity increases,
and this can only be at the expense of the water in the tissues and blood
stream. At the same time we may conclude that the sugar is passing
out of the peritoneal cavity into the blood stream and tissue spaces,
although this has not been established in these experiments by quanti-
tative examination of the fluid. }

In short an equilibrium is being established by osmosis and diffusion
between the two systems: the sugar solution which has been introduced
into the peritoneal cavity, and the contents of blood vessels and tissue
spaces. In considering the absorption of the solution after the equi-
librium has been reached, which is indicated by the highest points of
the curves, it seems evident that some other factor besides osmosis and
diffusion must be involved. The most plausible explanation for this
remarkable absorption, which takes place against the concentration
gradient, seems to be that the sugar molecules are drawn over into the

494 R. S. CUNNINGHAM

blood stream and tissue spaces by means of some difference in electrical —
potential; and this increase in concentration of the dextrose in the blood
forces the water out of the peritoneal cavity. The evidence obtained
from these experiments is insufficient to establish any hypothesis
regarding the nature of this exchange.

The nature of the change in the mesothelium has not yet been estab-
lished, and upon this depends whether or not the reaction of these
cells can be: considered as a definite contraindication to the adminis-—
tration of dextrose via the peritoneal cavity. If the change is funda-
mentally an injury, if the desquamating cells are badly injured or
_ dying, and if those remaining are unable to recover the denuded areas,
then adhesions are likely to form and inflammatory processes to develop,
if any organism should obtain access to the peritoneum. On the other
hand if the change in these cells is the result of stimulation, if the cells
are actively proliferating, if those cells which do separate are viable
and continue life as free macrophages, and if the remaining cells are
capable of renewing the denuded spaces so that only a few cell areas
will be empty at any time, then adhesions should not develop and the
protection against adventitious infection would seem likely to be as
great as ever. Theoretical possibilities must include combinations of
stimulation and injury, in which case any application would depend
upon the ratio between the two.

These questions are most difficult to settle experimentally; the evi-
dence obtained from these experiments tends to support the suggestion
that the changes are due to stimulation. The mesothelium, after ex-
posure to repeated doses of a 10 per cent solution of dextrose during a
period of fourteen days, does not show denuded areas larger than the
space normally occupied by two or three cells, there is no evidence of
injury or death, but rather of active proliferation in these cells, no ad-
hesions have occurred, and recovery, either by the return to normal
of the cells which have rounded up or their replacement by other cells,
is complete in six days. |

The distribution of this change over diaphragm, spleen and omentum,
while the remainder of the peritoneal surfaces is comparatively nor-
mal, would indicate that there is some special differentiation of the
mesothelium in these regions. And it is quite possible that the expla-
nation of the observed reaction should be sought in, this differentiation
rather than in some injurious effect produced on the cells by the dextrose.

BIBLIOGRAPHY

(1) BuackFAN AND Maxcy: Journ. Diseases of Child., 1918, xv, 19.
(2) CunNINGHAM: Anat. Rec., 1920, xviii, 229. |

Ve SO

aro S

INDEX TO VOLUME LIII

ABSORPTION from serous cavities,
488.
Adrenalin and tissue extracts, antag-
onism between, 343, 477.
Alkali reserve in surgical shock, 109.

BERGEIM, 0. See Minter, Bur-
_GEIM, Renruss and Hawk, 65.
Bioluminescence, physico-chemical
studies on, 137.

Blood cells, red, viability of, 1.

—— regeneration following simple ane-
mia and different diets, 151, 167, 206,
236, 263.

—— volume, adjustment of, after in-

jection of isotonic solutions, 323.
Brain volume, effect of salt ingestion
on, 464.

CARBON dioxide, permeability of

eel wall to, 457.

Cardiodynamics, myo- and, 377.

Cardio-vascular system, reciprocal
reactions in, 355.

Cerebro-spinal fluid pressure and brain

. volume, effect of salt ingestion on,
464.

CHILLINGworTH, F. P. See Hopxins
and CHILLINGWORTH, 283.

Cireulation, coronary, mechanical
impairment of, 283.

Coagulation of citrated plasma, chemi-
cal factors in, 25.

Couuip, J. B. Antagonism of depres-
sor action of small doses of adrenalin
by tissue extracts, 477.

Antagonism of inhibitory ac-

_.tion of adrenalin and depression of
cardiac vagus by a constituent of
certain tissue extracts, 343.

495

CunninGHAM, R. §. Studies in pla-
cental permeability. I. The differ-
ential resistance to certain solutions
offered by the placenta in the cat,
439.

——. Studies on absorption from

serous cavities. III. The effect of

dextrose upon the peritoneal meso-

thelium, 488.

J)AVIS, L. H. See Ross and Davis,
391.
Dextrose, effect of, on peritoneal meso-
thelium, 488.

MOTIONAL and metabolic § sta-
bility, 307.

FOLEY, F. B. and T. J. Purnam.
The effect of salt ingestion on
cerebro-spinal fluid pressure and
brain volume, 464.
FRANKLIN, A.C. See Martin, FRANK-
LIN and Hrexp, 421.

(,ASTRIC hunger contractions, 293.

—— response to foods, 65.

—— secretion, influence of sugars and
candies on, 65.

GESELL, R. Further observations on
thé relation of initial length and
initial tension of auricular fiber on
myo- and cardiodynamics, 377.

Growth, influence of alcoholic extract
of thyroid on, 101.

HAMMETT, F.S. Observations on
the relation between emotional
and metabolic stability, 307.
See Harar and Hammett, 312.

——

496

Harat, S. and F. S. Hammett. Four
factors causing changes in the type
of response of the isolated intestinal
segment of the albino rat (Mus nor-
vegicus albinus) to sodium carbo-
nate, 312.

Hawk, P. B. See Mituer, Bercerm,
ReuFvuss and Hawk, 65.

Hemolysin, natural, in rat, sex varia-
tion in, 483.

Hietp, C. See Martin, FRANKLIN
and Hirata, 421.

Hooper, C. W., F. S. Rospscueit and
G. H. Wuippte. Blood regeneration
following simple anemia: III. Influ-
ence of bread and milk, crackermeal,
rice and potato, casein and gliadin
in varying amounts and combina-
tions, 206.

V. The influence of Blaud’s pills
and hemoglobin, 263.

See Wu1prPLe, Hooper and Ros-

SCHEIT, 151, 167.

See WuippLe, RoBSCHEIT and
Hooper, 236.

Hopkins, R. and F. P. CuHILuine-
worTtTH. Physiologic changes pro-
duced by variations in lung dis-
tention. III. Impairment of -the
coronary circulation of the right
ventricle, 283.

Hyman, L. H. Physiological studies
on Planaria. IV. A further study of
oxygen consumption during starva-
tion, 399.

Hyperglycemia from ether,
pancreas in, 391.

Hypophysis and thyroid, functional
correlation of, 89.

a
7

[NTESTINAL absorption, influence
of pituitary extracts on, 43.
—— response to sodium ek AERTS 312.

JACOBS, M. H. The production of
intracellular acidity by - neutral

and alkaline solutions containing —

carbon dioxide, 457.

role of

INDEX

AMBE, H. and E. Komrya. The
transfusion experiment with red
blood corpuscles, 1

Kanpa, S. Physico-chemical studies
in bioluminescence. III. The pro-
duction of light by Luciola vitticollis
is an oxidation, 137.

Karpman, B. Effect of various sub-
stances upon the coagulation of
citrated plasma, 25.

Katz, L. N. See Wiaarrs and Karz,
49.

Komrya, E. See Kamse and Komrya, 1.

LARSON, J. A. Further evidence

on the functional correlation of

the hypophysis and the thyroid, 89.

Lung distention, changes produced by
variations in, 283.

MARTIN, E. G., A. C. FRANKLIN

and C. Piitierss: Vasomotor re-

flexes from receptor stimulation in
intact animals, 421.

MENDEL, L. B. See SmitH and Mzn-
DEL, 323.

Metabolic stability,
307.

Miturer, R. J., O. Bercem, M. E.
ReuFuss and P. B. Hawk. Gastric
response to foods. XIII. The in-
fluence of sugars and candies on gas-
tric secretion, 65.

Myo- and cardiodynamics, 377.

emotional and,

ERVES, accelerator, and ventricu-
lar systole, 49.
Nutritional value of alcohol extract of
thyroid, 101.

XYGEN consumption in starva-
tion, 399.

ANCREAS, rdéle of, in ether hyper-
glycemia, 391.

PattTerRsoN, T.L. Vagus and splanch-
nic influence on the gastric hunger
movements of the frog. Compara-
tive studies III, 293.

a a a ee Ne eee i ay LP r oie

INDEX 497

Peritoneal mesothelium, effect of dex-
trose on, 488.

Permeability of cell wall to carbon
dioxide, 457.

Pituitary extract and intestinal ab-
sorption, 43.

Placental permeability, studies in, 4389.

Planaria, physiological studies on, 399.

Plasma, citrated, chemical factors in
coagulation of, 25.

Polyneuritis, influence of alcoholic ex-
tract of thyroid on, 101.

Putnam, T. J. See Fouiny and Purt-
NAM, 464.

RAYMUND, B. The alkali reserve
in experimental surgical shock,
109.

Rees, M. H. The influence of pitui-
tary extracts on the absorption of
water from the small intestine, 43.

Reuruss, M. E. See Miturer, Berc-
BEIM, ReHFuss and Hawk, 65.

Rosscueit, F. 8. See Hoorrer, Ros-
SCHEIT and WHIPPLE, 206, 263.

—. See WuiprLe, Hooper and
RosscHEIT, 151, 167.

—. See Wuirpte, RosscuHeir and
Hooper, 236.

Rogers, F. T. On the regeneration
of the vagus nerve, 15.

Ross, E. L. and L. H. Davis. The
role of the pancreas in hyperglycemia
from ether, 391.

GALT ingestion, effect of, on cerebro-
spinal fluid pressure and brain
volume, 464.

Seaman, E. C. The influence of an
alcoholic extract of the thyroid
gland upon polyneuritic pigeons and
the metamorphosis of tadpoles, 101.

Sex variation in natural hemolysin in
the rat, 483.

Shock, surgical, alkali reserve in, 109.

Smitu, A. H. and L. B. Menpeu. The
adjustment of blood volume after
injection of isotonic solutions of
varied composition, 323.

Starvation, oxygen consumption in,
399. :

Suzuki, Y. Observations on a sex
difference in the presence of natural —
hemolysin in the rat, 483.

(THYROID, functional correlation of
hypophysis and, 89. ;
——, nutritional value of alcohol ex-
tract of, 101.
Tissue extracts, antagonism between
adrenalin and, 348, 477.

VAGus nerve, regeneration of, 15.

Vasomotor reflexes from receptor stim-
ulation, 421.

Ventricular systole, influence of accel-
erator nerves on duration of, 49.

Viability of red blood cells, 1. °

WHIPPLE, G. H., C. W. Hooper
and F. 8. Roscsuerrit. Blood re-
generation following simple anemia:

I. Mixed diet reaction, 151.

II. Fasting compared with sugar
feeding. Analysis of ‘“‘sparing
action of carbohydrates,’ 167.

——, F. §S. Rosscueir and C. W.
Hoorrer. Blood regeneration fol-
lowing simple anemia. IV. Influ-
ence of meat, liver and various ex-
tractives, alone or combined with
standard diets, 236.

——. See Hoopmr, Rosscueit and
WHIPPLE, 206, 263.

WickwirkE, E. W. Reciprocal reac-
tions in the cardio-vascular system,
355.

Wiaaers, C. J. and L. N. Katz. The
specific influence of the accelerator
nerves on the duration of ventricu-
lar systole, 49.

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THE
AMERICAN JOURNAL

PHYSIOLOGY

VOLUME LIV

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BALTIMORE, MD.
1920-1921

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CONTENTS

No. 1. NovemBer 1, 1920

Tue EFFrectT ON THE COMPOSITION OF THE BLOOD oF MAINTAINING AN IN-
CREASED BLOOD VOLUME BY THE INTRAVENOUS INJECTION OF A HYPER-
TONIC SOLUTION OF Gum ACACIA AND GLUCOSE IN NORMAL, ASPHYXIATED

AND SHOcKED Dogs. H.L. White and Joseph Erlanger................ I
Tue FuncTionaAL ACTIVITY OF THE CAPILLARIES AND VENULES. D. R.

PD Se a a erie Po eT yey GS RE Wh Re ee Nea ee 30

” SruDIES ON THE VISCERAL SENSORY Nervous System. I, LuNG AUTOMATISM

AND Luna REFLEXES.IN THE FROG (R. PIPIENS AND R. CATESBIANA).

A. J. Carlson and A. B. Luckhardt.............. Mei ce f Heed Ga ee aR AR 55
Tue Errect or ADRENALIN ON VENOUS BLOOD Pressure. Helene Connet.. 96
STUDIES ON THE VISCERAL SENSORY NeERVOwUs System. II. Lune Autroma-

TISM AND LUNG REFLEXES IN THE SALAMANDERS (NEcTURUS, AXOLOTL).

me wmurknardt ond A.J Carleen (o.oo ee Ee, COP ad 122
CHANGES IN AcID AND ALKALI TOLERANCE WITH AGE IN PLANARIANS. WITH ;

A NoTE on CataLasE Content, John W. MacArthur............... . 138 |
STUDIES ON THE ALKALINE RESERVE OF THE BLOOD OF THE INSANE. WN Shes

haru Suitsu. Res Ro SNe sh RMR See OP ; WON Ree Co awd Hen 147
GASTRIC Tons’ OF THE Empty STOMACH OF THE nde: COMPARATIVE

Ma Bs ete aieranies or. 6 ada POR he a 153

SruDIES ON THE SUBMAXILLARY GLAND. VI. ON THE DEPENDENCE OF
TissvuE ACTIVITY UPON VOLUME-FLOW OF BLOOD AND ON THE MECHANISM
ConTROLLING THis VOLUME-FLOW OF BLoop. Robert Gesell.......... 166

STUDIES ON THE SUBMAXILLARY GLAND. VII. On THE Errects or INCREASED
SALIVARY PRESSURE ON THE ELECTRICAL DEFLECTIONS, THE VOLUME-
FLOW OF BLOOD AND THE SECRETION OF THE SUBMAXILLARY GLAND OF THE
REE PE MMOLE see ws 5 ss on eih 8 Seale. Sige EA uns Rea WLR tad ope he 185

STUDIES ON THE Sams eay Guano. VIII. On THE poe ada oF ATROPIN
UPON VOLUME-FLOW OF BLOop, ELECTRICAL DEFLECTIONS AND OxIDA-
TIONS OF THE SUBMAXILLARY GLAND. Robert Gesell.................. 204

No. 2. DecemBeER 1, 1920

THE DisTRIBUTION AND QUANTITATIVE ACTION OF THE VAGI AS DETERMINED
BY THE ELECTRICAL CHANGES ARISING IN THE HEART UPON Vaaus STIM-
ey Fe Oe EE, CO PULCKENANK i's 5-5 ace Ded we tae Bik SIL ERE ec ces vie 217
Tue INFLUENCE OF GLANDS WITH INTERNAL SECRETIONS ON THE RESPIRA-
ToRY Excuancre. I. Errect oF THE’ SUBCUTANEOUS INJECTION OF
ADRENALIN ON NoRMAL AND THYROIDECTOMIZED Rapssits. David
Ee TT. LORRATE . s biing bs voice CARI AMD Dee oe Sige A ae ee 1... 248

lv CONTENTS

STUDIES ON THE VISCERAL SENSORY NeERvouwus System. III. Luna Automa-
TISM AND LUNG REFLEXES IN REpTILIa (TURTLES: CHRYSEMYS ELEGANS
AND MsLACOCLEMMYS LESUEURII. SNAKE: EUTENIA ELEGANS). A. J.
Carlson and A. B. Luckhardt.......... Ree sl ged way a ee ee . (eae 261

SruDIes OF THE RESPIRATORY MECHANISM IN CarRpIAC Dyspnea. I. THE
Low ALVEOLAR CarBoN DioxipE or Carpiac Dyspnea. John P.

* Peters, dv.and. Dand.P. Berr.e 55 eRe. Ai ee eee 307

SrubIES OF THE RESPIRATORY MECHANISM IN CarpIAcC Dyspnea. II. A
Nore ON THE EFrrectivE LUNG VOLUME IN CarpIAc DyspNnEA. John
P. Peters; Jri:and Dand Bs Bair cae ie 2s a i a V3 dole 335

STUDIES OF THE RESPIRATORY MECHANISM IN CARDIAC Dyspnea. III. Tue
EFrectTIvE VENTILATION IN CarpIac Dyspnea. D. P. Barr and John P.
Peters, Ff 6 scan eels bs cs ae Ae ae 345

Srupigs ON THE Brain Stem. IV. ON THE RELATION OF THE CEREBRAL
HEMISPHERES AND THALAMUS TO ARTERIAL BLOOD PRESSURE. » FP. * a8
Rogers. . Ai ee WCBAR oi. a¥ Vee 1 le Ue 2 355

iikteaiepneen Sein) IN ‘Dua Series IJ. . Tue INTERNAL PAn-
CREATIC FUNCTION IN RELATION TO Bopy Mass anp Mprapouism. 5.
Tue INFLUENCE OF FEVER AND INToxicatTion. Frederick M. Allen.... 375

EXPERIMENTAL STUDIES 1N DiapetTes. Series II. Tur INTERNAL PAN-
CREATIC FUNCTION IN RELATION To Bopy Mass anp Merasouism. 6.
Gas Baciuuus Inrections In Diasetic Docs. Mary B. Wishart and

LdaiW.. Pritchett) oo). i go-h Aa 5's sts petal «ae eee wees O02
Srupiges IN EXPERIMENTAL TrRAuUMATIC SHock. I. THE Basa Mareatioe

wien): Joseph) Caheubs coches oh sees Ae on oe 388
Strupies In EXPERIMENTAL TRavumMaTIc SHock. II. THe OxycEn CONTENT

oF THE Buoop. Joseph C. Aub and T. Donald Cunningham....... .... 408
Srupies IN ExpERIMENTAL TRAUMATIC SHocK. III. CommMicaL CHANGES

IN THE BLoop.: Joseph C. Aub and Hsien Wu... 2... ck ee ee 416

No. 3. January 1, 1921

EXPERIMENTAL Stupies IN DiaBetes. SERIES II. Tue INTERNAL Pan-
CREATIC FUNCTION IN RELATION TO Bopy Mass AND METABOLISM. 7.
Tue INFLUENCE OF CoLp. Frederick M. Allen. sind tee See

EXPERIMENTAL STUDIES IN DIABETES. SERIES’ ‘eT: “Te Twain ene Pan-
CREATIC FUNCTION IN RELATION TO Bopy Mass anp Merapo.uism. 8,
THE INFLUENCE OF EXTREMES OF AGE UPON THE PRODUCTION oF DIA-
betes. Fréderich M. Allen... ss.0.0c0 eeeeees . a a 2 439

EXPERIMENTAL Stupiges IN Diraspetes. SERIES I]. Tur INTERNAL PAn-
CREATIC Funcrion IN ReLaTion To Bopy Mass anp Mrrapouism. 9.
THE INFLUENCE OF PREGNANCY UPON EXPERIMENTAL DIABETES. Fred- .
erick M. Allen. oo ec nak &. aed coo os eee vagieeees < ad tna oe . 451

RHYTHMICITY OF THE PyLoRic SpHinctTeER. Homer Wheelon and J. Earl
DONE o.oo ss cine gy sinlcin ney © taphe ei impos nb bi: SNROMRD a ptm eet ers at 460
A DIFFERENCE BETWEEN THE MECHANISM OF HYPERGLYCEMIA PRODUCTION —
BY ETHER AND BY CHLOROFORM. Ellison L. Ross and L. H. Davis..... 474
DIGESTIBILITY OF SOME HypROGENATED O1ns. Arthur D. Holmes and Harry
WF DCB FR bs nn a VA OR RR a a woe ewe 6 ee ee
INDIR 05 6 oc SURGE Val eelev aes 0 he es ia GE nea Powel eae Pee

Se

Pretest

THE AMERICAN
JOURNAL OF PHYSIOLOGY

VOL. 54 NOVEMBER 1, 1920 No. 1

THE EFFECT ON THE COMPOSITION OF THE BLOOD OF
MAINTAINING AN INCREASED BLOOD VOLUME BY THE
INTRAVENOUS INJECTION OF A HYPERTONIC SOLUTION
OF GUM ACACIA AND GLUCOSE IN NORMAL, ASPHYXI-
ATED AND SHOCKED DOGS

H. L. WHITE and JOSEPH ERLANGER
From the Physiological Department of Washington University, St. Louis

Received for publication July 3, 1920

The present work was undertaken with the idea of investigating the
effect upon the composition of the blood of producing and then main-
taining for some hours an expansion of the blood volume through the
intravenous injection of a hypertonic crystalloid and colloid in com-
bined solution, in the hope of throwing some light on the mechanism of
the processes involved. The solution injected consisted of 18 per cent
glucose and 25 per cent gum acacia and is the one that has been found
useful in the treatment of traumatic shock (1). Studies of the blood
changes produced were carried out under conditions which were varied
in a way that might change the permeability of the vessel walls to the

~ tissue fluids and their various constituents.

The literature contains many references to studies, more or less com-
plete, of the blood changes following the intravenous injection of crys-
talloids, glucose, NaCl, Na.SO, and urea being the substances most
commonly used. Brasol (2) found that following intravenous injection
of a hypertonic glucose solution the blood was greatly diluted in 2
minutes but that the blood volume had returned to normal within 2
hours. He found that there was no relation between the amount of
glucose injected and the per cent of glucose found in the blood 2 minutes
later, that the blood sugar was normal 2 hours after the injection, that
part of the sugar which leaves the blood can be found in the tissues, but

1

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

2 ; H. L. WHITE AND JOSEPH ERLANGER

part cannot be found and is perhaps changed to glycogen, lactic acid or
“undergoes some other chemical metamorphosis,” and that the abso-
lute quantity of protein in the serum remains unchanged. He con-
cludes that the effects produced are purely the results of the increased
intravascular osmotic tension due to the injected glucose. Klicko-
‘wicz (3) obtained similar results as regards blood volume with strong —
solutions of Na,SO,. Leathes (4) finds that the “‘increase in the volume
of the blood caused by the injection of 5 grams of dextrose per kilo is
enormous and quite out of proportion to that caused by the volume of —
the injection. This increase takes place with remarkable rapidity; the
volume of the blood is nearly doubled by the time the injection is fin-
ished.’’ Gasser and Erlanger (5) followed the blood volume after in-
travenous injection of 5 cc. of 18 per cent glucose solution per kilo body
weight, the injection being made as rapidly as possible. They found
that the maximum dilution was attained within 0.5 to 2 minutes and
that the blood then began to concentrate, rapidly at first, and then more
slowly, and became normal within 5 to 45 minutes. Starling (6) finds
_ hydremic plethora following glucose injection and that the distended
vessels begin at once to unload the excess of water. Paton (7) finds
that when 4 grams of carbohydrate in 10 ee. of water per kilo body weight
are injected intravenously only a small per cent of the amount injected
can be recovered immediately from the blood, the time from the be-
ginning of the injection to the end of collecting the sample being 90 to —
100 seconds. Hamburger (8) finds that in the horse an intravenous in-
‘jection of 7 liters of 5 per cent Na2SO, causes a marked dilution of blood
serum in respect to its chloride and protein content, a return to normal
being accomplished very quickly, within 2 hours. Practically the same
effect on protein content of serum follows the injection of 5 liters of
0.5 per cent Na,SO, solution, while the chlorides are diluted as with a
hypertonic solution but do not return to normal. The blood volume~
was not followed in these cases, so no conclusions can be drawn as to
whether the return to normal was due to a passage of water out of the
blood stream or an entrance of solids into the blood stream. Fisher and
Wishart (9) found that blood dilutes as a result of alimentary hyper-
glycemia. Magnus (10) obtains varying results in his studies of changes
in blood composition following the intravenous injection of NaCl solu-_
tions, the results depending upon the osmotic tension of the injected
solution, although consistent results were not obtained with hypertonic
‘solutions. |

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BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION © 3

The common feature of the experiments discussed above is that the
substances injected were crystalloids and, with the exception of those
of Fisher and Wishart, the injection was rapid; and the increase in
blood volume, with hypertonic solutions, always is very rapid in ap-
pearance and of short duration. Woodyatt, Sansum and Wilder (11)
injected glucose intravenously over long periods of time at a uniform
rate and determined the tolerance rate as 0.8 to 0.9 gram per kilo per
hour. Erlanger and Woodyatt (12) injected glucose intravenously at
uniform rates varying between 0.57 and 4 grams per kilo per hour for
from 20 to 60 minutes into anesthetized animals in shock. The pulse
amplitude was uniformly markedly increased, indicating a condition of
plethora. A subtolerant dose was as effective as injections made at
more rapid rates. In a normal animal an injection lasting 30 minutes
at the rate of 1.78 gram per kilo per hour raised arterial pressure only

about 5 mm. Hg. but the pulse amplitude increased quite appreciably.

The fact that a considerable increase in blood volume is accompanied
by only a slight rise in arterial pressure is presumably due to diminu-
tion in viscosity of the blood:and to vasomotor accommodation. While
the blood volume was not followed in these cases it presumably soon
fell after cessation of injection to its pre-injection state.

With regard to the entrance of protein into the circulation, Morawitz

(13) found restoration of proteins most active in the first few hours

after severe hemorrhage. Whipple and co-workers (14) find that after
severe hemorrhage the regeneration of plasma proteins is much more
rapid on a liberal high protein diet than on a liberal bread and milk
diet, and least rapid on fasting. They infer from this that there is an
actual new formation of protein and make no mention of the possi-
bility of some of the protein being drawn in from the tissue fluids.
They also find that ‘after the initial depletion of serum proteins the
body can almost always regenerate 1 per cent of the total protein during
the first 24 hours following the plasmapharesis. This figure is remark-
ably constant and does not seem to be influenced by diet or fasting

: or the amount of shock. We may perhaps look upon
ee as the maximum effort of the body to replace these essential serum
proteins, an effort which surely depends upon the body protein as it is

_ present in fasting experiments. It will be the same whether there is a
marked breakdown of host protein, as evidenced by great increase in

urinary nitrogen, or whether this tissue autolysis is minimal.’’. That

_ they do not regard this maximum effort as accomplished by the passage

of protein directly into the blood from the tissues but rather as a true

4 H. L. WHITE AND JOSEPH ERLANGER

rebuilding is shown by their further statement that it “‘ probably repre-
sents the absolute maximum production under the greatest stimulus.”
They present further evidence that the restoration of plasma proteins
after hemorrhage is due to a new formation of proteins and that the
liver is the chief seat of this process in that restoration is greatly hin-
dered by phosphorus and chloroform poisoning and by an Eck fistula.

A rather careful review of the literature has failed to disclose any
references to studies of the body fluids made during a period of pro-
longed increase of blood volume due to the injection of hypertonic
solutions. It is evident that only two means are at our disposal for
maintaining an increased blood volume for a comparatively long period,
several hours for instance; with fluids other than blood or blood plasma.
One is by a continuous injection of a hypertonic or isotonic solution of
crystalloids at a rate more rapid than the rate of the blood’s fluid loss.
In this case the large amount of fluid injected would so dilute the blood
that no conclusions could be drawn as to the interchange of water and
solutes between tissue spaces and blood stream. The other method is to
inject a small volume of a strongly hypertonic solution which will not
only draw fluid into the blood stream but hold it there for some time.
To accomplish this result a solution containing both crystalloid and col-
loid is necessary (15). So far as we know the present work represents
the first effort made to study changes in the composition of the blood
induced in this way.

PROCEDURE

Dogs were used in all experiments. The animals were not prepared
by controlling their intake of food or water previous to the experiment.
Most of them were fed and watered on the morning of the experiment.
In the first seven cases the animal was given 1 grain of morphine an
hour before starting the operation; in the case of dog 8, 2 grains were
given by mistake. In the remainder of the cases the morphine was
omitted because of its disturbing effect on the blood and urinary sugar.
In the first four cases strict surgical asepsis was observed throughout
the experiment; in the remainder these precautions were relaxed, al-
though the fluid injected and the injecting apparatus were sterile. No
differences in results due to these technical differences were observed.
Three sets of experiments were performed; the first, a series of nine
experiments, on normal dogs; the second, a series of two, on asphyxiated
dogs; the third, a series of three, on dogs in shock. The procedure and
data of each series are presented separately.

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 5

The procedure in the first series was to anesthetize the dog with ether,
immediately draw a ‘‘standard” sample of blood from the femoral ar-
tery, then to start the injection into the femoral vein. The dose in all
cases was 5 cc. per kilo per hour of an 18 per cent glucose and 25 per cent
gum. acacia solution, the injection lasting 1 hour and proceeding at a
uniform rate. Immediately at the termination of the injection another
sample of blood was taken and the dog put back into its cage. Subse-
quent samples were taken at approximately 2-hour intervals until five
samples were obtained. Little or no anesthetic was required while
drawing the last three samples. On each sample determinations were
made of hemoglobin (16), plasma chlorides (17), plasma total nitrogen
and plasma non-protein nitrogen (18), and plasma sugar (18). The
nitrogen and chloride determinations were made in duplicate. Direct
Nesslerization was at first attempted for both the total and the non-
protein nitrogen determinations, appropriate dilutions being made, but
it was found that the presence of the large amount of carbohydrate in
the samples rendered digestion with the prescribed digestion mixture
so difficult that indirect Nesslerization had to be resorted to. In the
latter cases of the series the total nitrogen determinations were made by
the macro-Kjeldahl method since it was felt that the micro-Kjeldahl
with indirect Nesslerization was not sufficienty trustworthy. Figures
given in the tables under the heading ‘total N” are protein nitrogen
figures and are obtained by subtracting the non-protein nitrogen figure
from the total nitrogen figure as determined by the Kjeldahl. Several
dilutions of the filtrate for plasma sugar determinations were made,
the d lution being accepted whose reading was closest to 20, the standard
being set at 20 on the Dubosq or Bock-Benedict colorimeter. Freezing
point determinations were attempted in the first experiment, working
with 1.5-cc. of hirudinized plasma in a special tube in the Beckmann
apparatus, but it was found that consistent results could not be obtained
with this amount of plasma and these determinations were discontinued
as it seemed inadvisable to take more blood. In lieu of direct determi-
nations the crystalloid osmotic pressure of the plasma has been roughly
estimated by adding the osmotic pressures exerted by the NaCl and
glucose present. The calculations were made from published tables of
direct osmotic pressure determinations and of freezing point determi-
nations. By using various schemes for utilizing every available drop
of plasma it was possible to make all determinations with 13 cc. of
blood drawn at each sample. Allowance for blood drawn as samples

has been made in the calculations. Dogs 8 and 9 were bled 15 per cent

6 H. L. WHITE AND JOSEPH ERLANGER

of their blood volume at the time of taking the first sample. In the
last few cases the urine was also followed, the bladder being emptied as
completely as possible with a small coudé catheter at the time of draw-
ing the blood samples. The results of the first series are given in table 1.

The procedure in the second series was to anesthetize the dog with
ether (morphine being omitted in these and subsequent experiments),
insert a tracheal cannula, draw a standard sample of blood from the
femoral artery and empty the bladder. The animal was then made to
rebreathe air in a large spirometer until the CO, tension of the air in it
mounted to about 30 mm. Hg. as determined with Marriott’s (19) tubes
on samples drawn from the spirometer. The injection was then started,
the CO; tension being kept around 30 to 35 mm. Hg., fresh air being
admitted at intervals when the dog became markedly dyspneic. Dys-
pnea was moderate to marked throughout the injection. This procedure
was introduced in an effort to discover whether asphyxia altered the per-
meability of the vessel walls. At the conclusion of the injection the
second sample of blood and urine was drawn, rebreathing discontinued,
the animal put back into its cage and subsequent samples taken as in
the first series. The results of the second series are given in table 2

In the case of dog 10 the injection rate was not accurately timed, sek
first. Twenty-five cubic centimeters were run in during the first 20
minutes. The injection was then stopped for 10 minutes, the remainder
of the injection then proceeding at the proper rate. When the last
sample was drawn from dog 10 he struggled quite vigorously and it
was necessary to anesthetize him deeply, which is not usually necessary
for the last three samples. This anesthesia probably accounts for the
high blood sugar value in the last sample of dog 10.

The procedure in the third series was to anesthetize the animal, insert
a tracheal cannula and connect a manometer with a femoral artery,

draw a standard sample and then put the animal into a condition of —
shock. The condition induced was the so-called mechanical shock of

Janeway and Jackson (20), which they produced by temporary partial
occlusion of the vena cava regulated so as to keep the blood pressure at
30 to 40 mm. Hg. for 2 hours. The method of obstructing the cava

used in the present work was one described by Erlanger and Gasser |

(21), a clamp being used by which graded compression can be exerted
upon the cava between the liver and the diaphragm through a small
abdominal incision. The mean arterial pressure was kept at about 40
mm. Hg..for 120 to 135 minutes. At the end of this time the clamp was
removed, the second sample drawn, the injection given, a third sample

=.

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 7

then taken, and the dog kept as long as he would live, samples being |

drawn at about 2- or 24-hour intervals. Very little anesthetic was

required to keep the dog quiet after shock had been produced. This |

procedure was employed in an endeavor to ascertain whether the per-
meability of the vessel walls is altered in shock.

The results of the third series are given in table 3 and in the brief |

ease histories.

DISCUSSION OF RESULTS

Series I. Normal Dogs

1. Blood volume. It is evident that in all cases there is a marked |
immediate increase in blood volume, amounting to 11 to 16 per cent, |

which usually slowly returns toward normal. Some slight increase in

volume, about 2 per cent, usually persists even after 6 or 7 hours. The -

combined action of the glucose and acacia in effecting changes in blood
volume is interesting. The early high intravascular osmotic tension

which causes the rapid increase in volume is due principally to the glucose. -

The glucose, however, rapidly passes out into the tissue spaces (see
plasma sugar figures) but through the influence of the gum acacia the
water very slowly leaves the vessels. The persistent increase of blood
volume, 2 or more per cent, is less than the amount of water which must
be added to the amount of acacia presumably remaining in the blood
(22) in order to make it isosmotic to the plasma proteins. It will be
noticed, however, that in every case but one, dog 3, where the figures
are the same, the total amount of plasma proteins at the close of the
experiment is less than that at the start. Part of the injected acacia,
then, presumably is taking the place of the removed plasma protein
in holding water, and only the remainder can be employed in hold-
ing an excess of water above normal blood volume. Quantitative

determinations of acacia in the blood would be necessary in order to.

ascertain just how much acacia is available for maintaining an increased
blood volume. Any fraction of the persisting increase in intravascular
osmotic tension which might be due to the glucose could not play any
part in maintaining the increase in blood volume, since glucose now
exists in the tissue spaces in equal concentration, having passed out
from the blood stream, and is exerting an equal attraction there. In
fact, the blood sugar figures show that the excess of glucose has been
entirely removed from the blood stream, and presumably from the
tissue fluids, inside of 2 hours. The fate of the glucose will be discussed

_ Jater.

PA

H. L. WHITE AND JOSEPH ERLANGER

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10 H: L. WHITE AND JOSEPH BRLANGER >

The rather wide variations in the rate of the falling off from the initial
increase in blood volume possibly are to be attributed to variations in
vasomotor accommodation and in the preexisting water content of the
tissue spaces. Scott (23) finds the hemoglobin content increased im-
mediately by a rise in blood pressure produced by various procedures,
and lowered by a fall in pressure. He cites evidence indicating that the
hemoglobin method gives an accurate picture of relative blood volume;
and, as he says, ‘‘these results can only be explained by increased pres-

sure forcing fluid out of the blood to the tissue spaces and the passage —

of fluid back from the tissue spaces to the blood when the pressure is
lowered.”’ He did not determine the nature of the interchanged fluid.
At the time of taking the first sample dog 8 was bled approximately
15 per cent of his calculated blood volume, 15 per cent having been about
the average initial increase in blood volume following the injection.
This was done in order to ascertain whether the interchange of liquids
and solids between the blood and the tissues is modified when the pleth-
ora that is induced by the injection of a hypertonic solution is prevented
by preyious hemorrhage. Blood volume figures indicate that the liquid
interchange is about as in unbled animals. The question of the depar-
ture of the behavior of the solids in this animal from the behavior of the
interchanges of solids in animals without previous hemorrhage will be
considered below. In the case of dog 9 the 15 per cent depletion of the
blood is more than made up by the time of the termination of the
injection (blood volume raised to 106 per cent of normal); this volume
falls in 2 hours to 97 per cent, then very slowly to reach 93 per cent at
the end of 7 hours. The gum acacia is in all probability filling the place
of the removed plasma proteins in holding water. ;

2. Blood composition. The main point of interest is, What is the
nature of the fluid drawn into the blood stream and how does its com-

position change subsequently? A satisfactory answer to these questions |

should serve to elucidate the processes determining the interchanges.

a. Protein. Any conclusion as to the passage of protein into or out
of the blood stream must be based on calculations of the absolute amount
of plasma protein in the entire blood stream. If, for instance, it should
be found that the curve representing the percentage of plasma protein
ran parallel to the curve representing the reciprocal of blood volume,
this would not mean that the absolute protein content of the plasma
was unchanged, since practically all of the fluid drawn into the blood
stream enters the plasma and an increase of 15 per cent in blood volume
would mean an increase of over 23 per cent in plasma volume. The

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 11

method used here of calculating absolute amounts of plasma protein is

essentially that used by Magnus (10), some changes in the method being
introduced in order to allow for the hemoglobin withdrawn in the
samples. It assumes that the blood is 9 per cent of the body weight (24),
(25), (26), this figure being used instead of 7 per cent used by Magnus,
that the blood is 64 per cent plasma by volume and that all the water
drawn into the blood stream enters the plasma. While none of these
three assumptions is strictly correct, the first and third are very nearly
so and variations in the second will not involve gross errors since the
calculations for the additional samples of any one animal are based upon
the plasma volume figure obtained in the calculation of the standard.
blood of that animal. A sample calculation may. be given for purposes
of illustration.

Sample calculation. Dog 7. Body weight, 9.1 kilos

HEMOGLOBIN TOTAL NITROGEN*
Semplenumber| Reading | Vumenereant| Volumepercent | Sample | Grane N pet
1 20.0 —100 100 mea | 0.83
2 22.6 113 111 2 0.74
3 22.0 110 107. 3 0.76
4 21.4 107 102 4 0.77
5 21.0 105 98. 5 0.80

* The terms “‘N”’ or “Total N”’ refer to protein N, i.e., the non-protein N has

_ been subtracted from the Kjeldahl figure.

Method of correcting blood volume for hemoglobin withdrawn. For each sample
13 cc. of blood are removed. This is 1.6 per cent of the blood volume of this
animal, or 1.6 per cent of 819, which is 0.09 X 9100. Since, after the first sample,
only 98.4 per cent of the original amount of hemoglobin remains in the blood
stream the increase in volume of the blood is only 98.4 per cent of that indi-
cated by the blood’s dilution as shown by the hemoglobin readings in the
colorimeter. To illustrate, assume that 5 per cent of blood and therefore of
hemoglobin is withdrawn from a normal dog, assume that the normal or standard
sample reads 20 in the colorimeter, as in our case, and assume further that a
second sample drawn subsequent to the withdrawal of 5 per cent blood volume
reads 21 in the colorimeter. If we failed to consider the previous loss of the
5 per cent hemoglobin we would infer from this reading that the blood volume
had increased 5 per cent, was now 105 per cent of the normal. What has actu-
ally happened, however, is that the blood volume has been replaced just to its
original state, the diluent being of course hemoglobin-free, so that each unit

volume of blood now contains only 95 per cent of its normal amount of hemoglobin.

To find the true blood volume we should take 95 per cent of 105 or 100 per cent,

12 H. L. WHITE AND JOSEPH ERLANGER

in round numbers. To find the true blood volume in any case, therefore, we
should multiply the apparent blood volume by the percentage figure of hemo-
globin remaining in the blood stream at the time of drawing the sample.

Upon the basis of this treatment of the data we find that in dog 7the
blood volume at the time of drawing the last sample is actually slightly
decreased rather than increased, as the hemoglobin readings would
seem to indicate, i.e., the decrease in the percentage of hemoglobin in
the last sample is not great enough to account for all the hemoglobin
removed. We must assume, therefore, that the blood volume has
fallen slightly. The decrease, however, is not as great as the volume
lost by hemorrhage. This means that some of the fluid drawn in has
stayed in but not quite enough to equal the amount lost by hemorrhage.
In all the other normal animals the final blood volume even after cor-
rection for the withdrawal of blood was slightly above the initial or
normal volume.

Using the corrected blood volume figures we may proceed with the method of
calculating total plasma nitrogen. It may be noted here that the figures for
blood volume per cent given in all the tables have been corrected for the amount
of blood lost according to the method outlined above.

0.09 X 9100 = 819 cc., blood volume at beginning, blood 1.

0.64 X 819 = 524 cc., plasma 1.

0.88 X 5.24 = 4.35 grams N in plasma 1. ‘

Blood 2 is 111 per cent of blood 1, by volume. The 111 per cent increase has
been in the plasma. 0.11 X 819 = 90 cc. increase.

Plasma 1 = 524 cc. 524+ 90 = 614 cc., plasma 2.

0.74 X 6.14 = 4.54 grams N in plasma 2.

Blood 8 is 107 per cent of blood 1. The 7 per cent increase en been in the
plasma. 0.07 X 819 = 57 cc. 524 + 57 = 581 cc., plasma 3.

0.76 X 5.81 = 4.42 grams N in plasma 3.

By the same method we get 0.77 X 5.40 = 4.16 grams N in plasma 4.

0.80 X 5.08 = 4.06 grams N in plasma 5.

These figures indicate that by the time sample 5 was drawn protein had
passed out of the blood stream, i.e., less protein is in the plasma than was there
at the beginning. If, however, we add to each plasma N figure the amount of
plasma N taken out in that sample and in preceding samples we get the following
figures: The amount of N withdrawn with each sample is 0.64 < 13 X 0.008 = 0.07
gm. N, 0.008 being taken as the average amount of N per cc. plasma in this ani-
mal. Variations in different samples are not significant in calculations on 18 ce.
The effect is of course cumulative; 0.14 gm. will have been removed by the time
the third sample is drawn, 0.21 gm. by the time of the fourth, etc.

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BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 13
PROTEIN N
SAMPLE NUMBER
N remaining N withdrawn Total protein N

grams gram grams
1 4.35 0.0 4.35
2 4.54 0.07 4.61
3 4.42 0.14 4.56
4 4.16 0.21 4.37
5 4.06 0.28 4.34

_ These figures show that there is at first a slight increase in the amount
of plasma protein but that by the time of the fifth sample this has dis-
appeared, and that the final total plasma protein N is the same as the
original figure if the amount withdrawn is added. Different animals
show some variations from this behavior.

The figures obtained on the dogs without hemorrhage are not con-
stant. All show that there is a marked decrease in the concentration
of plasma protein accompanying the increased blood volume; but two
cases, dogs 3 and 7, show that this decrease is not quite so great as the
increase in plasma volume, i.e., a slight amount of protein enters the
blood stream; while two others, dogs 4 and 6, indicate that a slight
amount of protein passes out of the blood. Of these cases, however,

the figures for dog 7 are the most reliable, since they were obtained by

Kjeldahl determinations, using 2 cc. of plasma, while the others were
obtained by indirect Nesslerization, working with 0.05 ec. of plasma.
While the figures are not convincing, it appears that there is a slight in-
crease in the absolute amount of plasma protein immediately after the
injections. If the amounts of protein withdrawn in taking the samples

“are added to the amounts remaining we find a greater, though still quite
slight, increase and that the amount of plasma protein tends to become

constant in most cases at a level slightly above or about the same as
the original one.

Scott (27) showed that dilution of the blood in vitro with isotonic
Ringer’s solution causes protein to pass from the blood cells into the
plasma. He uses this observation to account for the increase in total
plasma protein following hemorrhage or the injection of isotonic Ringer’s
solution (28). By inferring that slight variations from the proper pro-
portions of the salts in the fluid added to the blood greatly inhibit this
transfer of protein, he accounts for the greater increase in plasma
protein after hemorrhage.

14 H. L. WHITE AND JOSEPH ERLANGER

In order to determine whether or not the increase in total plasma pro-
tein which we found could be due to a passage of protein from blood
cells to plasma the following experiment was performed: From an 8
kilo dog 140 cc. of blood were drawn into a vessel containing the mini-
mum amount, 0.12 gm. per 100 ce. blood, of potassium oxalate that
would prevent clotting. This was stirred and 45 cc. samples immedi-
ately put into each of three cylinders. All three were stirred continu-
ously while to one was added 2.5 ce. of the gum-glucose solution at a
constant rate over 40 minutes, to the second 2.5 cc. of gum-glucose solu-
tion + 5 ec. Ringer’s solution with the calcium omitted; the third was
merely stirred and used as a control. The second of the above men-
tioned procedures may be assumed to approximate the conditions ob-
taining in the intravenous injection in vivo of 5 ce. of the gum-glucose
solution per kilo body weight or per 90 cc: blood, the 5 cc. Ringer’s per
45 ec. blood being the average increase in blood volume over the amount
of fluid injected. After the addition of the solutions was completed
hematocrit determinations were made on each sample and Kjeldahl
nitrogen determinations made on each plasma. :

eve eee { Pas ce IN enviar:
Total Cells Plasma RLOON
ce. - gram
l (46.66. Blood) oo ee eee, Bis Eo a 6.5 27.4 0.259
2 (45 ce. blood + 2.5 cc. g.g.)......... 11.0} 4.1 6.9 28.2 0.260
3 (45 ec. blood + 2.5 cc. g.g. + 5 ee.
PRAT Bh ic ie tics Suk tain oan Aneta 11.4} 3.6 7.8 30.8 0.257

The results collected in the accompanying table show that there was

no exchange of protein between cells and plasma. It seems fair, there-
fore, to eliminate the blood cells as a source of the increased plasma
protein in our 7n vivo experiments.

Kjeldahl determinations were also made on the gum-glucose cud in
order to determine if it might be a source of N. It was found that 2
ec. of the solution is equivalent to 0.6 cc. tenth normal HCl. Expressed
in percentage of N our determination shows 0.168 per cent N in the gum
- acacia or 0.042 per cent N in the solution injected. Rideal (29) finds
gum acacia to contain only 0.031 to 0.082 per cent N. Other observers

have reported varying percentages of N, depending upon the source of
the gum. The form in which the N exists has never been determined,
so far as we have been able to learn. The amount of the solution used

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 15.

for a 10 kilo dog, 50 cc., would contain only 0.021 gm. N, according to
our determinations. Corpuscles and injected solution may therefore
be disregarded as a source of the increase in plasma N. Our results
lead us to the conclusion, therefore, that the injection of the acacia-
glucose solution leads in some way to the entrance of protein into the
circulation.

The changes in the percentage concentration of plasma protein fol-
lowing the injection are also of interest in that they indicate roughly the
changes in the colloidal osmotic tension of the blood. It is seen that
the concentration falls markedly immediately after the injection, consid- _
erably more than can be accounted for by the blood’s dilution due to
the injected fluid alone. Let us assume that each 5 cc. of the injected
solution contains 4.5 cc. of water. Since 4.5 cc. of water are injected
per kilo body weight or per 90 cc. of blood or per 58 cc. of plasma, the
plasma is diluted 7.8 per cent by the water injected. The plasma pro-
tein percentage, however, invariably falls more than this, averaging
about 15 per cent, showing that not only the fluid injected but the fluid
drawn in from the tissues dilutes the plasma in respect to its protein
content. The plasma protein content in the subsequent hours rises
toward normal but never reaches it, this state of affairs co-existing with
a slight persisting increase in blood volume. ‘The assumption seems jus-
tified that part of the colloidal osmotic tension of the plasma is being
supplied by the injected gum acacia.

Magnus reports two experiments with intravenous injection of con-
centrated (85 per cent) NaCl solution. In one case he finds the total
plasma proteins diminished by 7 per cent by this procedure, in the
other case increased by 5 per cent. These were on previously unbled
dogs and the results are comparable to our results on previously unbled
dogs.

Dog 8, which was bled 15 per cent of his blood volume before the injec-
tion, shows a decrease in total plasma protein at the second sample.
When, however, the amount of plasma protein withdrawn by hemor-
rhage is added to the total amount in the blood at the time the second
sample was drawn we find a slight increase in the plasma protein—some
protein has passed in.

b.. Sugar. It is seen that the initial blood sugar values in the first
8 cases are very high, 0.192 to 0.308 gm. per 100 ce. This is due to the
well known action of morphine as a respiratory depressant, the imper-
fect respiration causing a hyperglycemia. At the close of the injection
the figure is still higher, 0.247 to 0.564 gm. per 100 ec., due to the in-

16 H. L. WHITE AND JOSEPH ERLANGER

jected glucose. Within 2 hours the figure has usually fallen to or below
the initial value. The sugar figures on dog 9 are much the most valu-
able since here the disturbing effect of the morphine is-not present.
The initial figure is 0.133, the slight increase above normal probably
being due to the ether; the figure immediately at the termination of the
injection is 0.292; 2 hours later, 0.163; 2 hours later, 0.133; and the
last sample, 2 hours after this, contains 0.135 gm.. These figures show
that the sugar rapidly passes out of the blood, and is almost back to
normal in 2 hours. The fate of the sugar will be considered later.

Since the increased blood volume persists for several hours after the |
blood sugar has returned to normal it must be concluded that gum
acacia remains in the circulation and holds water there. The great
difficulty of digestion for nitrogen determinations of all the samples
taken subsequent to the injection, even those which had regained a nor-
mal blood sugar value, indicates that there was an abnormally large
amount of carbonaceous material in the blood and confirms the view
that gum acacia remains in the blood for some time. ;

c. Plasma chlorides. The plasma chlorides were followed because it
was felt that their behavior under the conditions of the experiment
would be typical of that of the inorganic crystalloids in general. The
percentage concentration of plasma chlorides always falls in the second
sample, but not in any way to the same extent that the blood is diluted;
in fact, the percentage fall in concentration is not even as great as the
percentage of dilution of plasma accomplished by the injected fluid. It
has been seen above that the plasma is diluted about 7.8 per cent by
the injected fluid. The percentage fall of plasma chloride concentration
is less than this, averaging about 6 per cent. In other words, the fluid
drawn into the blood stream carries with it chlorides in concentration
equal to that of plasma and in addition to this inward filtration of chlor-
ides an inward diffusion has started to supply chlorides for the injected
fluid. This diffusion continues for several hours, the percentage of
plasma chlorides steadily rising and becoming normal in 6 to 8 hours.

d. Non-protein nitrogen. It will be noted that the plasma N.P.N.
concentration remains constant with variations in plasma volume, in-
dicating that the vessel walls are more permeable to urea than to NaCl.
The constant concentration of N.P.N. means that not only does the
fluid drawn in have a N.P.N. concentration equal to that of plasma but
also that the diffusion into the blood stream of the N.P.N. necessary to
make up the injected fluid to the concentration of N.P.N. existing in
plasma is very rapid, being completed by the time of completion of the

et i Meee

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 1%5

injection. Urea’s greater solubility in lipoids, which are assumed to be
present in cell walls, may account for this. An alternative view is that.
the urea does not diffuse into the blood stream but is secreted into the

_ blood stream at the proper rate to maintain a constant concentration. -

Our methods and data do not afford us any means of deciding which of

- these processes actually occurs but the former seems the more probable: -

e. Rather rough estimations of the crystalloid osmotic tension of the
plasma samples, using the method of calculation mentioned above, show
that while there may be some attempt on the part of the organism to
keep this tension constant, the compensatory mechanism, if such exists,
is not adequate to keep pace with the factors tending to vary the osmotic

tension.

8. Urine. When morphine had been given sugar always appeared in
the urine in rather high percentage, 5 to 6 per cent. In two cases, dogs
6 and 8, a marked diuresis resulted; in two others, dogs 5 and 7, urinary
secretion was practically normal in volume. Dog 9, which had no mor-
phine, showed only a trace of sugar and his volume excretion was nor-:
mal or slightly increased. As to the fate of the injected glucose, it is.
evident that it does not stay in the blood and that only a small fraction.
is excreted in the urine, in fact only a trace when morphine is omitted.
It will be noted that the injection is so timed that the anesthetized ani-
mals receive glucose at the rate of 0.9 gm. per kilo per hour, the un-
anesthetized dog’s tolerance rate (11). No study of the tissue fluids or. .
glycogen depots was made but we may conclude with Brasol that. the -
remainder is ‘‘perhaps changed to glycogen, lactic acid or undergoes
some other chemical metamorphosis.”’. ‘The observation of Fisher and
Wishart (9) that the metabolism is increased 20 per cent accounts for
a part of it. : 6

It is evident, confirming the finding of Meek and Gasser (22), that
the gum acacia injected with the glucose does not diminish urinary
secretion, as is maintained by Kruse (30); in fact in the cases where.
hyperglycemia was extreme, with blood sugar values around 0.500 gm.
per 100 cc., glycosuria occurred with an accompanying diuresis. Two
cases with hyperglycemia and glycosuria, dogs 5 and 7, while not ex-.
hibiting a diuresis, certainly did not show.a suppression of urine. The:
urine was not followed in the first four cases. In the case of dog 9,..

_ where morphine was omitted with the result that hyperglycemia was.

not so marked and no significant glycosuria occurred, excretion. rate .
of water was normal or slightly increased. Knowlton (31) finds that:!
colloids exerting osmotic pressure, such as gum acacia, inhibit NaCl

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 1

18 H. L. WHITE AND JOSEPH ERLANGER

diuresis but have but little effect on Na,SO, diuresis. Glucose diuresis
belongs to the same class as that of NaCl, i.e., it is presumably due to
purely mechanical factors, such as hydremic plethora, rather than to a
direct action on the kidney cells. We might explain, then, the normal
excretion of urine following the injection of the gum-glucose solution,
in the absence of morphine, as due to a balance arrived at between the
inhibitory action of the acacia and the diuretic action of the glucose,
the later gaining the upper hand when, due to morphine, a marked
hyperglycemia is produced with a resultant glycosuria.

SERIES II. ASPHYXIATED DOGS

There is evidence that asphyxia increases the permeability of the
vessel walls. Bolton (32) holds that permeability is increased by stag-
nation of blood with decreased O, supply, since edema of the neck is
produced by ligature of the superior vena cava, despite the absence of
any rise in pressure in the jugulars and presumably in the capillaries.
Starling (33) also draws the conclusion that the edema under these con-
ditions must be due to increased permeability of walls which allows
protein to pass out into the tissues and hold water there.

The present experiments on asphyxiated animals were carried out
with these statements in mind. Study of the data (table 2) shows that,
owing probably to the asphyxia, the blood sugar figures are very high,
0.133 and 0.244 before and 0.800 and 0.770 immediately after the injec-
tion. In both cases the value 2 hours after the injection was below the
initial value, the asphyxia having long since worn off. Except for this
no significant departure from the behavior in normal animals occurs.
The asphyxia introduces so many complicating circulatory factors,
however, that we are not justified in concluding that the permeability
_ of the vessel walls is unchanged, although we have no proof that it is.
For example, protein might have been drawn in but promptly squeezed
out again due to the asphyxial rise in blood pressure. It will be noted
that even without morphine here the hyperglycemia is extreme and the
glycosuria marked but that the blood sugar rapidly falls to or below the
normal. The apparent continuance of the glycosuria after the blood
sugar has fallen to normal is probably due to the fact that the bladder
was not completely emptied at each catheterization, urine containing
sugar which had really been excreted during the period of hyperglycemia
being obtained at each sample.

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BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 21

SERIES III. SHOCKED DOGS

Dog 12. Body weight, 14.36 kgm. No morphine. Specimens from pancreas,
liver, spleen, large and small intestine and kidney for histological study. B. P.

at 9:55 is 160 mm. First sample at 10:10, B. P. then 150. Cava clamped at

10:25, B. P. immediately fellto40mm. 11:30, B. P.40, pulse 120, very little ether
required. 12:40, B. P. 40, clamp removed, B. P. rose slowly, reached 50 in 3 min-
utes, 55 in 6 minutes. Second sample, at 12:52, B. P. fell sharply to 38; injec-
tion started at 12:57. B.P. soon fell to 34, respirations of expiratory type, heart
very irregular. As injection proceeded respiration and heart action improved,
B. P. climbed to 40 in 15 minutes. 1:25, B. P. 60. 1:30, animal tried to vomit,
intestines extruded from wound, B. P. fell to 40, rose again to 54 in 5 minutes.
1:50, B. P.60. 1:55, B. P.65. 1:57, injection ended. 2:03, 3rd sample. Ani-
mal tried to vomit during taking of sample 3, B. P. fell to 42. 2:10, B. P. 34.
2:13, animal died. It will be noted that in this severe grade of shock the loss of
13 ec. of blood almost killed the dog (sample 2) and an additional loss of 13 cc.
did produce death (sample 3). |

Dog 13. Body weight, 18.05 kgm. 1:00 p.m., B. P. 125. 1:20, B. P. 128, 1st
sample drawn. 1:25, cava clamped, B. P. fell to 40 and was maintained there.
1:30, ether discontinued. 3:30, clamp removed, B. P. rose to 60 in 2 minutes,
amplitude of fluctuations very great. In 4 minutes B. P. averaged 80. 4:00,
2nd sample, B. P. 100, not affected by drawing 13 cc. blood. Injection started
at 4:08, 4:20,B.P.110. 4:30, B. P. 115, ether required. 4:40, B. P.120. 4:43,
B. P. 125. 5:08, 180. 5:08, injection ended. 5:12, 3rd sample, 5:15, B. P.
110, ether almost continuously since 4:30. 5:50, B. P.90. 6:00, B. P. 80. 6:10,
B. P. 64, pulse regular, ether discontinued. 6:25, B. P. 58. 6:40, B. P. 50.
6:55, B. P. 40. 6:57, B. P. 30. 6:59, 4th sample, animal died immediately.
This case illustrates well the type of shock in which the blood pressure is well
maintained for some time, masking the true severity of the shock; eventually
comes the rather sudden terminal circulatory and respiratory collapse.

Dog 15. Body weight,12.2kgm. 10:10, 1stsample. 10:20,B.P.140. 10:25,
cava clamped, B. P. fell to 40 and was maintained there. 12:25, clamp removed.
B. P. rose to 100 on removing clamp. 12:30, 2nd sample, B. P. 110. 12:35,
injection started, B. P. 110. 12:50, B. P. 1380. 1:25, B. P.125. 1:35, injection
ended. 1:37, 3rd sample, B. P. not affected by drawing sample. 2:30, B. P.
110. 3:35, B.P.100. 3:45, 4th sample, B. P. 90. 6:10, 5th sample, B. P. imme-
diately before sample = 80, immediately after = 65. 6:25, B. P. 60. 6:40,
B.P.55. 7:15, B.P.45. 8:20, 6th sample, B. P. 35 immediately before, dropped
to 30 immediately after, death in few minutes.

It is now believed practically universally that in traumatic shock
blood plasma leaves the circuldtion. Inasmuch as the concentration
of the proteins of the plasma remaining in the vessels is not appreciably
changed it must be assumed that in shock the permeability of the vessel
walls is increased. ‘The present series of experiments was planned in
order to ascertain whether the fluids drawn into the blood stream by
the hypertonic solution in fatally shocked animals would carry along

22 H. L. WHITE AND JOSEPH ERLANGER

with them more protein than this proceduré brings in when applied to
normal animals. Four animals were used, but one died in shock before
the injection was completed. The results from the other three are
given (table 3). Non-protein nitrogen was not followed in these cases
as blood was so valuable and it was felt that this determination could
best be omitted. The only respects in which the results of this series
differ from those of the normal series are in blood volume and protein
content.

1. Blood volume. In accordance with the results of numerous previ-
ous observers we find the blood volume greatly diminished in shock.
In this series the second or ‘‘shocked”’ sample was taken as the standard
for subsequent determinations of hemoglobin and blood volume. As a
result of the injection of the gum-glucose solution the blood volume is
markedly increased above its shock level, the increase even bringing the
blood volume above its initial normal level in all three cases. The
volume then gradually falls off until (in the one case which could be
followed for several hours, dog 15) at the end of 7 hours after the injec-

tion it reaches approximately its initial normal level. This was shortly

before the ‘animal died.

2. Plasma protein. The method of calculating the absolute amounts
of plasma protein may be illustrated here. The case of dog 12 may be
taken.

Body weight 14,360 grams

HEMOGLOBIN* TOTAL NITROGENT
: Volume per | yo) N ico
Sample Reading seat Magor, sae pera 4| Sample oe
grams
1 20.0 100 100 1 1.004
2 17.8 89 88 2 0.993
3 25.6 128 127 3 0.77

* Sample 1 is taken as the standard for reading sample 2, sample 2 as the
standard for reading sample 3 and any subsequent samples that may be obtained.

+ Since N.P.N. was not followed in shocked animals the figures given here as
‘‘total N’’ are the actual Kjeldahl figures.. Practically no change in the rela-
tions of the figures is produced by the subtraction of the small constant N.P.N.
figure from each total N figure, as was done in the first two series.

Calculation. 0.09 X 14,360 = 1292 cc., blood Li

0.64 X 1292 = 827 cc., plasma 1.

1.004 X 8.27 = 8.30 gm. N. in plasma 1.

Blood 2 has 88 per cent volume of blood 1, i.e. 12 per cent less.

:
¥

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 23

Assume this has all been taken from the plasma. This probably is not cor-
rect because of accumulation of corpuscles in the periphery. This peripheral
aggregation of corpuscles, however, would, as is mentioned below, make the
calculated exchange of protein even greater if it could be quantitatively
considered.

12 per cent of 1292 = 155. 827 — 155 = 672 cc., plasma 2.

6.72 < 0.993 = 6.67 gm. N in plasma 2.

Volume of blood 2 is 88 per cent of blood 1, i.e., 1137 ec.

Volume of blood 3 is 127 per cent that of blood 2.

The extra 27 per cent of 1137 or 307 cc. has entered the plasma,

Plasma 2 = 672 cc. 672 + 307 = 979 cc., plasma 3.

9.79 X 0.77 = 7.54 gm. N in plasma 3. Summarizing in the form of a table

we have:
SAMPLE NUMBER PLASMA N REMAINING | PLASMA N WITHDRAWN TOTAL PLASMA N
grams gram grams
1 8.30 8.30
2 6.67 0.08 6.75
3 . 7.54 0.16 7.70

We find that the absolute amount of plasma protein is greatly dimin-
ished in shock, its concentration remaining practically constant or being
very slightly diminished. This is in agreement with the refractometric
findings of Gasser, Erlanger and Meek (34). With the increase in
blood volume determined by the injection the absolute amount of pro-
tein rises markedly above its shock level but does not reach the nor-
mal level. The concentration falls considerably but not to the same

degree as plasma volume is increased. In other words, the fluid drawn

in is not so rich in protein as is plasma but neither is it protein-free.
Due to the method of following the blood volume these figures probably
do not indicate the real magnitude of the changes in total protein. As
is well known, the slowed circulation of shock causes red blood cells
to accumulate in the capillary area so that the number in the arte-
rial blood, in which the hemoglobin was followed, is relatively small
and the estimated blood volume therefore high. And it is to be pre-
sumed that the improved circulation following the injection of the gum-
glucose solution will return some of the jammed corpuscles to the cir-
culation, causing the blood volume estimation to be too low.
Following the blood changes after the injection, it is found that pro-
tein continues to increase even while water is passing back out, the pro-
tein concentration rising faster than the plasma volume falls. What
may happen is that when the injection is given the fluid drawn in brings

24 H. L. WHITE AND JOSEPH ERLANGER

with it protein through the abnormally permeable walls, but in lower
concentration than it occurs in plasma. Then, as the blood pressure
rises, the circulation improves and lymph flow is reéstablished. The
lymph flow from the liver and intestines is probably accelerated to a
far greater degree than that from the extremities, for two reasons: a,
In these animals the circulation of both posterior extremities was prac-
tically done away with since both femoral arteries and veins were ligated.
b, Since the capillaries of the liver and intestines are the most permeable
it is probable that most of the protein that disappeared from the plasma

in the process of the development of shock passed out into the tissue

fluids of the liver and intestines. As the circulation to these organs is
now improved the normal lymph flow is reéstablished and this lymph

flow sweeps along with it back into the blood stream through the

thoracic duct the plasma protein which had accumulated in the tissue
spaces of the liver and intestines during the induction of shock. Thus
protein is entering the blood stream even while blood volume is falling.

Such an interpretation of the results accounts for the rapid initial in-

crease in blood volume with an increase in absolute amount, although a
decreased percentage, of plasma protein, for the subsequent falling off

in volume and for the concomitant continued increase in the absolute

amount of protein. The prospect of technical difficulties in collecting

lymph over a long period of time has kept us from making direct —

observations on this latter point. |
- In the shocked animals, as in the normal and asphyxiated, the final

sample shows a percentage concentration of plasma protein lower than

that of the initial sample and at the same time the blood volume at the
time of the final sample is greater than initially; or, as in the case of

. dog 15, who was followed for 8 hours, when the blood volume has in

‘time fallen to slightly below its initial normal level, the percentage con-
centration of plasma protein has fallen to a considerably greater extent.
This must mean that here too gum acacia is taking the place of plasma
protein in holding water in the circulation. |

Starling (35) says “Absorption by the blood vessels as a result, say /

of artificial hemorrhage, if determined entirely by the osmotic attrac-
tion of the plasma colloids for the extravascular fluids, can only bring
about a passage of water and salts into the blood vessels.

According to my explanation this (absorbed) fluid should be pure salt
solution. That it is more dilute than plasma is clearly shown by experi-
ments but our data do not yet suffice to determine whether the incoming
fluid is a weak solution of protein, such as that contained in the tissue

oo) ee ae

——

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 25

spaces, or is a pure salt solution. If it is proved by quantitative results
to contain protein, then some other factor, such as back filtration or
active absorption by the endothelial cells of the blood vessels, must be
involyed in addition to the colloid constituents of the circulating blood.”’
The present data point strongly to the conclusion that some protein
does pass in directly in the case of shock although it might not be im-

_ possible to explain all of our data on the assumption that the protein
is carried back into the blood stream by the lymph. The rapidity
with which it occurs, however, speaks against this.

S01 ~
2 ‘
a rie .
a ‘ ee yer ST
£ \ se heal toh Dog7
45 Be ies) eet a iu
\ cemeae
0.7 | | 1 L ! | J se !
Or oO’ / be Re) + o, 6 y }

Fig. 1. Changes in total amount (solid line) and per cent (broken line) of
plasma protein N in a normal animal, dog 7, and in an asphyxiated animal, dog
10. Time of taking Ist sample is indicated by 0, the end of the injection by 0’.

8. Urine. The urine in the case of dog 13, a shocked animal, con-
A tains no sugar in spite of the hyperglycemia which far exceeds the nor-
4 mal overflow threshold. This dog was in rather severe shock and the
absence of glycosuria is probably due to the fact that no urine was se-
+ - ereted after shock developed. The fact that additional samples of
4 urine could be drawn probably means that the bladder had not been
ie completely emptied of its pre-shock urine. Dog 15 was in better con-
: dition and excreted urine containing sugar.

26 H. L. WHITE AND JOSEPH ERLANGER

4. Non-toxicity of the acacia-glucose solution. It was our intention in
these experiments to produce a grade of shock that would prove fatal
in a few hours and in this we succeeded. Some of the animals were -
almost moribund when the solution was administered. In no case

1010.0

0.995

oF 40

Gm Total N.

2 % Total N

°
a
~3
on
bas
=
=

Fig. 2. Changes in total amount (solid line) and per cent (broken line) of
plasma total N in a shocked animal, dog 13. Letters designate times of pro-
cedures, as follows: a—cava clamped; b—clamp removed; c—injection started;
d—injection ended. Time of taking 1st sample is designated by 0, while /, 2,
etc., designate hours after taking 1st sample up until end of injection. The end
of the injection, when the 3rd sample is taken, is designated by 0’, while 1’, 2’,
etc., designate hours after end of injection.

were any untoward effects observed as a result of the injection; the
blood pressure, heart action and respiration always were benefited.
The rectal temperature was practically unchanged. In one case a
slight albuminuria was observed in one sample, but microscopic exami-

Ee ee ee
7 ° Sie ct ;

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION 27

nation revealed large numbers of spermatozoa. The albuminuria*had
disappeared in the next sample. Casts, hematuria or hemaglobinuria

were never observed. No hemolysis occurred as a result of the injec-

tions, some slight laking being present in several plasma samples but
it was no more evident in samples taken after the injection than in
those taken before. This laking is attributed to the ether. These
findings in regard to the non-toxicity of gum acacia are in accord with
those of Bayliss (36), who discusses the rather misleading statements of
Kruse (30) as to the toxic effects observed after the injection of acacia.

SUMMARY AND CONCLUSIONS

A strongly hypertonic glucose and gum acacia solution was injected
intravenously into normal, asphyxiated and shocked dogs, and the
resultant changes in blood volume and composition were studied.

The immediate effect was a marked increase in blood volume; in
normal and asphyxiated animals the blood volume then gradually fell
toward but did not completely return to normal in several hours.

The blood volume, markedly diminished in shock, is increased to
above its normal level by the injection and then gradually falls to or
below its normal level.

The absolute plasma protein is increased slightly or not at allin normal
animals and in asphyxiated animals; in an animal which had been bled
there was a slight increase when the amount withdrawn was allowed
for. The absolute amount of plasma protein is markedly diminished
in shock, is increased by the injection and the increase continues for
some time after the injection. It is believed that at least a part of the
increase in plasma protein following the injection in shock is due to a
passage of protein in through the vessel walls.

Gum acacia seems to take the place of plasma protein in. holding
water in the circulation.

There is a marked hyperglycemia immediately after the injection in
normal animals; this is accentuated by morphine and asphyxia. The
blood sugar value falls to or nearly to normal within 2 hours. In
shocked animals the blood sugar behaves much as in normal animals.
There is only a trace of sugar excreted by normal animals excepting
when morphine or asphyxia cause marked glycosuria. Shocked animals
without morphine excrete some sugar unless, as a result of the shock,
there is a suppression of urine.

28 H. L. WHITE AND JOSEPH ERLANGER

The fluid drawn into the blood stream brings with it chlorides in con- ©
centration equal to the chloride concentration of plasma but the dif-
fusion into the blood stream of sufficient additional chlorides to bring
the chloride concentration of injected fluid up to that of plasma i is not
complete for several hours.

The entrance of urea into the plasma takes place with such facility
that the non-protein nitrogen concentration of the plasma remains |
constant.

There is no suppression of urine in normal animals as a result of the
injection, if anything the rate of secretion is slightly increased.

The crystalloid osmotic tension of the plasma does not remain con-
stant.

No hemolysis, hematuria, hemoglobinuria, albuminuria, cylindruria,
fluctuations in body temperature or any other untoward effects were
observed as a result of the injections.

The authors wish to thank Dr. W. H. Olmsted, of the Department of
Internal Medicine, for his kindness in extending the facilities of his
laboratory for carrying out many of the determinations and for several
valuable suggestions on points of analytical technique.

BIBLIOGRAPHY

(1) ERLANGER AND GassEeR: Ann. Surg., lxix, 389.

(2) Brasou: Arch. f. Physiol., 1884, 211.

(8) Kutcxowicz: Arch. f. Anat. u. Physiol., 1886.

(4) Leatues: Journ. Physiol., 1896, xix, 1.

(5) GASSER AND ERLANGER: This Journal, 1919, 1, 104.

(6) Starting: Journ. Physiol., 1899, xxiv, 317.

(7) Paton: Journ. Physiol., 1899, xxiv, 419.

(8) HamBurGcEeR: Osmotischer Druck u. Ionenlehre, B. II, 7.

(9) FisHerR AND WisHaARrT: Journ. Biol. Chem., 1912, xili, 49.

(10) Magnus: Arch. f. Exper. Path. u. Pharm., xliv, 68.

(11) Woopyatt, Sansum AnD WiLp=ER: Journ. pene Med. Assoc., 1915, lxv, 2067.
WILDER AND Sansum: Arch. Int. Med., 1917, xix, 311.
SaNsuM AND WoopyattT: Journ. Biol. Chem, 1917, xxx, 155.

(12) ERLANGER AND Woopyatt: Journ. Amer. Med, _Assoc., 1917, lxix, 1410.

(13) Morawitz: Oppenheimer’s Handb. d. Biochem., II, 2, 78.

(14) Kerr, Hurwitz anp WHIPPLE: This Journal, 1918, shed! 356.

(15) ERLANGER AND GassER: This Journal, 1919, 1, 119.

(16) Scorr: This Journal, 1916, xl, 128.

(17) Van StyKE AND DonueEavy: Journ. Biol. Chem., 1919, xxxvii, 551.

(18) Foutin anp Wu: Journ. Biol. Chem., 1919, xxxviii, 81.

(19) Marriorr: Journ. Amer. Med. Assoc., 1916, Ixvi, 1594.

BLOOD ANALYSES FOLLOWING ACACIA-GLUCOSE INJECTION

(20) JANEWAY AND Jackson: Soc. Exper. Biol. Med., xii, 193.
(21) ERLANGER AND Gasser: This Journal, 1919, xlix, 151.
(22) Merx anp GassER: This Journal, 1918, xlv, 548.

(23) Scort: This Journal, 1917, xliv, 298.

(24) Merx anp Gasspr: This Journal, 1918, xlvii, 302.

(25) Dawson, Evans AND Wuippte: This Journal, 1920, li, 232.
(26) McQuarrig AND Davis: This Journal, 1920, li, 257.

(27) Scorr: Journ. Physiol., 1915-16, 1,128.

(28) Scorr: Journ. Physiol., 1915-16, 1, 157.

(29) Ripgau: Pharm. Journ., 1892, 1073.

(30) Kruse: This Journal, 1919, xlix, 137.

(31) Knowtton: Journ. Physiol., 1911-12, xliii, 219.

(32) Bouton: Proc. Roy. Soc., Ixxix, 267.

(83) Srartine: Fluids of the body, 164.

(34) GassER, ERLANGER AND Merk: This Journal, 1919, 1, 31.
(35) Staruina: Fluids of the body, 102.

(36) Bayuiss: Journ. Pharm. Exper. Therap., 1920, xv, 29.

29

THE FUNCTIONAL ACTIVITY OF THE CAPILLARIES AND
VENULES

D. R. HOOKER
From the Physiological Laboratory of the Johns Hopkins University

Received for publication July 10, 1920

INTRODUCTION

The significance of the blood stream in the capillary bed for the nutri-
tive processes of the body is well recognized. But it is only in recent
times that emphasis has been laid on the capillaries as a factor in the
dynamics of the circulation. It is the purpose of this paper to present
evidence in support of the belief that the capillaries and venules, as well
as the arterioles, respond to direct chemical stimulation and also to in-
direct stimulation through the intervention of nerve fibers. If this
proof is established, the functional activity of the capillary and venous
beds must henceforth increase in both theoretical and practical impor-
‘tance. In the first place local tissue needs may by direct chemical
action control the capillary blood streaming through the part as shown
so clearly by Krogh (1) for muscle. In the second place, the capillaries
and venous fields being under the influence of the central nervous sys-
tem, it follows that local vascular reflexes (2) and systemic vascular
reflexes undoubtedly depend upon the codperation of the capillary
and venule with the arteriole; that, in other words, the peripheral
resistance, both functional and static, includes arteriole, capillary and
venule. And in the third place the effective blood volume, both fluid
and corpuscular, must be subject to alteration and regulation to a
very significant degree.

Most of the work on the function of the capillaries hitherto reported,
except the recent paper by Krogh just mentioned, has been done on the
frog in which the transparency of the tissues permits microscopic
visualization of these vessels. It is possible, however, using Lom-
bard’s method of a drop of oil on the skin (3) to extend such studies to
themammal. Furthermore, most of the recent evidence in the mammal —
bears upon dilatation of capillaries. It may be assumed that a vessel

30

FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 31

which is shown to dilate must also contract, but the direct evidence has
hitherto been lacking. Nor is there much evidence that the blood
capillary and the venule are under nervous control.

HISTORICAL

The first evidence that capillaries have the power independently to
change their caliber was published in 1858. In that year Lister (4), de-
scribing the early stages of inflammation, pictured with camera lucida
drawings a very great increase in the caliber of the capillaries in in-
flamed tissue. This observation was followed by the studies of Stricker
published some seven years later (5). Stricker observed the capil-
laries in the nictitating membrane of the frog to dilate and to constrict.
The constriction was less evident than the dilatation and appeared to
be due to two processes, one a nuclear swelling and the other an actual
contraction of the endothelial protoplasm. These results were ob-

tained in tissue removed from the body and therefore deprived of its

blood supply, and Stricker himself is not convinced that they are not
due to lethal changes in the. tissue. This possibility is supported by
figure 4 in his paper, which pictures a change in shape of a capillary
vessel which must be extremely unusual if compatible with functional
activity. In a second paper published the following year Stricker (6)
describes further experiments with the nictitating membrane of the
frog removed from the body and examined in the aqueous humor
under the microscope. Many of these preparations were entirely
unsatisfactory but a few of them gave beautiful responses to stimula-
tion by ammonia vapor. When the tissue was exposed to ammonia.
vapor for three or four seconds and then examined under the micro-
scope, Stricker saw the capillary lumina almost disappear and then
dilate wide enough for a corpuscle to pass. This phenomenon occurred
about twice in fifteen minutes. Further, Stricker observed a varicosity
on a capillary which moved forward, suggesting peristaltic activity
on the part of the endothelial tube. When the capillaries thus under
observation contracted they became practically invisible but could be
readily seen again after the contraction had passed off. Stricker
likewise employed electrical stimulation. The procedure which he
used is not clear but with it he obtained repeatedly good results on
various specimens. When stimulated, the capillaries would contract
almost instantaneously and dilatation would follow a moment or more|
after the cessation of the stimulus. After afew applications of the stim-'

$2 D. R. HOOKER {

ulus no further response could be obtained. Even better results were ;
had in the case of the capillaries of the tail of the living tadpole. These
vessels were found to respond to mechanical, chemical and electrical
stimulation. Of these, chemical stimulation was apparently by far the
most effective. Stricker regarded these changes as due to a turges-
_cence of the capillary endothelium such that, without change of th
outside diameter of the vessel, the lumen was altered in size due to
thickening of the wall (7). ) os
Although Cohnheim (8), among others, contested Stricker’s view |
that the capillaries possess inherent contractility in the sense just
defined, confirmatory evidence was quickly forthcoming. Stricker’s
work was followed by that of Golubew (9), who confirmed the obser-
vation that capillaries change their size under varying conditions, and
advanced the observation that certain spindle elements on the capillary
walls contract into spheres, thus occluding the lumina of the vessels
/ and so functioning as a constriction. This notion of the mechanism
| of capillary function was'supported a few years later by Tarchanoff (10).
- This author used alcohol, ether, ammonia, ferric chloride and acetic
_ acid as well as heat. These procedures caused the so-called spindle
' elements to swell and so to occlude the lumina, but it was not un-
common to find that the vessels failed to dilate after the striae was
removed. :
_ In 1878 Severini (11) published a monographic study of the capil-
laries in which he states, among other things, that the application of
oxygen causes the capillaries to contract, while the application of COs
causes them to dilate. Severini appears to be of the opinion that these
gases act chiefly on the spindle elements described by Golubew. The
observations were made upon the frog but also upon the capillaries in
the mesentery of the guinea pig. Tarchanoff was unable to confirm
these observations of Severini’s, nor was Roy, working with von
Mehring in 1879, able to confirm them. In 1879 Roy and Brown (12)
published their very important paper describing observations on the
| capillary blood pressure in the frog. These authors were convinced
| that the capillaries have the power of independent contractility and
| that their caliber is not, as was believed by many, due to passive
changes. They found that there was little difference in the size of the
capillaries of the frog’s web before and after the leg was amputated.
Dilatation of the capillaries was also observed on the application of
chloroform, and it was also noted that the capillaries dilated as the
result of Goltz’s “klopfversuch.’”’ These authors were of the opinion

ee eee ee ee eee

FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 33

that it is the whole capillary and not the spindle elements of Golubew
which contract. They observed that local anemia produced by com-
‘pression of the area under observation results in a subsequent hyper-

emia which is accompanied by a dilatation of arterioles, capillaries and
venules. This latter phenomenon occurred after section of the sciatic
nerve when stimulation of the nerve caused a strong contraction of the
arterioles. It is interesting that they observed only a contraction of
arterioles on nerve stimulation. Since this procedure failed to elicit
changes in the capillaries, they conclude that the functional activity
noted in the latter vessels must be due to some local mechanism and
they favor the conception that it is due to a direct chemical action upon
the endothelium rather than upon the functional existence of what
they refer to as peripheral vasomotor ganglia. The latter conception
accords with what we now speak of as axon reflexes, which Krogh has
recently invoked to explain localized dilatation of capillaries following
punctate stimulation (13). More recently Bied] (1894) saw the periph-
eral vessels (arterioles, capillaries and venules) all contract in the
frog’s mesentery on the application of salt solution at 45°C., and dilate
again when the heat was removed (14).

Mayer in 1885 (15) again observed the endothelium of the capil-
laries to contract under electrical stimulation. The evidence that the
lymphatic vessels throughout the body are contractile is very strong.
The phenomenon was observed by Mayer and by Elliott Clark (16)
and others in the tail of batrachian larvae. Histological evidence
makes it perfectly clear that the lymphatic capillaries are supplied with
nerve fibers. These fibers have been observed and pictured a number
of times (Kytmanof, 17), and Camus and Gley (18) have obtained
graphic records of the functional response of the larger lymphatics to
electrical stimulation of nerves. .Sabin, in a comprehensive article on
the origin and development’ of the lymphatic system (19), has collected
these data in a convincing manner. The lymphatic and _ blood
capillaries are essentially similar in histological structure. The evi-
dence that the blood capillaries are innervated is not so well established
as is the case with the lymphatics, nevertheless there appears to be
little doubt of the fact. Shaffer states that nerve fibers may be
found to follow each individual blood capillary in the rabbit’s mesen-
tery (20). Anatomical knowledge therefore furnishes adequate grounds
for the expectation, supported by experimental work on the frog,
that the mammalian blood capillaries will be shown to be contractile
and under the ennirol of the nervous system.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

34 D. R. HOOKER

In 1903 Steinach and Kahn (21) published an extensive paper on the
contractility of the capillaries. Their observations were directed almost.
wholly to the effect of direct electrical stimulation in the-frog. They
used the excised nictitating membrane from the frog and observed,
contrary to the findings of Stricker and Biedl, an actual collapse of
the whole capillary tube. According to these writers the collapse is.
/ due to activity, not of the endothelium as such, but of the perivascular

cells described by Rouget (22) and Mayer (15) so that the endothelial
capillary tube is collapsed by a passive infolding of the tissue. The
‘venules were also observed to contract.

Analogous results were demonstrated in the omental capillaries of
kittens and guinea pigs. The technical procedure used for these ani- |
mals is not described and capillaries of less than 10 » were not seen to |
contract. They believe that the vessels which they actually saw
contract belong, however, to the category of capillaries.

Finally, in a frog preparation which is described in great detail, they
obtained contraction in the capillaries of the nictitating membrane upon
stimulation of the sympathetic fibers which leave the cord in the third,
fourth and fifth spinal roots. The latency of contraction was four to
five seconds, sometimes twenty seconds, as compared with a latency of
one to three seconds by direct stimulation. It was not uncommon to
observe rhythmic contractility of the capillaries after the stimulus had
ceased. Galvanic was more efficacious than faradic stimulation and it
appears that an exceedingly powerful stimulus was required—twelve
Daniell cells. ,

In recent times attention has been again focussed on the functional
significance of the capillary bed largely as the result of the work of
Dale and his collaborators in the study of histamine shock (23). Dale
has advanced the hypothesis that histamine is an endothelial poison
which paralyzes the capillary wall so that a marked dilatation occurs.
With this dilatation there results a pooling of blood in the capillaries
sufficient to account for the marked fall in blood pressure found in
conditions of shock. As the result of the work of Bayliss (24) and
Cannon (25) and of Abel and Kubota (26), it would appear that a his-
tamine-like substance may be produced in the body as the result of
tissue injury sufficient, under certain conditions, to account for the

primary symptoms of shock. So that today the opinion is quite widely
held that histamine or a histamine-like body plays an important part
in functional processes of the body. This applies not only to patho-
logical states such as shock, but Abel and Kubota have advanced the

\

eee ov Bee

FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 30

conception that such a substance is significant in normal physiological
processes regulating the distribution of the blood.

‘In an important contribution to this subject, Krogh (1) has recently
shown that an enormous increase in patent. capillaries may be demon-
strated in active muscle as compared with resting muscle. This in-
crease may indeed amount to more than 700 per cent. Krogh noted
that electrical stimulation and massage open up many new capillaries
and that electrical stimulation enlarges the capillaries already patent.
He regards the mechanism for the regulation of the capillary eapacity
as being in the capillaries themselves; that is, as due to some chemical
regulation or else as associated with an axon reflex mediated through
the sensory nerve fibers. He observed that scratching the tongue of
an urethanized frog with a glass needle caused a local hyperemia with
dilatation of the capillaries and arterioles (13). A closed capillary thus

- made to dilate opens from the venous end with the appearance of a

reversed corpuscular flow. When the dilatation reaches the arterial
end of the capillary, the blood stream suddenly assumes the normal
direction. Since cocaine was found to abolish this reaction, . Krogh

was forced to the assumption that it was an axon reflex. He found »

that cocaine also abolishes the dilatation caused by the application of |
iodine but .does not affect the dilatation caused by acids. Section of
nerves supplying the part under investigation at first did not affect
the response to chemical and mechanical stimulation. For some days

after the nerve section the tissues were hyperemic and the capillaries

responded normally to stimulation. Later on, however, the hyperemia
disappeared and the capillaries reacted only with a very localized re-
sponse. From these and other observations Krogh concludes that the
capillaries dilate and contract independently of the general blood
pressure, and that the spread of response in the capillaries is due to a
local axon reflex, probably along the sensory fibers.

Another line of evidence for the contractility of the capillary is
found in the tache originally described by Marey (27) in the human
being. If a blunt-pointed instrument is drawn across the skin a white
line is left which turns to red in a few seconds. In certain cases the
red line may be bordered with white and develop a definite urticarial
wheal. Clinically this condition is regarded as indicative of vasomotor
insufficiency. Marey explained it as due to a localized contraction and
dilatation of the capillaries. Bloch (28) thought there was no evi-
dence that the capillaries actually contracted. According to him the
red represented capillary dilatation while the neighboring pallor was

36 . D. R. HOOKER ;
due to a drainage of blood into the vessels which dilated because of the
mechanical injury.

Ryan (29) has sought to standardize this test for apolicaiien in
various forms of fatigue. Cotton, Slade and Lewis (30) found the
reaction well developed in cases of soldiers with irritable heart and
adduced new evidence in support of the belief that it is a capillary
phenomenon. The red line and neighboring pallor may still be dem-
onstrated after the circulation in the arm has been shut off with a
‘sphygmomanometer cuff. Hence they conclude that the arterioles
and venules cannot participate in the production of the red line be-
cause there is no excess pressure available to dilate these vessels. If
the arm be raised before the pressure cuff is applied the reaction cannot
be demonstrated because the skin is depleted of blood. On the other
hand, if the arm be lowered before the pressure is applied, the reaction
is vivid because there is an excess of blood in the skin.

These investigators incidentally show that epinephrin constricts the
capillaries. When epinephrin is injected into the skin the resulting
pallor is to be regarded as due to a contraction of the arterioles which
shuts off the blood from the distal capillaries. If such an injection be
made, however, after the blood flow in the arm has ceased as the result
of occlusion by a blood pressure cuff, pallor is still produced.- Since
constriction of the arterioles would now have no effect in stopping the
flow of blood into the capillaries, the pallor which results must be due
to constriction of-the capillaries themselves. This observation that
the local application of epinephrin constricts the capillaries is especially
interesting in conjunction with the work of Sollmann (31) who showed
that histamine also locally applied develops an urticarial wheal. —

Very recently Thaysen (32) has reported quite remarkable oscilla-
tions in the red cell count in a case of polycythemia. On one occasion
he recorded an increase from 5.3 million to 10.6 million in the course of
twelve hours. His investigations and tests showed that these fluctua-
tions were caused by varying contraction and dilatation of the eapil-
laries and precapillaries of the skin. This writer is reported to believe
that careful observations would reveal similar results in other cases of
polycythemia in which the vasomotor system is so unstable that the
condition might be called one of vasomotor or capillary ataxia. These
observations are an extreme instance of the well recognized difficulty in
obtaining consistent red cell counts in many clinical cases. It would
be of very great interest and importance to know if such fluctuations
are indeed due to alterations in capillary tone.

FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 37

METHOD

The technique followed in the experiments which form the basis
of this paper was very simple. Cats were used exclusively. After
anesthesia was established the animal was placed on a flat holder so |
that the back of the head and ears rested on the same plane with the
vertebral column. The ear to be observed was shaved and thoroughly
cleaned and dried. By means of a heavy thread through the upper
lip the head was so held that an ear, flattened out against the board.
This flattening of the surface was further accentuated by sealing the-
ear to the board with collodion. A microscope giving a magnification
of about 70 X was adapted for adjustment over the animal holder and
a strong artificial light arranged to give direct illumination of the
area at an angle of approximately 45 degrees completed the equipment.
Castor oil was flooded over the field at the outset and occasionally during
the observations. :

With such an arrangement a flat vascular plexus can easily be found
_ preferably in the neighborhood of the tip of the ear in which the cor-
puscular flow in the finer vessels can be readily followed for an indefi-
nite period. In this region the skin lies loosely attached to the connec-
tive tissue which forms the framework of the ear. There is no muscle
other than that in the blood vessels which might indirectly influence
the vascular area under observation. Animals with little pigment in
the skin are preferable but it is far from impossible to obtain good —
visualization of the capillary network and venules even when a con-
siderable amount of pigment is present. One sees vessels of various —
size, capillaries with red cells streaming through in single file up to
larger vessels in which the corpuscles are packed in a thick column.
The picture is essentially like that seen in the transparent tissues of
the frog. Vessels exhibiting pulsation are infrequent; probably the
arteries and arterioles take a deeper course while the capillaries, venules
and veins lie quite superficially, as is indicated to the unaided eye.
The capillaries ramify freely particularly about the hair follicles and
considerable areas are readily found in which the network forms a
horizontal plane and therefore is easily visualized without change of
focus. It should be emphasized that with the magnification employed
one does not see the capillary wall; it is only by the presence of the
red blood cells that the capillaries are recognized. Consequently
when the vessels are emptied of blood the field becomes a blank so far
as the smaller vessels are concerned.

38 D. R. HOOKER

Attention may be directed to the fact that our present conception
that the red blood cells course through the capillaries in single file rests
upon observations of the capillary circulation in the frog.” In this ani-
mal the corpuscles are extremely large as compared with the same
cells in the mammal (frog 22x16, cat 6u, man 8) so that the
reason for the prevalent opinion is obvious. In the mammalian eapil-
lary circulation, including that of man (33), corpuscles may sometimes
be seen moving in this manner but it is much more usual to find the cap-
illaries with lumina sufficient to allow more than one corpuscle to pass
‘atatime. This difference is doubtless due to the higher rate of metab-
olism in the mammal. There is likewise a much more rapid movement
of the corpuscles in the warm than in the cold blooded animals. There-
fore to restrict the capillary, in the mammal, to blood vessels in which
the corpuscles move slowly and in single file is not strictly consonant
with the facts. |

Post-mortem behavior of vessels. If in the preparation as above de-
scribed ether be poured down the tracheotomy tube and the animal be
thus killed, the movement of blood at first comes to a sudden stop.
Then the corpuscles clump slightly and shortly begin to move forward
in the normal direction. This movement, at first noticeable in the
capillaries, extends to the venules and there is a slow and gradual
progression of blood toward the larger veins. The appearance is as if
/ no blood entered the capillaries from the arterial side and that a milk-
| ing process, akin to peristalsis, swept the corpuscles onward toward
- the vein. When the process is complete it may be found that here and
there in the capillary net a few clumped corpuscles are locked as if
the constriction of the vessel had failed to carry on the last of its
contents. :

These events occupy varying lengths of time in different animals.
They may develop completely in a few minutes or they may last half
an hour or more. The completeness with which the vessels empty is
also a varying factor. Sometimes, particularly in old and debilitated
animals, the blood may not be moved at all; sometimes the field is
swept absolutely free of blood but more often a few clumps of cor-
puscles are left stranded, particularly inthe venules. The stagnation
of these clumped corpuscles is significant in that they give direct evi-
dence of the contraction of the venules since their diameter is readily
appreciated to be less than was that of the same section of the vessel
prior to death. |

Se ae ne ee

FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 39

The condition of the vessels as thus described prevails for some
time, fifteen minutes or more. Then a remarkable change occurs for
the vessels begin to relax and fill. Close observation reveals that
relaxation first develops on the venous side and that the blood flows,
slowly at first, then quite rapidly, from vein to venule to capillary,
and that by the end of approximately an hour the vascular area is
filled again and filled full with the indication that capillaries invisible

_ before death are now widely open.

One further change remains to be mentioned: an indefinite time after

' the relaxation and filling of the capillaries just described, the vascular

net is once more emptied of blood. This change is roughly coincident
with the onset of skeletal muscle rigor and after it is once developed —
there is apparently no tendency to a reversal, at least after four days
there was in one instance no sign of blood in the smaller vessels.

The foregoing description of the post-mortem appearances of the
peripheral vascular bed is substantiated by the photograph reproduced
in figure 1. In the technique of photographing these vessels two major
difficulties had to be overcome, especially when applied to the living
animal. The first was to provide sufficient illumination without heat
to cut down the exposure time so as to avoid accidental movements.
This was accomplished by the use of an arc light projection lantern
without the bellows and projecting lenses. The lantern was tilted at
approximately forty-five degrees and so placed that the light rays were
concentrated almost to a focus on the part to be photographed at a
distance of about 60 cm. from the source of light. The second diffi-

. culty was to overcome the movements of the ear due to respiration

which, however slight, were sufficient when magnified to spoil the
picture in the requisite exposure of thirty seconds. In some animals
the respiration was quiet enough not to be a disturbing factor but in
the majority of cases a clear picture could not be obtained. In the
latter, resort was therefore had to artificial respiration for a couple
of minutes before the picture was taken. The apnoea which resulted
was adequate to the requirements. |

For observation alone the nictitating membrane has some advantages
over the ear. In the cat it usually offers a perfectly white ground on
which the vessels stand out with exquisite definition. Arterioles, capil-
laries and venules are readily recognized and less illumination is re-
quired. Probably the vessels have less covering tissue over them.
Salt solution instead of oil must of course be used to keep the tissue
moist. A thread passed through the cartilaginous edge may serve to
spread the membrane over the eye ball.

40 : D. R. HOOKER

This area of tissue is, however, ill-adapted for photographs. It suf- —
fers more than the ear from respiratory movements and the arterial —
pulsation both in the vessels of the tissue itself and in the underlying
eye ball cannot be overcome. In addition to these difficulties activity
in the smooth muscle of the membrane itself cannot be controlled with
the result that the focal plane cannot be maintained. Finally, and
most important for the work in hand this tissue cannot be easily brought
below the heart level. It follows that passive drainage of the vessels
may therefore result if the circulation stops.

The area photographed was magnified ninety times in most of the °
pictures taken. A larger magnification than this led to trouble be-
cause slight differences of position of the plate, which could not be
avoided, resulted in a poor focus. The vessels were focussed on the
grownd glass plate of the camera at the beginning of an experiment.
and as a rule this focus was not changed. A focal error frequently
crept in, however, due it is presumed to changes in turgidity of the
underlying tissues so that the results so far as the relative size of a
vessel is concerned are not wholly trustworthy in the case of capil-
laries. Conspicuous changes in the size of the larger vessels may be
relied upon because such changes can be readily recognized by the eye ©
when using the microscope without the camera. As to the capillaries,
their presence or absence in the picture should be the sole criterion.
If a capillary has disappeared or if its continuity is broken it is proper
to assume that it has constricted because, as has been stated, the vessels
under inspection lay below the heart level and only active constriction
could empty them of corpuscular elements.

Using the procedure above outlined, photographic records were
obtained from eight cats in which no preliminary steps were taken other
than etherization and tracheotomy. The animals were kept under
ether until a satisfactory control picture was taken and then sufficient
ether was poured down the trachea to cause prompt death. Of these
eight animals three failed to show a primary vascular constriction
shortly after death and were not observed further, and six showed com-
plete or partially emptied vessels. The latter group all showed a sub-
sequent peripheral vascular dilatation followed later by emptying. In
addition a number of other animals were observed, but not photo-
graphed, with confirmatory results.

Figure 1 is selected to exemplify these results. If shows the four
stages of vascular change. Three minutes after death the venules are —
constricted and the capillaries largely emptied. Forty-five minutes

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41

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

42 D. R. HOOKER

after death the venules are widely dilated and many capillaries are
visible. After another ~period of forty minutes all the blood has been

swept out of the vessels and only the shadows of the largest vessels

can be found. The data at hand indicate that this last change is per-

manent since no further alteration was observed to occur for a period
of four days. |

_ It may be pointed out here, as was emphasized by Bayliss (20), that
there is no inherent difficulty in the conception that the protoplasm con-

stituting the capillary endothelium undergoes change of shape and so

mediates a constriction of the vascular lumen. The motility of amoebae
and of the white blood cells of higher animals is dependent upon such a
change of form and many other similar instances could. be mentioned.
That the vascular phenomena above described are not dependent
upon the innervation of the vessels was shown in three animals in which
death by ether was produced subsequent to section of the cervical
sympathetic. In each of these cats the primary constriction was well
developed in from one to four minutes. This was followed by dilata-
tion and subsequent constriction in two of the three. In the one which

failed to show dilatation the vessels were largely empty three and a ~

half hours after death.

Death by ether subsequent to the intravenous injection of a dose of
ergamine phosphate sufficient to cause permanent or transient “shock”
(23) is not followed by the same vascular changes. The intact animal

or the animal after section of the cervical sympathetic exhibits a pri- ©

mary constriction in the capillaries and venules followed by dilatation

and subsequent permanent constriction, as above indicated. If the
dose of ergamine is a fatal one or if the animal be killed with ether.

after partial or complete recovery from ‘‘shock,” the primary constric-
tion is inconspicuous or wholly absent. In four animals thus ob-
served, two showed no primary constriction whatever and two gave a
mere suggestion of it. Furthermore in but one of these animals (a

kitten killed by the ergamine) did the constriction subsequent to dila-

tation develop. In all but this last animal the vessels remained

dilated and filled with blood as long as observes (in one case nineteen

hours).
These results then clearly support the view advanced by Dale that
/ ergamine phosphate (histamine) is a capillary poison. Additional evi-

dence is found in a single animal in which after histamine and nerve —

section the vessels similarly failed of the usual response after ether
death. In other words, the histamine effect is not dependent upon the
integrity of the vascular nerves,

Ce ee eg ee oe

FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 43

The results thus far presented show that the peripheral vascular bed

passes through a number of active changes subsequent to death and

that these changes depend upon a local or peripheral function since they
are not affected by nerve section and are largely done away with by
the injection of an endothelial poison (histamine). Almost at once
after death the capillaries, venules and, presumably, the arterioles
constrict with the result that the peripheral field is swept more or less

completely free of blood. Since the corpuscles can be seen in transit)
toward the veins, the result is suggestive of peristaltic constrictions.

running along the vascular tubes. This condition must be looked |

upon as the first consequence of asphyxia and may well be a significant\,
j factor in the asphyxial rise of arterial and venous blood pressure as \
ordinarily recorded whereby the lesser and minute vessels throughout

the body discharge their contents into the larger channels. This pass-
ing constriction gives way shortly to a marked dilatation such as is
usually associated with the collection of asphyxial and catabolic prod-
ucts. Some time later and roughly coincident with the onset of skel-
etal muscle rigor a second constriction develops which is apparently
permanent in character. Whether this change is due to a rigor con-
traction of smooth muscle and endothelium, our present knowledge of
these tissues is insufficient to determine. In no case were observations
continued long enough (never more than four days) for skeletal muscle
rigor to pass off, consequently the assertion that the change, assuming
it due to rigor, is permanent is somewhat arbitrary. Post-mortem
tissue changes, including laking of the blood, may in this length of
time, however, have so clouded the field that the vessels could not be
seen even though they were filled with blood. Also the blood may
clot and so fail to run into the vessels even after their lumina have
become patent. It will be noted that this observation does not accord
with the livor mortis seen so frequently by pathologists. :

Experiments with nerve stimulation. A second point of even greater
interest and importance brought out in this research is the effect of
nerve stimulation upon the peripheral vascular bed. In this set of
observations the animals were usually anesthetized with urethane
and the cervical sympathetic nerve dissected out for stimulation. In
no instance was it possible to determine, by inspection or by photo-
graphs, that section of this nerve altered in any way the caliber of the
vessels of the ear. On the other hand, electrical stimulation of the
nerve gave unmistakable evidence of constriction in both capillaries
and venules and subsequent to stimulation an over-dilatation was

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FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 45:

recognized. ‘These results were so sharp that it was considered neces-
sary to perform but three experiments in each of which the observation
was repeated many times. No indication of fatigue was noted since the
results were as good five hours after an experiment was begun as at
the start. Figure 2 gives the photographs obtained in one of these
experiments.

Fig. 3. Constriction of capillaries and venules following the injection of 3 ce.
1: 50,000 epinephrin. Magnification 90 x. Cat. The cervical sympathetic nerve

had been cut. a, Before, and b, shortly after the injection of epinephrin. The

negatives have not been retouched.

This figure shows clearly the constriction and disappearance of the |
capillaries and venules during electrical stimulation of the cervical

sympathetic and their subsequent over-dilatation two minutes after the
stimulus was removed. It happens that no larger venule was present in
this field but these were repeatedly seen to respond just as conspicu-
ously as shown here for the capillaries. The response of the venules
may be appreciated, although not very clearly, in the next figure
(fig. 3), which shows the effect of epinephrin injection.

a ST

46 D. R. HOOKER

Experiments with epinephrin. Further evidence of the sympathetic
innervation of the capillaries and venules was developed from the in-
jection of epinephrin. This substance, selective in action for contrac-
tile tissues with sympathetic nerve supply, gave results comparable
with those obtained with electrical stimulation of the cervical sympa-

thetic. The effect was the same both before and after section of the

sympathetic. Figure 3 is made from photographs taken before and

just after the intravenous injection of 3 cc. of 1: 50,000 epinephrin in

a cat three and a half hours after the cervical sympathetie had been
cut.

The injection of histamine destroys this mechanism. In a cat in |

which nerve stimulation had given sharp constriction, 6 mgm. ergamine

a

phosphate were injected. As soon as the resultant dilatation of the
peripheral vessels was developed, the cervical sympathetic was again —

stimulated. The strongest stimulating current available failed to elicit
the slightest response.

The findings here presented are contrary to our nese belief that.
the active functional peripheral resistance is to be found wholly in the

smaller arterioles with. smooth muscle in their coats. The evidence

given indicates that nerve impulses along vasomotor fibers may play
upon the caliber not only of the arterioles but on that of the eapillari
and venules as well. We must therefore modify our conception of th
peripheral resistance in the matter of functional activity to include the
~ whole peripheral vascular bed including therewith the arterioles,
capillaries and venules.

It will be obvious, furthermore, that if the conception in regard to
the peripheral resistance here advanced is substantiated, the body pos-
sesses a remarkable mechanism for the regulation of the distribution
of the blood. For by alterations in the tonic capacity of the capillaries
and venules, which is under the control of the central nervous system,
circulating corpuscles as well as plasma can be mobilized to a very con-
siderable degree in accordance with the physiological needs of the
various tissues both local and general. It would seem also to follow
that blood volume and plasma volume determinations must be subject
to the same physical mechanism (34).

If, as is highly probable, specific nerve fibers supply the different
parts of the peripheral vascular bed (arterioles, capillaries and venules)
the play of functional adjustments must be exceedingly complex.
Hitherto it has been possible to invoke chemical processes alone to
account for many of the exquisite adaptations recognized to occur in

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FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES A?

physiological adjustments. In the light of the facts here presented

“we may conceive of a highly organized nervous mechanism adapted.

to quick and efficient response superimposed upon the primitive chemi-
cal methods available to the organism. There is doubtless a happy
coaptation between these two major processes of control butonthe body
surface, exposed to noxious environmental factors, and in the voluntary
muscles where quick adjustments of blood supply are constantly de-
manded, we might expect the nervous regulation to play a significant
teleological rdle. In the glands and deeper body tissues generally, on

the other hand, where reaction time is of less significance, responses

may largely depend upon chemical factors for their instigation.

If the body has at its control such a highly organized device for the
disposition and partition of the blood by which extensive capillary
beds may be largely emptied of or packed with corpuscular elements or
plasma, an explanation is readily found for the uncertainties of blood
cell counts and blood volume determinations. Individual capillaries
may be opened up or closed or they may be gorged with stagnant
inactive corpuscles, as is possible to demonstrate on the finger (32).
Mediation of these and similar changes would be accomplished by
activation of the arteriole, capillary or venule functioning individually

or collectively. It is natural to infer that normally such forces are well

balanced and counteract one another so that the volume and corpuscu-
lar composition of the blood is held relatively stable, but in time of
physiological stress or in disease the alterations which develop might
assume considerable proportions. ‘The significance of this mechanism
for the nutrition of the tissues will likewise be apparent since it may
be presumed to exercise control over the rate at which plasma passes
through the capillaries and into the tissue spaces.

The present work does not include a demonstration of nervous regu-

lation of vasodilatation in the capillaries and venules but the evidence

justifies the assumption of such an hypothesis. On the other hand,

the primary constriction followed by dilatation which occurs after |
death indicates that chemical regulation may function both in constric- |

tion and dilatation. The effects of the injection of epinephrin and of
histamine likewise substantiate this conception. These findings accord

with the recent clean-cut. results obtained by Krogh (1) on the in-

crease in number of patent capillaries in active muscle and the similar
results recognized to occur in the early stages of inflammatory processes
(4). The work of Gaskell (35), Bayliss (36) and the writer (37) on the
chemical regulation of peripheral resistance is thus to be interpreted

<; ss —

48 D. R. HOOKER

that chemical factors constrict as well as dilate the finer vessels other
than the arterioles.

To further substantiate the fact that nervous inipuleok saan pro-
duce a constriction of capillaries and venules, attention may again be ~
—ealled to the condition of these experiments. ‘The vascular bed under
observation lay some 2 cm. below heart level. An occlusive econstric-
tion in the arterioles could not therefore passively drain the vessels
distal to the constriction and supporting a hydrostatic column. I+ is
conceivable that, if these vessels were under tension due to a vis a
ergo, they might decrease in size when their filling pressure was shut
off but they would not empty. Indeed it was found in an experiment
in which the carotid was occluded long enough to bring the blood stream
to a standstill in the peripheral vessels, that the capillaries showed no
appreciable decrease in size and that the corpuscles did not tend to _
clump. » The latter point is small but significant because when the/
capillaries are made to contract, as by nerve stimulation, the cor-
puscles invariably tend to gather together and move along in clumps.

Lister (4) in that splendid paper to which reference has already been
made on the ‘Early Stages of Inflammation’’ published in 1858, de-
scribes an experiment on the frog which is of decided interest in this
connection. He was studying with the microscope the behavior of the
peripheral vessels in the web of the foot under various conditions and
in this experiment he observed that irritation of the cord caused the
capillaries and venules to disappear from view. He ascribes this result
to an active constriction of the aterioles sufficient to block the passage
of the red cells but insufficient to stop the flow of plasma so that the
corpuscles floating in the capillaries and venules are washed onward
from the field. The red blood cells of the frog are of course relatively
large and could presumably be blocked in the manner indicated, but 4
I am inclined to believe that Lister actually saw a constriction of the
vessels in question. In the first series of experiments described in the
present paper the evidence is clear that, after the heart has ceased to
beat, the capillaries and venules can empty themselves not once but ,
twice against an appreciable hydrostatic resistance. The movement
and disposition of the corpuscles under these conditions is not to be .
distinguished from their behavior under the influence of nerve stimula-
tion; the stream stops quite abruptly, the corpuscles congregate in |
masses and then progress slowly and without definite regularity. This
forward movement occurs against a hydrostatic resistance and in spite
of the fact that the corpuscles would tend in a stagnant plasma, be-
cause of their specific gravity, to settle and adhere to the vessel wall.

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FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 49

Doctor Connet has recently shown ina research done in this labora-
tory (38) that the injection of epinephrin raises the systemic venous
blood pressure. In this work she was able to exclude the slowing of the

heart which has hitherto been regarded as sufficient to account for the |

phenomenon, and reached the conclusion that the substance acts by a

direct effect upon the veins in the intact animal just as it has been re- |

peatedly shown to act upon isolated vein preparations. It seems

highly probable in view of the results presented here that the rise in |

venous pressure following the injection of epinephrin demonstrated
by Doctor Connet is associated with a constriction of the capillaries
and venules as well as of the veins.

Experiments with histamine. With a method available for the study
of the capillaries in the mammal it was quite natural that one should
be led to a study of the effect of histamine. The attractive hypothesis
advanced by Dale (23) that histamine ‘“shock’’ (and probably trau-
matic ‘‘shock’’) is due to a specific toxic action upon the capillaries
rested upon indirect evidence. It was possible with the preparation
at hand to put this hypothesis to a direct test.

References have already been made to the toxic action of histamine
on the capillaries and venulesin the experiments previously described :-—
after the injection of histamine post-mortem constriction of these
vessels was absent or very slight and no constriction could be obtained
by nerve stimulation. In addition to these experiments a number of

- experiments were performed, seven in all, in which attention was

directed primarily to the histamine effect. Ergamine phosphate, 6

mem. per kilo in salt solution, was injected intravenously in accorda ce

with Dale’s technique.
The results were uniformly clean-cut and decisive. Within a few

‘minutes after the injection the capillaries and venules were filled with

stagnant blood-and definitely dilated. The dilatation was distinctly
more conspicuous in the venules. These changes developed in con-
junction with the fall in arterial blood pressure and in one experiment
in which it was followed with a fall in venous pressure.

Doctor Rich has obtained similar results in the capillaries of the: mam-
malian omentum (39) by a different method. Rich found that flooding
the peritoneal cavity with Zenker’s fluid gave prompt fixation of the
tissue so that it could be removed and studied under the microscope.

‘If the tissue was thus fixed immediately after the intravenousinfusion of

histamine, marked capillary and venous dilatation and engorgement
could be demonstrated. This vascular change was entirely absent in

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FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 51

control experiments in which salt solution was infused instead of
histamine.

Usually, although not invariably in my experiments, this dilatation
was preceded by what must be interpreted to be a constriction, under

_ the experimental conditions. This constriction lasted a variable but

brief period of time, frequently so short that it could not be photo-
graphed satisfactorily. This reaction is especially well exemplified in
figure 4 in which the photograph which shows the constriction was
taken ten minutes after the injection of ergamine when the arterial
pressure was 24mm.Hg. The arterial pressure was still at 24 mm. Hg.
thirty-five minutes later when the last photograph shown in this figure
was obtained.

This transitory constriction of the capillaries and venules may not
occur throughout the body since Rich was unable to find any evidence
of it in the capillaries of the omentum. It may be due to a primary
central effect of the poison or, what seems more probable at the moment,
it may represent one of those curious reactions according to which a
drug or substance depressive i in effect at first acts as a stimulus. Such

a transitory reversal of effect is not uncommon in perfusing the iso-

lated heart with inorganic salts and I have observed a similar effect in
perfusing the respiratory center (40). Burridge (41) has suggested,

_ in the case of the heart, that the condition is associated with the state

of aggregation of the colloids.
Although it thus appears possible that histamine may under certain

- conditions produce its primary ‘‘shock”’ effect while the capillaries and

venules of the ear are constricted, these experiments as a whole undoubt-
edly lend strong support to Dale’ s hypothesis to explain: histamine
“shock.”

In conclusion it will not be out of place to state that the preparation
used in these experiments offers an excellent method of demonstrating
the capillary circulation in the mammal to students. A low power
microscope (ocular 1 and objective 3), adapted by removal of the stage
to fit over, the edge of an animal holder and a good light are all the
apparatus that is required. The capillary circulation can be similarly
observed in the rabbit and presumably also in the dog although the
latter animal has not been investigated. The rabbit’s ear is, however,
distinctly more susceptible to inflammatory processes than is that of
the cat.

52 D. R. HOOKER

SUMMARY AND CONCLUSIONS

A method is described whereby the peripheral circulation (particu-
larly the capillaries and venules) in the cat’s ear may be observed and
photographed. It is thus possible to study in the living mammal the
capillary circulation, investigation of which in the intact animal has
hitherto been limited to the frog. Making use of this method, the
following experimental results were obtained:

1. After ether death the peripheral vessels at first constrict so that
they are largely emptied of blood. This constriction which occurs usu-
ally within a few minutes lasts but a short time and is followed by a

marked dilatation and engorgement. Subsequently and roughly coinci-

_ dent with the development of skeletal muscle rigor, the vessels are again
emptied of blood. The latter condition prevails indefinitely (fourdays
at room temperature).

These changes are much less conspicuous or entirely absent if the
animal is previously injected with histamine. They are not affected
by section of the vasomotor nerve fibers (cervical sympathetic).

It is thus concluded that these post-mortem vascular reactions are
independent of the central nervous system and that they are abolished
by an endothelial poison. si

It is suggested that the first constriction represents a response to
asphyxia and may be a significant factor in the asphyxial rise of arterial
and venous blood pressure.

2. Section of the vasomotor fibers to the part (cervical sympathetic)
did not cause an appreciable dilatation of the vessels but electrical stim-
ulation of these fibers gave clear evidence, by causing constriction,
that the capillaries and venules are undersympathetic nervous control.

This fact was further substantiated by the injection of epinephrin-

which caused a similar vascular response.

The reaction to nerve stimulation could not be obtained in an animal
poisoned with histamine. .

These results indicate that the functional peripheral resistance is
not limited to the arterioles brit includes the capillaries and venules as
well. It is inferred that this resistance is subject to chemical’ as
well as nervous control. This concept involves a reorganization of our
present beliefs concerning the peripheral resistance and implies the
existence of an efficient physical mechanism for the distribution and
regulation of the circulating blood volume and of the supply of nutri-
ment to the tissues,

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FUNCTIONAL ACTIVITY OF CAPILLARIES AND VENULES 53

3. The injection of histamine (ergamine phosphate) causes a prompt
and permanent dilatation of both capillaries and venules with on
tion of the corpuscular stream.

This reaction appears to be characteristic and thus confirms Dale’s
hypothesis that histamine ‘“‘shock’”’ (and presumably traumatic
“shock’”’) is largely dependent upon the reaction of the capillaries.
It should be noted, however, that the present results extend this reaction
to include the venules as well as the capillaries.

Prior to the dilatation which appears to be the characteristic histamine
effect, there is usually a short period when the vessels in the ear are
constricted. Neither this transitory effect nor the general histamine
effect is influenced by nerve section. Both effects are thus due to the
direct action of the substance on the vessels.

It is probable that vessels elsewhere in the body do not show this
passing constriction since it may persist for some minutes after the
systemic arterial pressure is at shock level and give place.to rear capone
while the blood pressure level remains unchanged. :

BIBLIOGRAPHY

(1) Kroau: Journ. Physiol., 1919, lii, 457.

(2) Hit: Schafer’s Text, book of physiology, London, 1900, ii, 166.

(3) Lomsparp: This Journal, 1912, xxix, 335.

(4) Lister: Phil. Trans., 1858, exlviii, 645.

(5) Srricker: Sitzungsb. d. kais. Akad. d. Wissensch., 1865, li, 1

(6) Stricker: Untersuch.z. Naturl.d. Mensch. u.d. Thiere, Giessen, 1866-70, x.
(7) Stricker: Vorlesungen iiber die allgemeine und experimentelle Tv AinemEte,

Wien, 1883, 675.

(8) Connuermm: Arch. f. path. Anat., 1867, xl, 42.

(9) Gotusew: Arch. f. mikros. Anat., 1869, v, 49.

(10) Tarcuanorr: Pfliiger’s Arch., 1874, ix, 407.

(11) Severinti: Ricerche sulla innervazione dei vasi sanguigni, Perugia, 1878.
(12) Roy anp Brown: Journ. Physiol., 1879, ii, 323.
(13) Kroeu: Journ. Physiol., 1919, liii, p. xlvii.
(14) Brevi: Quoted from Steinacu AND Kaun (see 21).
(15) Mayer: Quoted from Sain (see 19).

_ (16) Cuarx: Anat. Rec., 1909, iii, 183.

(17) Kyrmanor: Anat. Anzeiger, 1901, xix, 369.
(18) Camus anp Guey: Arch. d. Physiol., 1894, vi, 454.
(19) Sasin: Johns Hopkins Hosp. Rept., 1916, xvii, 347.
(20) SHArreR: Quain’s Anatomy, vol. II, part I, p. 346. London, 1912.
(21) Srernacu anp Kaun: Pfliiger’s Arch., 1903, xevii, 105.
(22) Rovexrt: Arch. d. Physiol., 1873, v, 603.
(23) Date anp Laipuaw: Journ. Physiol., 1919, lii, 355.
’ Dae anv Ricnwarps: Journ. Physiol., 1918, lii, 110.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 1

4 >;

54 D. R. HOOKER

(24) Bayuiss: Intravenous injections in wound shock, London, 1918, 108.
(25) Cannon: Journ. Amer. Med. Assoc., 1919, Ixxiii, 174.
(26) Aspen anp Kusora: Journ. Pharm. Exper. Therap., 1919, xiii, 243.

(27) Marry: Ann. d. sci. nat., 4° serie, ix, cashier 2, 1858. Quoted from La Cir- ©

culation du Sang, Paria, 1881, 377.
(28) Buock: Arch. d: Physiol. norm. et path., 1873, v, 681.
(29) Ryan: This Journal, 1918, xlv, 537.

(30) Corton, SuapE AnD Lewis: Heart, 1917, vi, 227.

(31) Sotumann: Journ. Pharm. Exper. Therap., 1917, x, 147.
(32) Tuayson: Ugeskrift f. Laeger, 1920, Ixxxii, 473. Quoted from Journ. Amer.
dein Med. Assoc., 1920, Ixxv, 70.

(33) DaNzER AND Hoownn: This Journal, 1920, lii, 136.

(34) Smita: This Journal, 1920, li, 221.

(35) GaskEeLu: Journ. Physiol., 1880, iii, 48.

(36) Bayiiss: Journ. Physiol., 1901, xxvi, p. xxxii.

(37) Hooxerr: This Journal, 1911,. xxviii, 361.

(88) Connet: This Journal, 1920, liv, 96.

(89) Ricu: Personal communication.

(40) Hooxrr: This Journal, 1915, xxxviii, 200.

(41) Burripee: Quart. Journ. Exper. Physiol., 1915, viii, 331.

STUDIES ON THE VISCERAL SENSORY NERVOUS SYSTEM
I. Lune Automatism AND Lune REFLEXES IN THE FROG (R. ‘PIPIENS
: AND R. CATESBIANA)

A. J. CARLSON anp A. B. LUCKHARDT
From the Hull Physiological Laboratory, University of Chicago

Received for publication July 12, 1920

This report is the beginning of an investigation and analysis of the
reflexes evoked by the visceral sensory nerves in all the groups of verte-

brates available for study. The inception to this line of work was the

observation on man (7) that strong contractions of the empty stomach
produce reflex effects on the cardiac and the vasomotor centers. ‘To
date we have studied the reflexes from the visceral afferent system .
involving the skeletal musculature, the respiratory mechanism, the
gastro-intestinal tract, the heart and blood vessels, and the urinary
bladder. In some cases our reflex results compelled us to re-investigate
the motor mechanisms of the organ involved in the reflex response.

This is true especially of the lungs.

We have today fairly comprehensive and accurate knowledge of the
efferent nervous mechanism of the viscera, thanks to the work of

Gaskell, Langley and others.

On the sensory or afferent side our information is made up largely
of gaps and guesses, despite its probable importance in functional
integrations in health and disease. This phase of physiology has been
studied especially with reference to conscious visceral sensations, to
witness only the work of surgeons and internists on direct and referred
visceral pain, and of physiologists and psychologists on the sensibility
(conscious) of the alimentary canal. To our knowledge a thorough-
going investigation of the sub-conscious reflexes evoked from the vis-
ceral sensory nerves in health and disease has not been made. In

- Gaskell’s recent monograph (8) on the involuntary nervous system

the afferent component of this system is not even mentioned, and in

Sherrington’s article on the ‘Sympathetic Nervous System’ in the

1911 edition of the Enclyclopedia Britannica the afferent component
55

56 A. J. CARLSON AND A. B. LUCKHARDT

is dismissed with the following sentence: ‘Of the afferent fibers of the
sympathetic little is known save that they are, relatively to the efferent,
few in number, and that they, like the afferents of the cerebro-spinal
system, are axones of nerve cells seated in the spinal ganglia.”’

EXPERIMENTAL METHODS

1. The lung contractions were registered by means of water manom-
eters (diameter 8-10 mm.) connected with small glass cannulae
inserted and tied in the tips of the lungs. For the most delicate lung
contractions these water manometers were not sufficiently sensitive,
and in the study of these phases a very delicate tambour was employed.
In fixing the cannula in the tip of the lung care must be taken so that
the direct handling of the lung is minimum and gentle, as direct and
rough handling induces prolonged tonic contractions that may involve
the whole lung. In animals in poor physiological conditions, the lungs
are usually quite atonic, and these contractions due to direct handling
(mechanical stimulation) are less in evidence.

In experiments with the glottis open and the frog preparation breath-
ing spontaneously no artificial pressure can be maintained in the lungs,
because if the lungs are collapsed through cannulae in the lung tips,
the frog promptly fills the lung again up to the original pressure. If
this original pressure is slightly exceeded by inflation through the can-
nula the glottis is promptly opened and the pressure reduced. In the
experiments involving the closure of the glottis the lungs were prac-
tically always collapsed and empty at the conclusion of this operation.
In the subsequent inflation of the lungs we always took pains not to
exceed the normal pressure maintained by the frog (1-3 em. water).

Most of the previous investigators of the physiology of the respir-
atory movements in the frog have used various methods for graphic
registration of the throat and flank movements (Martin (16), Weden-
skii (26), Langendorff (14), Sherrington (23), Baglioni (3), Soprana
(24), Nikolides (20), (21)). Brown (5) and Willem (27) recorded intra-
pulmonic pressure by means of cannulae in the tip of the lungs. Mochi
(17), (18), (19) placed the body of the frog, except head and throat,
in a plethysmograph and closed the plethysmograph by sectioning the
frog’s skin around the neck and tying to the plethysmograph tube.
It seems to us that the method of Mochi introduces more trauma and
abnormal physiological conditions than a slit through the abdominal
wall for placing cannula in the tip of the lungs.

i Or |. ee Ca ee.

= A ee EE ne a ele ee ee ee

VISCERAL SENSORY NERVOUS SYSTEM 57

2. For the registration of the variations in the intrapulmonic pressure
during normal respiration the animals were usually decerebrated, and
slits made through the abdominal wall over the lung tips, of sufficient
size to insert cannulae in the lung tips. This incision through the
abdominal wall was made with or without local application of cocaine.
The animals were then placed, usually without restraint, on a board
or preferably in a small dark box. In most cases animals thus pre-
pared would sit quietly for long periods, unless disturbed by external
stimulations. In a few animals the abdominal incisions were made
under local anesthesia without previous decerebration.

8. In the experiments where it was necessary to separate the lungs
completely from the influence of skeletal muscle contractions several
methods of procedure were used:

a. After decerebration and fixing the cannulae in the lung tips, the

‘animals being placed in normal position (ventral side down) on the

board or in the dark box, the abdominal muscles were cut away and
the spinal cord pithed below the brachial plexus. In such a preparation
movements of the head, strong respiratory movements (swallowing)
or movements of the front legs will alter the intrapulmonic pressure,
but the rapidity of these movements is much greater usually than
the lung contractions so that the latter can be readily differentiated
from the passive effects of the former movements, and in favorable
preparations the former movements may be absent over considerable
periods, thus giving the lung contractions free play. |

b. Without previous decerebration the spinal cord was cut just below
the medulla and pithed the entire length caudad, the animal placed
on the board dorsal side down, the abdominal wall opened for its entire
length by a median incision, and the lungs completely isolated, except
for their anatomical connections with the pharynx and esophagus.
Animals thus prepared continue to breathe spontaneously for consider-
able periods if care is taken not to injure the lungs or pharynx and
prevent exsanguination. Head and pharyngeal movements are still
capable of influencing the intrapulmonic pressure mechanically. The
isolated lungs were prevented from drying by a thin layer of absorbent
cotton kept moist with Ringer’s solution.

4. Closure of the glottis. Our greatest technical difficulty consisted
in proper closure of the glottis in experiments where this procedure was
essential. The frog has no trachea and bronchi. The glottis opens
directly into a rather large tracheal sac which communicates with the
base of each lung. This tracheal sac is so closely adherent to the tis-

58 A. J. CARLSON AND A. B. LUCKHARDT

sues at the base of the heart that we found it impracticable to close
the lungs by ligation or compression of this sac without an amount of
injury to the heart nerves, and main blood vessels that resulted in quick
failure of the circulation. The following experiments were tried with
out practical success:

(1) Ligation of base of lungs at their junction with the seach sac,
leaving the lung blood vessels and nerves outside the ligature. This
failed because of the impossibility of accomplishing the latter without
puncturing the lung wall, or if successful the ligation produced enough
anatomical distortion to interfere with the lung circulation. |

(2) Closing the tracheal sac or either lung by small wads of cotton
pushed through the glottis. This failed mainly because the glottis
opening is smaller than the diameter of the tracheal sac or its com-
munication with the lungs. Hence the cotton wad passed sooner or
later into the lung cavities.

(3) It was noted, when the median incision exposing and isolating
the lungs was carried forward to the level of the base of the heart
only, that lateral and dorsal tension exerted by pull on the front legs
would prevent air from entering or leaving the lungs by the normal
breathing movements. This was evidently due to collapse of the
tracheal sac by external compression. This gave us the clew to a
method of closing the glottis and blocking the air communication be-
tween the two lungs with the least possible trauma or physiological
violence. A cotton plug of suitable size, with or without a coating of
vaseline, was inserted through the mouth and pushed down the esopha-
gus to the level of the glottis and the tracheal sac. The pressure thus
exerted on these structures from the esophagus not only closed the
glottis but usually compressed the tracheal sac sufficiently to prevent
air communication between the two lungs, especially if in addition
the front legs were put under slight dorso-lateral tension.

This mode of procedure sufficed for the degrees of lung contractions

accompanying the normal respiratory movements, or induced by reflex —

stimulation. But it proved inadequate in case of the extreme tetanus
of the lungs following section of the vago-sympathetic nerves or pithing
of the medulla. These strong contractions always forced the glottis
open against the cotton plug in the esophagus. Hence in all the experi-
ments on this phase of lung physiology the glottis had to be closed
more firmly. This was done by clamping the rim of the glottis with
a slender artery forceps. The mouth being held open, a slender hook
was passed through the glottis, under gentle forward traction the rim

oe ee

VISCERAL SENSORY NERVOUS: SYSTEM: — §>

of the glottis was compressed with a slender. artery forceps, a small
cotton plug pushed into the esophagus and left in situ together with
the forceps. Care must be taken not.to place the foreeps-too far down
on the tracheal sac and pharyngeal tissues, as in that case the pulmo-:
nary branches of the vagi as well as the lung blood vessels are included:
in the grip, or placed on such tension that vagus-action on aie lung resin
lung circulation are interfered with.

The essential drawback to this procedure is the Hanns produced
or rather the violent mechanical stimulation of the sensory nerves in’
the glottis, larynx and pharynx by the compression. Placing the
artery forceps in the region described produced something like
profound prostration or ‘“‘shock’’ in the preparation. Respiratory move-
ments cease for a considerable period, and in the case of animals other-
wise in poor condition may not return at all. It is scarcely necessary
to add that the results reported, using this method of closure of the-
glottis, are based on the vigorous seksi in desis mpoptancous
respiration returned.

§. The mucus in the lung cavities can, of course, not be eliminated:
from the lungs in the normal way under any condition of glottis ob-
struction. Furthermore, the unavoidable trauma to lungs and tracheal:
sac in preparation may actually increase the mucous secretion. This
lung mucus is a hindrance and a source of error in registering lung tonus.
and contractions by our method, as strong contractions may force
some mucus into the cannula in the lung tips, and this will interfere
with the prompt and accurate response of the water manometer to-
slight variations in the intrapulmonic pressure: It is needless to say:
that preparations must be discarded in which the lung mucus interferes
with accurate recording of lung contractions. Ns

6. Administration of the drugs. All the drugs used, unless cadionwiee
noted, were mixed with varying quantities of Ringer’s solution and:
injected slowly into the abdominal vein. A few injections were made
directly into the heart.

7. Prevention of asphyxia after closure of the glottis. It was, of course,
essential to maintain circulation and lung ventilation even after closure
of the glottis, so that abnormal reflexes and local lung reactions would:
not be set up by asphyxia. We endeavored to maintain good circula-
tion by ligation of the main blood vessels sectioned in the preparation
of the animal and by occasional intravenous injections of small quan-
tities of Ringer’s solution. The frog’s heart is apparently very sensitive
to the mechanical factors of filling, as it ceases to beat entirely or beats

60 A. J. CARLSON AND A. B. LUCKHARDT

very feebly when the blood pressure is very low, but resumes an ade-
quate rhythm on replacing the lost blood with Ringer’s solution. |

’ It is well known that the frog in water carries out a considerable
proportion of its gaseous exchange through the skin. Under the
temperature conditions prevailing in the laboratory during this work
the frogs (R. pipiens) would remain under water for 18 to 25 minute
periods, come to the surface and make a few vigorous respirations and
submerge again for the same length of time. Evidently the filling the
lungs with air, supplemented with the skin respiration, met the respir-
atory needs for 20 to 30 minutes. On the basis of these facts, we
always kept the skin of our frog preparations moist with water, and
gave occasional artificial respirations, except in cases of extreme lung
tétanus when the latter procedure would have been useless.

&. All the tracings reproduced with this report were taken with the
same speed of the kymograph. The time record is not always attached
to the tracings reproduced, for reasons of economy of print paper. But
the reader interested in any question involving the time element as a
matter of importance can readily transfer the time tracing, given in a
few of the tracings, to the others; 25 cm. of tracing (original size) = 17
ininutes.

LUNG TONUS AND LUNG CONTRACTIONS DURING NORMAL RESPIRATION

1. The anatomy of the frog’s lung is well known. ‘The reader will
recall that the lung is a paired muscular sac, numerous septa on the
interior surface dividing this into small spaces or alveoli. The septa
extend only a few millimeters from the lung wall, so that the larger
part of the lung cavity is a large single air space. There are no bronchi
and no true trachea, the tracheal sac having essentially the same struc-
ture as the rest of the lungs, and probably carries out the same meio
atory function.

Smooth musculature covers the entire wall of the lungs and extends
into the smallest septa on the inner surface. More or less definite
external muscle strands follow the course of the main pulmonary blood
vessels on the lung surface.

The arrangement of the lung musculature is such that contraction
(even of the septal musculature) will reduce the size of the lung cavity,
or raise the intrapulmonic pressure in case the air in the lung is not
free to escape.

VISCERAL SENSORY NERVOUS SYSTEM 61

_- The action of the septal musculature would be analogous to that
of the bronchial constrictor muscle of the mammalian lung. So far
as we know, the mammalian lung has no spain? 55 to the lung wall
musculature in the frog.

After having discovered the striking ‘ieunioaal motor automatism
of the frog’s lung we become especially interested in the local nervous

tissue in the lung of this animal group. According to the histological
investigations of Arnold (1), Smirnow (25), Cuccate and Wolff (28),

there are numerous ganglia, as well as isolated ganglion cells (multi-

polar and bipolar) along the course of the main vago-sympathetic

nerve trunks on the surface of the lungs. There are medullated and
non-medullated nerve fibers in these nerve trunks. A plexus of fine
non-medullated nerve fibers surrounds the strands of lung musculature.

_ The ganglion cells and these nerve plexuses are most abundant at the

base of the lungs. Arnold points out that the ganglia and ganglion
cells in the frog’s lung are histologically identical with those of the

_frog’s heart. They are also probably identical, both as to histology

and function, with the ganglionic plexuses (Auerbach) in the wall of
the gut, especially as the lung is a diverticulum from the esophagus.

2. The external respiratory mechanism of the amphibians differs
from that of all other air-breathing animals in that the air enters the
lungs under positive pressure due to the act of swallowing. One might
therefore surmise that in the amphibia the respiratory center in the
brain is anatomically and physiologically identical with the center
for deglutition.

The sequence and codrdination of the respiratory acts (buccal move-
ments, closing of nares, expiration and inspiration or swallowing air)
have been correctly analyzed and described especially by Langendorff
(12), (13), Baglioni, Brown (5) and others, and most recently by Wil-
lem (27). We have nothing new to add on that point, and can con-
tribute no new facts bearing on the old and new speculations as to
phylogenetic and physiological significance of the buccal movements
which proceed rhythmically between the actual renewal of air in the
lungs (swallowing).

The types of the respiratory rhythm as revealed by the intrapulmonic
pressure in normal and in decerebrated frogs are shown in figure 1.
The filling of the lungs by swallowing (upstroke) is preceded by opening
of the glottis and escape: of some air into the buccal cavity. According
to most of the competent and recent investigators, the nares are closed
during the whole act so that while the air escapes from the lung it

62 A. J. CARLSON AND A. B. LUCKHARDT

does not actually escape through the nares. Rebreathing is therefore
a marked feature of‘the frog’s lung respiration. The buccal movements
going on between the swallowing acts and with the nares open, bring
fresh air into the buccal cavity.

In exceptional cases there is‘a perfect synchrony between the buccal
movements and the actual air swallowing (fig. 1, B). But usually

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‘

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ii sl sy

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Fig. 1. Records of intrapulmonic pressure in frogs. Tracings A, D, E, taken
by water manometer; tracings B and C by air transmission and tambour. Trac-
ings A to D, Rana pipiens, animals decerebrated and the tip of one lung exposed
by small abdominal incision. No anesthetics, animals sitting quietly in nor-
mal posture. Tracing EH from bull frog (R. catesbiana), tip of lung exposed by
small abdominal incision after cocaine, animal sitting quietly in a darkened moist
box; time, 5 seconds. Showing varying types of respiration in the frog.

All the tracings n this article are reduced to about ¢ of the original.

several buccal movements are made between each swallowing act
(fig. 1, A), and a striking feature of the air swallowing rhythm in most
frogs is a periodicity similar to the Cheyne-Stokes breathing inmammals.
This has been observed by most of the former investigators on the
respiratory movements in’ the frog (Luschinger and Sakalow (15),
Langendorff (12), (13), Wedenski 26, Sherrington (23), ete.). The

a Re

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poe,

9

VISCERAL SENSORY NERVOUS SYSTEM 63

frog may go on over long periods swallowing as much air as that which
previously escaped through the open glottis, thus maintaining a con-
stant general level of intrapulmonic pressure of 1 to 2 cm. of water.
This type may periodically change into one in which during a few
powerful air swallowings the amount of air forced in greatly exceeds
the quantity that escaped between the time of glottis opening and the
swallowing act. In consequence of this the lungs expand and the
intrapulmonic pressure rises from the general level of 1 to 3 cm. of water
up to a level of 6 to 9 cm. of water. All respiratory movements then
cease for periods varying from 5 to 60 seconds and the act is renewed,

that is, the quantity of air let out of the lung in each respiratory act

is greater than that forced in; the lung shrinks and the intrapulmonic
pressure falls to its former general level of 1 to 2 cm. of water (fig. 1,
C, D, E).

According to our experience, this is the usual type of respiration in
the frog. As previously noted by Sherrington and others, decerebra-
tion or other methods of preparation are not responsible for inducing
it. It is probably a normal rhythm developed in connection with the

habitual under-water existence of the animal. During the respiratory

pause with high intrapulmonic pressure this pressure, as recorded by
the ordinary water manometer, may show an initial rise and then re-
main at a fairly constant level until the next respiratory act, but if

_the pause is long the intrapulmonic pressure gradually falls, due, not

to escape of air through the glottis, but to relaxation of the tonus of
the lung musculature.
8. Active lung contractions and lung inhibitions associated with the
respiratory movements. : .
a. Contractions. The reader’s attention is invited to the tracings
reproduced in figures 2 and 3. It will be noted, especially on the upper

tracing in figure 2, that at the end of the last respiration followed by

a Cheyne-Stokes pause, there is a latent period of 1 or 2 seconds fol-
lowed by a rise in the intrapulmonic pressure that may exceed the

‘maximum upstroke of the final inspiration. When the pause is suffi-

ciently long and registration apparatus sufficiently delicate, it will be
seen that this rise in pressure is due to a contraction lasting from 10
to 15 seconds. These contractions are evidently due to the activity
of the lung musculature, for we have observed them in animals after
isolation of the lungs, fixation or resection of the abdominal and shoulder
muscles or destruction of the entire spinal cord below the medulla.
Moreover, the changes in the intrapulmonic pressure due to active

A. J. CARLSON AND A. B. LUCKHARDT

64

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VISCERAL SENSORY NERVOUS SYSTEM 65

contractions or relaxation of the skeletal musculature are more rapid.
The contractions are also too slow to be due to passive elastic rebound
of the connective tissue of the lung. They are, however, very similar
to the quick spontaneous contractions that are seen at times in the
hypertonic frog lung after cutting the vagi or complete destruction
of brain and spinal cord (fig. 7). The tracing in figure 3 illustrates the
fact that these lung contractions may follow single respiratory move-
ments, if the pause between two successive swallowings is of sufficient
duration.

It would seem that these contractions of the lung musculature fol-
lowing the active inspiration or attempts at inspiration have not been
seen by previous workers, except possibly Graham Brown (5). On

Fig. 4. Water manometer tracings of the contractions of the lung musculature
in the frog (Rana pipiens) that follow upon the external respiratory movements.
Spinal cord transected and destroyed below the medulla. Lungs isolated and
cannulated (tip). Glottis closed with forceps so that there is no communication
between the two lungs. A, upper tracing equals left lung; lower tracing equals
right lung. B, same. X = pharyngeal respiration or attempt at swallowing
air.

some of the tracings published by Brown there is an increased pressure
in the lungs shortly before expiration, and Brown suggests that this
is due to muscular contractions in the lungs.

~The lung contractions do not depend on the change in tension on
the lung tissues following a forceful inspiration. The glottis may be
closed and the pressure in the lungs raised to that of the normal of
1 to 3 em. of water, and in such preparations each attempt at respira-
tion, single or a series, is followed by lung contractions (fig. 4). In
preparations with the glottis closed the contractions are usually more
prolonged than those seen in figures 2 and 3.

We are thus forced to the conclusion that in the frog the normal
inspiratory movements lead to active contractions of the lung mus-

66 A. J. CARLSON AND A. B. LUCKHARDT

culature through associated innervation, either of the motor fibers to
the lung or through central depression of the inhibitory fibers control-
ling a peripheral automatism.

b. Inhibition. In preparatioris with the glottis closed, and in intra-
pulmonic pressure approximately normal (1-3 em. of water), the active
respiratory movements lower the intrapulmonic pressure, evidently by
inhibition of the lung muscle tonus (fig. 5). With the glottis closed
no air can enter or leave the lungs. The respiratory movements of
the throat and pharynx, especially if they are vigorous, induce slight
fluctuations in the intrapulmonic pressure of equal rapidity with the
pharyngeal movements. Vigorous movements of the head may induce,

Fig. 5. Water manometer tracings of the intrapulmonic pressure in frogs
(R. pipiens). Spinal cord cut and pithed below medulla. Cannula in tip of lung,
and glottis closed (imperfectly) by cotton and collodion. A, simultaneous record
from both lungs ofthe bull frog. B, record from lung of R. pipiens. Showing
inhibition of the tonus of the lung musculature during the active respiratory
movements. The rapid fluctuations (respiration) on the tracings are due to
movements of larynx and head, and not to entrance and exit of air into the lungs.

possibly, stronger positive pressure in the closed lungs synchronously
with these movements. It is possible but not probable that the lower-
ing of lung muscle tonus during the.rapid and rigorous swallowing
movements are due to mechanical stimulation of the lung from this
source, since the inhibition cannot be produced by similar fluctuations
in intrapulmonic pressure artificially induced, and direct mechanical
stimulation of the lungs when strong enough to have an effect causes
contractions.

The return of the lung muscle tonus following the period of inhibition
is quite similar to the lung contractions described in previous sections
and illustrated in figures 2,3 and 4. So far as we know, this inhibition
of the lung musculature has not been noted by previous investigators.

A. ger te

VISCERAL SENSORY NERVOUS SYSTEM 67

The utility of the correlation is obvious, the relaxation of the lung
musculature during inspiration being favorable to the filling of the
lungs by the swallowing act.

THE PERIPHERAL MOTOR AUTOMATISM OF THE LUNGS AND THE INFLUENCE
OF THE VAGI AN D°THE CERVICAL SYMPATHETIC NERVES ON
THIS AUTOMATISM.

1. In preparations with the glottis closed, lungs isolated from the
influence of skeletal muscle contractions, section of the vago-sym-
pathetic nerves in the neck, destruction of the medulla, or ligation of
the base of the lungs induces immediately a permanent hypertonus or

incomplete tetanus of the lung neuro-muscular mechanism (figs. 6, 8 and

9). By permanent, we refer, of course, only to the time of observation
in these crucial experiments, that is, 2 hours. We found it difficult
to maintain the preparation in good physiological condition over longer
periods, especially with both lungs contracted down so that the lumen
is completely obliterated thus preventing lung ventilation. The circu-
lation also fails gradually.

The hypertonus of the lungs is usually at its maximum shortly after
the isolation from the central nervous system, and there may be a
gradual fall of the tonus during the observation period of 1 to 2 hours.
This gradual fall is probably due to the failure of maintaining adequate
circulation in the lungs, that is, to asphyxia.

This remarkable lung reaction is obtained in all frogs in good physi-
ological condition, and the better the condition of the frog the stronger
the lung tetanus on sections of the vagi. In frogs in poor condition (in-
fected, starved or moribund from any cause) destruction of the medulla
or vagi section causes little or no lung tetanus. In such preparations
stimulation of the peripheral end of the cut vagi also fails to influence
the lung tonus. Poor physiological conditions in the frog are evidently
associated with lung atony, just as these conditions usually involve
atony and absence of stomach rhythm in all species of animals so far
studied.

We have occasionally found preparations showing marked lung tonus
before section of the vagi or destruction of the medulla, that is, a tonus
greater than that of normal respiration. We are inclined to ascribe
this to the following causes: a, temporary depression of the medulla
or nervous “‘shock’”’ due to cutting the spinal cord, strong mechanical
stimulation of many sensory nerves; b, direct mechanical injury to the

A. J. CARLSON AND A. B. LUCKHARDT

68

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VISCERAL SENSORY ‘NERVOUS: SYSTEM. 69°

Fig. 7. Water manometer tracing of the intrapulmonic pressure in the frog
(R. pipiens). Showing various types of peripheral automatism after isolation
of the lungs from the central nervous system (section vago-sympathetic nerves or
pithing of brain). Time, 5 seconds.

SATESTIPCMIRTEALSAFAGRUTTRIFEESUMECTSAGGAITARSOTECEINGGVEREA) GESTED REI TEES Baw atiane (Fasit
mane tt 7 fa al YY yt : fh Preps PAT bey oter

Fig. 8. Water manometer tracing of the intrapulmonic pressure in the frog’s
lung (R. pipiens), showing the incomplete tetany of the lung following destruc-
tion of the brain. Spinal cord cut and pithed below the medulla. Abdomen
opened and lungs freed from influence of skeletal muscles. Cannula in tip of
lung. Glottis closed by forceps, leaving vagi nerves and lung circulation intact.
a, Transection of upper jaw. 6, Crushing of brain (including medulla) by artery
forceps. Time, 5 seconds.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 1

70. A. J. CARLSON AND A. B. LUCKHARDT

lungs; c, partial asphyxia of medulla and lungs from failure of the
circulation (unavoidable hemorrhage, etc.). In such preparations the
section of the vagi or the destruction of the brain may-cause a slight
increase of the lung tonus (fig. 9). The lung of the bull frog passes
into prolonged hypertonus on direct mechanical stimulation more read-
ily than does the lung of the common grass frog (R. pipiens).

In our most vigorous preparation the lung tetanus following iso-
lation from the central nervous system is extreme, that is, all of the
air is driven out of the lung cavity into the water manometer and the
lung cavity is completely obliterated. Even the traces of mucus are

potters
FE ait TT Umi

Fig. 9. Water manometer record of the intrapulmonic pressure in the frog’s
lung, showing moderate tetanus or tonus of the lung on destruction of the brain.
Spinal cord cut and pithed below the medulla; frog fixed on dorsal side, and lungs
isolated from influence of skeletal musculature. Cannula in tip of lungs; glottis
closed by forceps, also shutting off air communication between the two lungs.
Signal.= crushing of brain with forceps. Signal line = the zero line of water
pressure for right lung (lower tracing). Time, 5 seconds.

forced into the cannula in the tip of the lungs. The maximum. intra-
pulmonic pressure thus developed is from. 6 to 10 cm. of water above
the pressure existing during normal respiration, that is, a total pressure
of from 7 to 12 em. of water. This is not the maximum pressure the
lung tetanus is capable of developing. If the lungs are connected with
a mer cury manometer so that séme air still remains in the lung cavity,
even. under, the maximum lung tetanus following vagi section, the
intrapulmonie pressure rises to.the surprising height of 20 to 40 mm. Hg.
(25-50 cm. water).

2. The nature of the peripheral lung tetanus. In most of our prepara-
tions the water manometer tracings of the lung tonus following isolation

reer

VISCERAL SENSORY NERVOUS SYSTEM 71

of the lungs from the central nervous system show a straight line
indicating a continuous tonic or complete tetanic contraction (fig.
9). .The more vigorous preparations exhibit various types of rhythmic
contractions superimposed on the continuous hypertonus, at least dur-
ing the first 15 to 60 minutes following the lung isolation. But even
in these preparations the rhythm fails before the complete failure of.
the tonic state of contraction in later stages of the record. It would
thus seem that the appearance of rhythm on the hypertonic state of
the lung is a question of physiological condition of the lung motor
mechanism. |

The usual type of the rhythmic lung contractions is shown in figures
6 and 8, that is, a slow rhythm, the contraction and relaxation requiring
2 to 3 minutes. In general, the more vigorous the preparation the
more rapid the rhythmic contractions. In other preparations contrac-
tions last only 20 to 30 seconds, and occasionally this faster rhythm
may be superimposed on the slower rhythm (fig. 7), both rhythms being
in turn superimposed on the continuous hypertonus. Whether these
varying types of contractions involve different musculatures cannot be
made out by the present method of experimentation. The continuous
hypertonus as well as the strong slow rhythm seem to involve the whole
lung. The more rapid rhythm is too feeble to be detected by direct
inspection of the lung. Nothing that could be interpreted as peristaltic
contractions similar to those exhibited by other visceral structures has
so far been noted by us, although our water manometer tracings of
the lung hypertonus as slow rhythmic contractions suggest many
points of similarity to the contractions of the empty stomach as recorded
by the balloon method. This may be of significance in connection with
the fact that the lung is a diverticulum from the foregut (esophagus).

It appears that this striking lung tetanus or hypertonus following
isolation of the lungs from the central nervous system has not been
observed by previous investigators, evidently because none have sec-
tioned the vagi after closing the glottis under conditions permitting
recording the neuro-muscular tonus of thelungs. Several workers have
studied the influence of vagi section on the external respiratory move-
ments of the frog. Mochi states that the lungs remain permanently
collapsed and empty when the medulla and vagi are left intact but all
the brain anterior to-the medulla removed. This is probably an error.
Langendorff claims, at least, that the external respiratory movements
persist after removal of the brain anterior to the medulla. According
to Martin (16) and Mochi (17), (18), (19), stimulation of the optic

Fig. 10. Water manometer tracings of the intrapulmonic pressure in the frog
(R. pipiens). Spinal cord cut and destroyed below medulla; lungs isolated and
cannula fixed in tip of lungs. Glottis closed.

A: Right lung: a, section of vago-sympathetic nerve; b, stimulation of periph-
eral end of cut nerve with moderately strong tetanizing current.

B: Left lung: left vago-sympathetiec nerve sectioned. Stimulation of periph-
eral end of cut nerve with weak, a, and strong, b, tetanizing current.

C: Left lung, after section of left vago-sympathetic stimulation of peripheral
end of cut nerve with very strong, a, and moderately strong, b, tetanizing current.

D: Left lung, in very strong tonus after section of left vago-sympathetic
nerve. Stimulation of the peripheral end of the cut nerve with weak, a, and
strong, b, tetanizing current.

E: Upper tracing = left lung; lower tracing = right lung. Signal = stimu-
lation of the peripheral end of the cut nerves. Showing inhibitions of lung tonus
and contractions on stimulation of the peripheral end of the vago-sympathetic
nerve.

72

NES TS ke eI a ey aA ea
;

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VISCERAL SENSORY NERVOUS SYSTEM 73

lobes accelerates the respiratory movements. This may indicate. a
subsidiary respiratory center anterior to the medulla. At any rate
mere decerebration does not induce lung tetanus in our experience.
Martin (16) states that destruction of the brain and spinal cord
leaves the lungs entirely empty of air, but he does not make out or
recognize that this is due to a persistent lung tetanus. Babak (2)
quotes a number of authors as having shown that after vagi section
or lung extirpation the frogs swallow air, periodically, into the stomach,
and the air may actually escape by the cloaca. Berti and Marzemin
(4) state that section of the vagi peripheral to the superior laryngeal
branch results in irregular attempts at lung respiration on elevation
of the temperature. Nikolides (20), (21) states that vagi section slows
the respiratory movements making them at the same time irregular
and stronger. Heinemann (9), one of the earliest observers, states -

that section of both vagi leads in the course of several days to such

abnormal filling of the lungs that some of the viscera are pushed out
through the cloaca. But when he opened the abdomen of these frogs
the lungs were found collapsed or only partly filled. It is possible that
Heinemann’s frogs actually swallowed air into the stomach and intes-
tines because of persistently constricted lungs. Soprana (24) states
that vagotomized frogs breathe slower and deeper, but die more quickly
from asphyxia on elevation of the temperature. According to Pari
(22), the vagotomized frog is unable to fill the lungs, the lungs remain
collapsed for weeks, and the air is forced into the stomach. It is pos-
sible that Pari’s permanently collapsed lungs were in reality in a con-
stricted state. But the method of observation did not suffice to record
the fact. |
It is certain that the force of the air swallowing would fail to cause
air to enter the lungs against the maximum state of lung contraction
found by us after section of the vagi nerves. But we do not know
how long this hypertonus persists in the surviving animal, as all our
experiments to date have been crucial. And even if the hypertonus,
tonus or tetanus remained as long as the frog continued in good con-
dition, failure of lung respiration may soon operate to place the frog

below par in general, in which case there may be depression of the

peripheral lung automatism already noted by us in animals in poor
condition. The process of physiological readjustment of the peripheral
lung motor mechanism may also come into play, similar to the readjust-
ment that gradually takes place in the case of the heart and the respir-
atory center in the medulla after vagi section.

74 A. J. CARLSON AND A. B. LUCKHARDT

3. The action of the vagi and the cervical sympathetic nerves on the
lung motor mechanism. a. The cervical sympathetic nerves. Section of
the cervical sympathetic before its union with the vagus has no effect
on the lung tonus (fig. 11), in marked contrast to the effects produced
by section of the combined vago-sympathetic nerve. Electrical stimu-
lation of the peripheral end of the cut cervical sympathetic causes
slight contraction of the lung on the same side. It is difficult to stimu-
late the cervical sympathetic nerves, under the conditions of our experi-
ments, and at the same time avoid escape of the current to the vagus

Fig. 11. Water manometer tracings of the intrapulmonic pressure in the frog’s
lungs (R. pipiens). Frogs decerebrated, animals fixed on dorsal side, lungs iso-
lated from influence of skeletal muscle contraction. Cannula in tip of lungs.
Glottis closed.

A: Upper tracing, left lung; lower, right lung. a, section right cervical sym-
pathetic; b, section of right vagus; c, section of left cervical sympathetic; d,
section of left vagus.

B: Upper racing, left lung; lower, right lung. a, section of right cervical
sympathetic nerve; signal, stimulation of peripheral end of right cervical sym- ~
pathetic. In this preparation the left cervical sympathetic and both vagi were
intact.

C: Record from left lung, showing lung contractions on stimulation of the
peripheral end of the cervical sympathetic nerve with strong tetanizing current,
the vagus being intact.

Showing motor fibers to the lungs in the cervical sympathetic nerve, but no
effect on lung tonus from section of these motor fibers.

VISCERAL SENSORY NERVOUS SYSTEM 75

ganglion, or to other sensory nerves, thus inducing reflex effects. Our
usual technique was to section the large brachial nerve close to the
vertebral column, taking care not to injure the slender sympathetic
trunk passing under it; also section the root of the hypoglossal, and
after again sectioning these nerves peripherally, handle the cervical
sympathetic by the stump of the brachial to which it is attached. We

-also made it a point to apply the fine pointed electrodes to the cervical

sympathetic trunk at least 3 mm. distant from the vagus root. But
even with the best of precautions escape of current to adjacent struc-
tures could not always be prevented. And we are inclined to explain

the bilateral lung effects produced by the stimulation of one sympa-

thetic (fig. 11, B) as due to escape, and consequent reflexes. It is to
be noted further that it requires relatively strong tetanizing currents
applied to the sympathetic trunk to secure the lung contractions.
Inhibitory effects on the lung were never obtained from the cervical
sympathetic nerves.

Our conclusion is that the cervical sympathetic trunk carries motor
(but no inhibitory) fibers to the lungs via the vagi. Under the con-
ditions of our experiments the section of these motor fibers has no effect
on the lung tonus, showing that this motor mechanism is not in tonic
activity, and that the section of the nerves is not a sufficient stimulus
for even a transient contraction. tir BD

b. The vagi nerves. We have seen that section of the vagus induces
permanent hypertonus in the lung of the same side. Stimulation of
the peripheral end of the cut vagus with a tetanizing current causes
an inhibition of this tonus followed by a return to the former state.
The vagus stimulation is thus able to completely abolish (temporarily)
the tonus induced by the vagi section. In the preparations showing
no lung hypertonus on vagi section owing to peripheral lung atony
vagus stimulation usually causes no lung inhibition.

In several such preparations we observed that the vagi also failed
to influence the heart rhythm. We can state that the failure of the
vagi to act in the normal manner on the lungs and heart in these prepa-
rations was not due to mechanical injury to the vagi or to the heart
and lungs. The significance of this coincidence requires further in-
vestigation. It is well known to laboratory workers in physiology that
one frequently encounters frogs in which the vagi stimulation fails to
influence the heart. This inhibitory action of the vagus on the lung
is obtained with the minimum and up to relatively strong tetanizing
currents. The stronger stimuli produce at times motor after-effects,

-76 A. J. CARLSON AND A. B. LUCKHARDT

-and very strong tetanizing currents may produce contraction only or
a brief initial contraction followed by inhibition. This latter result
was obtained by strong stimuli, especially in preparations showing
less than the maximum lung tonus following vagus section.

Stimulation of the peripheral vagus inhibits not only the tonus but

also the spontaneous rhythmic contractions that may be superimposed -

on the lung hypertonus following isolation from the central nervous
system (fig. 10, £).

_.It-is thus clear that the vagi and the cervical sympathetic nerves
in ‘the frog bear the same physiological relations to the lungs as they
do to the heart, that is, motor fibers in the latter and inhibitory fibers

in the: former to both organs. We shall show later in the section on

the action of drugs on the lungs, that some of the motor fibers to the

lungs are true vagi fibers, and do not belong to the cervical sympathetic |

complex.

4. The peripheral lung automatism. We are now in position to analyze
more definitely the origin of the motor hypertonus of the lungs after
-vagi section. It is not due to temporary stimulation of motor fibers.
We have shown that section of the sympathetic nerve fibers has no
effect. | There are some motor fibers to the lungs of pure vagus origin.
But. cutting of these fibers produces no effect on the lungs, after previous
paralysis of the inhibitory vagi fibers by large doses of nicotine. It is
not due to mechanical trauma to the lungs. Ligation of the base of
the lung may induce lung tetanus by direct trauma or by asphyxia,
lung circulation being cut off. ‘But stopping the circulation by excising
the heart does not. cause lung tetanus, and section of the vagi is done
without touching the lungs or the adjacent structures. Moreover, the
indirect mechanical disturbance of the pharynx and the base of the
lungs is much greater from isolation of the vagi or the cervical sympa-
thetic nerves, and these Jatter procedures do not bring on lung tetanus.
The lung hypertonus is’ not an asphyxia phenomenon. Excessive lung
ventilation, normal or artificial, will not prevent or abort it, if the vagi
are sectioned or the brain destroyed. The lung hypermotility is not

a temporary motor reflex state induced via the medulla by the powerful ©

afferent impulses induced by the extensive operative trauma, for the

lung is found collapsed and maximally contracted in frogs with the:

brain destroyed, without previous operative injury of any kind, and
we iknow of no other reflex.state that may persist for hours after lesion
of,:the. reflex..path. .It is-not unlikely, however, that any condition
inducing a peripheral lung hypertonus of a degree interfering with the

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VISCERAL SENSORY NERVOUS SYSTEM 77

respiratory functions of the lungs would augment the inhibitory action
of the medulla on the lungs, probably through sensory fibers from the
pulmonary branches of the vagi, a mechanism analogous to that of
the depressor nerves and the eardio-inhibitory center.

In the experiments with pithing the medulla we at times obtained
a slight temporary inhibition of the lung tonus prior to the typical
lung tetanus. The temporary inhibition is evidently due to a transient
stimulation of the medulla inhibitory center by the act of destruction.
It is not probable that the subsequent lung hypertonus is due to a
more lasting traumatic stimulation of the motor fibers, analogous to the
effect produced on the heart by strong stimulation of the two sets
of fibers in the vagi nerve-trunks. This possibility is disproved by the

fact that nicotine paralyzes the lung inhibitory nervemechanism, leaving

the motor nerve mechanism intact, and nicotine causes a lung tetanus
which is not augmented by subsequent destruction of the medulla or
section of the vagi.

Hence we must conclude that in the frog the vagi inhibitory fibers
to the lungs are in constant or tonic ‘activity, holding the peripheral
motor automatism in check, and that on removal of this check the
lungs go into a persistent tetanus or hypertonus. In other words, the
lungs of the frog behave like the heart of many species of animals on

- section of the vagi; the heart beats faster, the lungs become hypertonic

to a degree that nullifies their function.

These observations place the lung of the frog in the same category
as the heart and the alimentary tract as regards independent peripheral
motor automatism. In all these structures we have the same motor
tissues, viz., nerve cells, nerve plexuses and musculature. We. are
therefore confronted by the same problems as regards the nature of
the mechanism of the lung automatism that have engaged the attention
of the physiologists in connection with the heart and the gut. A
question of equal importance is the persistence or modification of this
primitive lung automatism, in health and disease, in other groups of
lunged animals. |

CHANGES IN INTRAPULMONIC PRESSURE AS A RESULT OF THE
STIMULATION OF VARIOUS AFFERENT NERVES

No investigations have been made on reflexes into the lung muscula-
ture. Most of the previous workers have been engaged in a study of
the external respiratory phenomena of the frog. Wendenski (26) noted

LUCKHARDT

CARLSON AND A. B.

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VISCERAL SENSORY NERVOUS SYSTEM 79

“expiratory tetany’? following weak stimulation of sensory fibers in
the vagi. His method as well as that of other workers was not designed
to note actual contractions.of the lung itself, since in every case previous
experimenters worked with an open glottis. However, tracings 15 and
16 of this article, taken from doubly vagotomized frogs, show very
slight inspiratory and expiratory excursions of the flanks and long
tonus variations obviously due to the strong tonus of the lung and the
tonus contractions in the lung as seen by us.

Sensory stimulation of any sort, be it electrical or mechanical, has
a powerful effect on the external respiration of the frog by either reduc-
ing the intrapulmonic pressure by reflex opening of the glottis, or if
the latter is closed by hemostat or pressure by vaselinized cotton at the
time of application of stimulus, by reflex lung contraction which will
cause the intrapulmonic pressure to rise.

Figure 12 A shows at X the sudden opening of the glottis in de-
cerebrated bull frog, holding air under considerable pressure, following
gentle stroking of the skin of the hind leg. The first part of the tracing
shows voluntary respirations (swallowing of air) with maintenance of
high intrapulmonic pressure. The prompt collapse of the lung is fol-
lowed immediately by marked respiratory effects which raised the intra-
pulmonic pressure to its original level. Stimulation of the skin in
another preparation similarly prepared (fig. 12 C) not only increased
the volume of the respiratory gulps, which were occurring regularly
and continuously prior to the stimulation, but induced the animal in
every instance to fill the lungs to the maximum capacity in the fashion
described in the second section of this paper.

Figure 12 B shows a similar collapse of the lungs in Rana pipiens due
to opening of the glottis following electrical stimulation of the urinary
bladder with a moderately streng tetanizing current.

Tracing D, figure 12, was obtained from a frog with brain intact. In
this animal the simple approach of the finger or person at a led to
collapsed lung followed by more or less marked efforts at refilling.

In all tracings reproduced in figure:12 the glottis was open. These
preparations, therefore, were not favorable for a study of the pulmonary
activity itself (lung contractions or inhibitions) following the stimu-
lation of various afferent nerves. In order to maintain the volume of
air constant we closed the glottis by a mosquito forceps or vaselinized
cotton and raised the intrapulmonary pressure to a point maintained
by the animal under normal conditions and then noted the effect - the
stimulation of the afferent nerves.

80 A. J. CARLSON AND A. B. LUCKHARDT

_ The ligation of the vagus on one side (mechanical stimulation) in

many instances induced reflex contraction of the opposite lung. In

the present state of our. knowledge it is impossible to state whether
record of such a contraction as shown in figure 13 A at X is due to reflex
stimulation of the lung through the motor fibers of the vagus or due

to a temporary inhibition of the tonic inhibitory control over the lung

via the vagi, leaving the peripheral automatic mechanism in the lung
unchecked. A possible answer to this question might be obtained by
noting the effect of such stimulation in animals in which the tonic
inhibitory mechanism has been previously paralyzed by nicotine. If
under these conditions stimulation of the sensory nerves yields the
same results the recorded contraction is the result, not of a temporary
inhibition of the tonic inhibitory mechanism, but due to the reflex
stimulation of the pulmonary motor fibers through the vagi (and sympa-
thetics). Consideration of the law of reciprocal innervation would
suggest that under normal conditions both mechanisms are involved
in the phenomenon whose graphic record is that of a rise in date baal
monic pressure.

Irrespective of the nieolaniun’ or mechanisms involved in . ultimately
effecting contractions of the lungs, we can confidently say that stimu-
lation of the skin of the upper mandible, mild mechanical irritation of

the anterior nares, mechanical stimulation of the bladder and cloaca, or —

electrical stimulation of the urinary bladder, mesentery, small intestine,
pyloric end of stomach, esophagus and central end of the brachial nerve

effect reflex contractions of the lung. These points are shown indi- —

vidually in figure 13 and figure 14, which with the accompanying
legends are self-explanatory. The mode of preparation of the animals

used. in both series is virtually the same with this exception: In the

preparations, records of which are illustrated in figure 13, the glottis
was clamped by means of a mosquito forceps; in the tracings reproduced
in figure 14 the glottis was kept occluded by pressure over it by a ae
of vaselinized cotton.

Of these records two deserve: particular attention. In Sguri: 13, D,
is recorded the powerful reflex lung contractions obtained by closure of
the glottis by a hemostat. A decided inhibition preceded the con-
traction. |

Tracing E in the same ihe Meas an sieaimuinlly strong cee con-

traction of both lungs as a result of farnebing the skin of the lower F

mandible.

Fig. 13. Water manometer tracing of intrapulmonic pressure in the frog (R.
pipiens). Spinal cord cut and destroyed below medulla. Cannula in tip of
lungs, abdomen opened and the lungs isolated from abdominal and shoulder
musculature. Glottis closed by clamp except in tracing D.

A: Left lung; 2, ligation of right vago-sympathtic nerve, showing reflex
contraction of left lung on vagus stimulation.

B: Upper tracing, left lung; lower, right lung. X, mechanical stimulation
of the skin of the upper mandible, showing reflex lung contraction.

C Upper tracing, left lung; lower, right lung. XX, mechanical stimulation
of the nares; X, mechanical stimulation of the cornea, showing reflex lung con-
tractions.

D: X, closure of the glottis with artery forceps, showing temporary reflex
inhibition of lung tonus followed by strong contraction.

E: Upper tracing, left lung; lower equals right lung. XXX, crushing skin of
lower mandible, showing exceptionally strong reflex lung contractions.

81

82 A. J. CARLSON AND A. B. LUCKHARDT

In this.series of experiments stimulation of the fundie end of the
stomach (fig. 14, D, ‘‘c’’) with the electrical current yielded no reflex con-
traction of the lung at a time when stimulation of the=pyloric end of
the stomach and cardiac region of the esophagus with the same strength
of current gave uniformly striking results.

a a b a . a.

Fig. 14. Water manometer tracings of the intrapulmonic pressure in the frog’s
lung, showing reflex contractions of the lung musculature. Frogs decerebrated.
Abdomen opened, lungs isolated from influence of skeletal muscle contractions.
Cannula in tip of lungs, and glottis closed with a plug of vaselined cotton pushed
into the pharynx.

A: a, mechanical stimulation (stroking) skin of hind leg; b, pinching toes of
hind leg.

B: a, mechanical stimulation of urinary bladder; 6, electrical stimulation of
urinary bladder; c, mechanical stimulation of cloaca.

C: Electrical stimulation; a, large intestine; b, mesentery; c, small intestine.

D: Electrical stimulation;-a, small intestine; b, pyloric end of stomach; ec,
fundus of stomach; d, esophagus (cardiac region).

E: a, electrical stimulation of central end of brachial nerve plexus. X,
spontaneous respiration (quick up stroke).

VISCERAL SENSORY NERVOUS SYSTEM 83

In a summarizing sentence we might therefore state that the stimu-
lation of every sensory nerve (afferent visceral or cutaneous) gives rise
reflexly to lung contractions.

THE ACTION OF CERTAIN DRUGS ON THE MOTOR MECHANISM OF THE LUNG

Our interest in the action of drugs on the neuro-muscular mechanism
of frog’s lung had its inception during our study of the physiological
action of the vagus on the lung musculature following electrical stimu-
lation of the nerve. Such stimulation gave at outset variable results
until we noted that the effects depended to some extent on the strength
of the tetanizing current employed, as noted above. At any rate,
we had good reason to suspect that the vagus carried both motor and
inhibitory fibers to the lung motor mechanism. At this juncture it
occurred to us that the use of drugs might be helpful in clarifying the

_ situation.

Nicotine. Mindful of the action of nicotine in abolishing the in-
hibitory effect of vagus stimulation on the heart without affecting the
motor action, we assumed that the drug might act similarly with respect
to the inhibitory fibers in the vagus to the lung. If this were so, elec-
trical stimulation of the vagus following the injection of nicotine might
give clearer evidence of motor fibers in this nerve than before nicotini-
zation. Since, furthermore, the paralysis of the ganglion cells in the
course of the inhibitory fibers to the heart effected by this drug is
preceded most commonly by stimulation, the same effect ‘might be
anticipated in the case of the inhibitory fibers to the lungs. If this

were true nicotine ought to cause, on injection, an inhibition similar

to, if not identical with, the inhibition of the heart commonly observed
as the marked effect of stimulation of the vagus before the injection
of the drug, especially if the tonic central inhibitory control exercised
over the lungs via the vagi had been abolished by either sectioning of
the nerves or destruction of the medulla.

The results obtained exceeded our expectations. Inspection of figure
15 (at g) shows that 1 mgm. nicotine when intravenously injected effects
a pulmonary inhibition in the lungs released from the tonic inhibitory
influence of vagus by section of these nerves (at a and. b) which compares
favorably with the inhibition obtained by previously stimulating the
nerves with a tetanizing current of moderate intensity (see e and f).

If, on the other hand, the nicotine is injected intravenously in an
animal following ligation and section of but one vagus, as in figure 16,

A. J. CARLSON AND A. B. LUCKHARDT

84

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VISCERAL SENSORY NERVOUS SYSTEM 85

the immediate effect on the lungs is an inkibiien of the tonus of the
lung which has been denervated, and an escape from the tonic inhibitory
influence exercised by the vagus on the lung which is still connected
with its center, as at c, where the left lung (upper tracing) shows a rise
in tonus occurring in the course of an inhibiton of the right lung, the
latter comparing favorably with the inhibition effected by previous
stimulation of the vagus (b). In this experiment the left vagus has
been cut physiologically by the drug. If at the time of this drug‘cutting
the vagus through central action is exercising its maximum inhibition
on the lung, the effect of the primary stimulating action of the drug
would not appear since the lung at the time of drug stimulation is
already under maximum inhibitory control. As a matter of fact, 1

the majority of preparations this is apparently the case, the drug ah
tine simply releasing the peripheral automatic mechanism from the
maximum tonic inhibitory effect of the center through the’ vagus.
This release is certainly complete for section of the vagus to this lung
is without further effect on its tonus. This latter fact would further-
more indicate that the more or less prompt rise of intrapulmonary

pressure following section of the vagus without nicotine was due, not

to the mechanical stimulation of the motor fibers contained :in this
nerve, but to the removal of the tonic inhibitory control. That the
vagus nerve contains such motor fibers can be shown very satisfactorily
in any preparation that has been nicotinized. In figure 16 electrical
stimulation of the nerves after nicotine (as at g, right vagus, and h,
left vagus) gives now marked contraction of the lung instead: of the
usual inhibition before nicotine (6).

Figure 17, A, is offered as another example of this phenomenon.
Following the release of the left lung from tonic inhibitory control
exercised over it through central vagal control by section of the left
vagus at a, 2 mgm. nicotine were injected at b with the result that the
right lung was now released from its inhibition by the ‘‘cutting”’ action
of the drug and the left lung was inhibited by the primary stimulation
action of the drug on the inhibitory mechanism of the left lung. Figure
17, B, shows the change in effect as a result of electrical stimulation

of the vagus nerve following the injection of nicotine. In this experi-

ment stimulation of the vagus at 6 caused pronounced inhibition. The
injection of nicotine at c was followed by the usual inhibition in the
lung which has been released from the tonic inhibitory control by
section of the nerve at a. Subsequent, however, to this nicotinization,
electrical stimulation at d caused marked contraction of the lung instead
of inhibition.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

A. J. CARLSON AND A. B. LUCKHARDT

86

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VISCERAL SENSORY NERVOUS SYSTEM 87

The results are uniformly clear-cut and decisive. By means of this
drug nicotine it is possible to differentiate between two types of efferent
pulmonary fibers occurring in the vagus, i.e., inhibitory fibers and motor
fibers. The former exercise in the normal frog a powerful inhibitory
control over the lung and are either more numerous or more readily
susceptible to electrical stimulation than the motor fibers. It is only
after the abolition of the inhibitory control of the lung by nicotine that

Fig. 17. Water manometer tracings of the intrapulmonic pressure in the frog.
(R. pipiens). Spinal cord cut and destroyed below the medulla. .Glottis closed.
Cannula in tip of lungs. Lungs isolated from influence by skeletal musculature.

A: Lower tracing, right lung; upper, left lung. a, Ligation of left vagus; b,
injection of 2 mgm. nicotine in 10 cc. Ringer’s solution into abdominal vein.
Showing abolition of the tonic vagus inhibition of the lung neuro-muscular mech-
anism by nicotine.

B: Tracing from right lung. a, Ligation of right vagus; 6, stimulation of
peripheral end of right vagus with weak tetanizing current; c, injection of 2
mgm. nicotine in 10 ce. Ringer’s solution into abdominal vein; d, stimulation of
peripheral end of right vagus with same strength of tetanizing current as at b.
Showing inhibition of lung tonus and paralysis of the vagi inhibitory fibers by
nicotine.

C: Tracing from left lung. a, Injection of 5 mgm. nicotine in 5 ce. of Ringer’s
solution into the heart; b, injection of 1 cc, 1-1000. histamine into the heart.
Showing stimulation of the lung by large doses of nicotine and stimulation by
histamine after paralysis of the inhibitory nerves by nicotine.

88. A: J. CARLSON AND A. B. LUCKHARDT

electrical stimulation yields a more or less marked motor effect. Nor
are these. motor fibers of sympathetic origin running in the trunk of
the. vagus; for, granting the presence of some motor fibers in this nerve
to the lung, the motor response on stimulation of the sympathetic
after. nicotine is smaller in a given animal than the response from the
vagus. itself, as noted earlier in the paper. In short, the vagus nerve

contains two sets of fibers to the pulmonary motor mechanism of which ©

the inhibitory exerts a tonic predominant control; the motor fibers are
apparently not in a stale of tonic activity for sectioning of the vagus
after cutting the inhibitory fibers in this nerve by nicotine has no further
effect on the intrapulmonic pressure. It would appear on the basis
of our pharmacological studies that the inhibitory fibers of the vagus
have interpolated in their course to the automatic tissues nerve cells
on which the drug acts; the motor fibers on the other hand run directly
to the automatic tissue.

Large doses of nicotine. Whereas the constant effect of the intrave-
nous injection of small doses (2 mgm.) of nicotine in the previously
denervated lung (by vagotomy) is inhibition, injection of large doses
(5 mgm. or more) causes pronounced contraction of the lung. .This

is well shown in figure 17, C, where the injection of 5 mgm. caused more —

or less abrupt contraction followed by slow relaxation. It is probable
that the nicotine in this dosage acts as a direct stimulant to the smooth
musculature.

Atropine. This drug, even when given in large doses, dood not para-
lyze the endings of the inhibitory fibers to the lungs as it paralyzes
the cardio-inhibitory nerve endings. Figure 18, A, is a tracing from
a frog which had received 1 cc. of a 0.1 per cent solution of atropine
sulfate 45 minutes previous to experimentation. Pithing of the brain
at a was followed by the typical escape of the lung from tonic central
inhibitory control. The failure of atropine to paralyze the inhibitory
nerve ending in the lung is shown further by the fact that even mechani-
cal stimulation of the nerve at b gave powerful inhibition, as did elec-
trical stimulation at c. As an after-effect of mechanical or electrical
stimulation of the nerve there were pronounced motor effects. The
results obtained from the right lung of another frog were somewhat
different. Here (fig. 17, B) ligation of the vagus caused the usual
escape of the lung from the inhibition. On the other hand, mechanical
stimulation due to handling of the vagus nerve (at b) was followed by
contraction. Stimulation of this nerve at c with a mild tetanizing
current was in this instance followed by contraction instead of the
usual inhibition.

89

SENSORY NERVOUS SYSTEM

VISCERAL

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90 A. J. CARLSON AND A. B. LUCKHARDT

To meet the objection that in these cases the dosage was too small
and that the effects of the drug had worn off before experimentation
was begun, we prepared another frog as follows: After decerebration,
insertion of the cannula into the tips of both lungs, and clamping of
the glottis, we sectioned the left vagus and obtained the usual escape
of the lung from the tonic. central inhibition control. Stimulation of
this nerve gave the usual inhibition. The intravenous injection of 3
mgm. atropine did not effect the result of stimulation of the vagus.
We now injected within 1 hour’s time in successive doses, 1, 2 and 4
mgm. atropine sulphate. These injections did not change the usual
reaction obtained from stimulation of the peripheral vagus. Following
the injection of the last 4 mgm., the right lung escaped from central
inhibitory control in a manner indistinguishable from section of its
vagus nerve. Apparently this huge dose of atropine paralyzed the
center, for section of the right vagus was without further effect. Stimu-
lation of its peripheral end, however, yielded even now inhibition fol-
lowed by contraction of the lung, indicating that the chief action on
the lung of atropine in huge doses is not peripheral. Since decided
motor after-effects result from stimulation of vagus to the lung in a
heavily atropinized frog, we might conclude that the drug likewise
has some effect in paralyzing the inhibitory nerve endings unless we
assume that it sensitizes the motor nerve endings in the vagus. At
any rate the peripheral action of atropine and nicotine are quantita-
tively decidedly different if one compares the dosage in milligrams and
the results effected thereby.

Both drugs paralyze the center and likewise act on the peripheral
mechanism. Nicotine accomplishes both results quickly and decisively
in smaller doses, while atropine acts on the center only in large doses
and only renders paretic the inhibitory terminations of the vagus.
The frog is apparently quite tolerant to atropine. Eight milligrams
injected intravenously into a decerebrated frog at one time suspends
external respiration promptly (as does 1 mgm. of nicotine). Ex-
amination of the lungs shows them contracted. But almost complete
recovery sets in within an hour and at this time the lungs assume their
normal size and function. ! |

Atropine in this dosage does not paralyze the terminals of the in-

hibitory fibers of the lung as it does the vagal nerve endings in the

heart. In all of these atropinized preparations stimulation of the vagus
nerve was without effect on the heart.

P
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VISCERAL SENSORY NERVOUS SYSTEM Ot

Adrenalin. In the frog the irrigation of the lung itself or injection
of adrenalin chloride into the circulation causes inhibition of the, auto-.
matic quick rhythm of the lungs which appears spontaneously as noted
in a previous section of this paper or inhibits the hypertonic activity
of the lung following section of the vagus. As illustrations of me
shows inhibition of the quick rhythm Ae: adr erat was applied to the
lung directly at X; the latter, inhibition of the hypertonic state of the
lung following vagotomy.

Fig. 19. Water manometer faenge of the intrapulmonie pressure. in mi ‘frog
(R. pipiens). Spinal cord cut and destroyed below medulla. Cannula in’ tip
of lungs. Vagi nerves cut, and lungs isolated from influence of skeletal: muscle
contraction. | ecee

A: X, application of 75 cc. adrenalin chloride (1-100) ; in Ringer’s ‘dbasicms ae
surface of lung. is

B: X, injection of 7 vo cc. adrenalin binarides in 2 ce. . Ringer’ s solution into the
heart.

C: Upper tracing, left lung; lower tracing, right lung. Intravenous injections
of histamine in 2 cc. Ringer’s solution; a, 0:01 cc.; b, 0.02 ec.; c;} 0.06 ce. 1+1000
histamine hydrochloride.

Showing inhibition of lung tonus by ‘epinephein and wl uaddbinis by histamine.

Histamine. Figure 19, C, and figure 17, C ‘at b show the effect’ ‘of
histamine-HCl on the ‘tivo iiudeulen apparatus of the lung’ Wwhidn
‘injected intravenously in varying concentrations. In moderately ‘small
or large doses it causes invariably a slight: contraction of the lung which
in arnplitude bears no relationship to the dosage. Our experience, as
a matter of fact, leads us to believe that successive doses of the: drug
given within a relatively short period of time’ have less''and less effet
because, possibly, of the cumulative poisonous property of this drug.

92 A. J. CARLSON AND A. B. LUCKHARDT

SUMMARY

1. The actual respiratory movements (opening of glottis and swal-
lowing of air) are accompanied by relaxation of the tonus of the lung
musculature, due either to greater action of the inhibitory fibers in
the vagi or central inhibition of the motor nerve mechanism, on the
assumption that the latter is in tonic activity. This inhibition occurs
during the respiratory movements even when no air can enter or leave
the lungs. It is therefore of central origin, an effect codrdinated with
the true respiratory act. From the point of view of utility the in-
hibition may be designated as a “receptive relaxation.’”? The buccal
movements that go on during the period between actual air swallowing
do not seem to influence the lung tonus.

2. At the end of the respiratory act there is an active contraction of
the lung musculature, after a latent period of 5 to 6 seconds. This
contraction is of variable duration (10 to 20 seconds) depending on
the respiratory rate and the condition of the lungs. The contraction
is usually followed by a gradual tonus relaxation up to the next respir-
atory act. Occasionally this gradual tonus relaxation is absent.

These active lung contractions are best seen during the pause of the

Cheyne-Stokes type of breathing, which appears to be normal for the
frog. The contraction is cut short by the next swallowing act, so that
_when the animal is breathing rapidly, the active lung contractions are
“not in evidence, the lung musculature being in a continuous state of
“receptive relaxation.””? The active lung contractions following a
respiratory act can be accounted for by a lowering of the inhibitory
“influence, thus permitting the peripheral automatism to come into
greater play. We have not been able to determine whether motor
‘innervation via vagi and sympathetic nerves also play a réle as con-
_tractions follow the respiratory act even when: no air enters or leaves
the lungs. It is not a reflex initiated by the stimulation of pulmonary
sensory fibers through lung distention. It is probably entirely central
‘in origin and referable to the respiratory center, the inspiratory dis-
. charge of this center having the immediate effect of.a temporary stimu-
lation of the inhibitory mechanism for the lung tonus, the lung con-
. traction of the end of the inspiration merely signifying excess back
-swing of the central inhibitory control on its return to whe more or less
constant level.

8. Section of the cervical sympathetic fibers vey no effect on the
lung tonus but stimulation of these fibers before .they join the vagi

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VISCERAL SENSORY NERVOUS SYSTEM 93

nerves causes lung contractions. The cervical sympathetic nerves
contain a few motor fibers, but no inhibitory fibers to the lungs. These
motor fibers in the sympathetic do not appear to be in tonic activity,
but our experiments on this point are not final.

4. Section of the vagi nerves or destruction of the medulla’ causes
a permanent hypertonus or incomplete tetanus of the lungs. This
is due to removal of a tonic inhibitory check on the peripheral lung
motor mechanism (peripheral neuro-muscular automatism). Ligation
of the pulmonary branches of the vagi produces the same effect. The
only part of the central nervous system necessary for this tonic
inhibitory control is the medulla. Section and destruction of the entire
spinal cord below the medulla has no permanent effect on the tonus
mechanism. Decerebration has likewise no permanent effect on it.
Destruction of the midbrain causes a temporary diminution of the
lung inhibition probably through a “shock’”’ state of the medulla. The
afferent aspect of this tonic lung inhibition requires further study.
The most important afferent pathway is probably the pulmonary
branches of the vagi.

The contractions of the lungs following vagi section are powerful
enough to develop a pressure of from 20 to 40 mm. Hg., and if the
glottis is not artificially closed all the air in the lung cavity is forced
out, the lungs contract down to a solid mass and are thus rendered
useless as organs of respiration. All our data on this point are those
of acute experiments lasting only 2 to 3 hours. The possible readjust-
ment of this peripheral lung automatism to meet the needs of the

animal after double vagotomy is being investigated by long time

experiments.
5. Stimulation of the peripheral end of the vagi inhibits the lung

tonus induced by yagi section. Strong tetanizing currents applied to

the peripheral vagus trunk usually cause strong contractions following
the primary inhibition. Nicotine paralyzes the lung inhibitory fibers
of the vagi apparently without injury to the motor fibers, so that after
nicotine vagus stimulation causes lung contractions only. These con-

tractions are stronger than can be caused by stimulation of the cervical

sympathetic nerve. Hence, on the basis of the usual interpretation
of nervous action, the vagi carry both inhibitory and motor fibers to
the lungs, the former predominating and being tonically active like
the cardio-inhibitory mechanism in many animals. .

By stimulation of the peripheral end of the vagus we have so far
failed to cause a greater tonus relaxation in the lungs than that which

94 A. J. CARLSON AND A. B. LUCKHARDT

existed before vagi section. This means either that the tonic vagi —

inhibitory action is ordinarily maximal, or that the simultaneous stimu-
lation of the motor fibers in the vagi trunks neutralizes a part of the
inhibitory effects.

The efferent actions of the vagi and the cervical sympathetic nerves
on the lungs are unilateral. Only occasionally have we seen effects
on the lung of the opposite, in case of sympathetic stimulation. This
was probably due to escape of current, and not to actual nerve crossing.

6. Reflex contraction of the lungs are induced by the stimulation
of the afferent fibers in the vagi, by stimulation of the cutaneous nerves,
the sensory fibers in the nares and the cornea, and the sensory fibers
in the visceral organs. These lung reflexes could be brought about
either by augmented action of the motor nerve mechanism or by de-
pression of the tonic inhibitory mechanism. On the basis of the usual
conceptions of reciprocal innervation both factors are probably involved.
It is thus clear that practically all afferent nerves have reflex connection

with the medullary nuclei controlling the lung tonus and contractions. —

7. As stated above, it is possible to differentiate between the motor
and inhibitory fibers in the vagus trunk by means of nicotine. This
drug in moderate doses- (2 mgm.) paralyzes not only the respiratory
center but also the peripheral inhibitory mechanism so that subsequent
stimulation of the vagus’ causes more or less powerful lung contractions
in place of the usual inhibition resulting from stimulation of this nerve
before nicotinization. If nicotine is injected in a frog that has suffered
bilateral vagotomy with the usual escape of the tonic inhibitory control
of the corresponding lung, nicotine effects a marked inhibition of this
lung and after a slight interval an escape from central control of the
other lung. The escape of the one lung does not occur until the primary
and temporary inhibition (stimulation) of the peripheral mechanism
in the other is about over. This probably means that the lung still
connected with the center before nicotinization is under maximal
central inhibitory control since the drug in this instance produces no
primary inhibition. Injection of nicotine in any case destroys the
central tonic inhibitory control of the lungs similar to destruction of
the medulla or double vagotomy. In large doses nicotine causes con-
traction of the lung musculature probably by direct stimulation of the
muscular elements. /

8. Atropine in doses large enough to paralyze the cardio-inhibitory
fibers of the vagus has no effect on the terminations of inhibitory fibers
in the lungs. In fact, even huge intravenous doses (8 mgm.) do not

ee

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VISCERAL SENSORY NERVOUS SYSTEM 95

completely paralyze these terminations. Such doses paralyze chiefly
the medullary centers which send out the inhibitory impulses. As
a result the lungs contract. But even this center recovers within an
hour and again assumes its tonic inhibitory control over the lungs.

9. Histamine in small or large doses (0.01 to 0.07 cc. 1:1000 sol.)
causes temporary contraction of the lung musculature.

10. Epinephrin inhibits both the peripheral automatic tonus and the
peripheral automatic rhythm if one is present. :

BIBLIOGRAPHY

(1) Arnoutp: Virchow’s Arch., 1863, xxviii, 433.
(2) Baspax: Handb. d. Vergl. Physiol., 1914, i, 729.

(3) Baeuiont: Arch. f. Physiol., 1900, Suppl. Band, 33.

(4) Berti er Marzenint: Arch. d. Fisiol., 1910, viii, 389.

(5) Brown: Arch. f. d. gesammt. Physiol., 1909, exxx, 193.
(6) Bour: Skand. Arch. f. Physiol., 1899, x, 74.

(7) Cartson: This Journal, 1913, xxxi, 318.

(8) GasKELL: The involuntary nervous system, London, 1916.
(9) Hernemann: Virchow’s Arch., 1861, xxii, 1.
(10) Kerra: Nature, 1904, lxix, 511.

(11) K6niestern: Arch. f. d. gesammt. Physiol., 1903, xcev, 616.

(12) LANGENDORFF AND SerBert: Arch. f. Physiol., 1881, 241.

(13) Laneenporrr: Arch. f. Physiol., 1887, 285.

(14) Laneenporrr: Arch. f. Physiol., 1888, 304.

(15) Lucusincer anp Soxotow: Arch. f. d. gesammt. Physiol., xxiii, 283.

(16) Martin: Journ. Physiol., 1878, i, 137.

(17) Mocut: Arch. Ital. d. Biol., 1910, liii, 472.

(18) Mocut: Zeitschr. f. Biol. Tech. u. Metb., 1912, ii, 115.

(19) Mocutr: Folia Neurolial, 1912, vi, 769.

(20) Nixouwes: Centralbl. f. Physiol., 1908, xxii, 753.

(21) Nrxouiwes: Arch. f. Physiol., 1910, 197.

(22) Pari: Arch. d. Fisiol., 1906, iii, 283.

(23) SHerRineton: Journ. Physiol., 1891, xii, 292.

(24) Soprana: Arch. Ital. d. Biol., 1904, xlii, 151.

(25) Smrrnow: Anat. Anzeiger, 1888, ili, 258; Cuccatt: Int. Monatschr. f. Anat.
u. Physiol., 1888, v, 194.

(26) Wepenski: Arch. f. d. gesammt. Physiol., 1881, xxv, 129.

(27) Wittem: Arch. néerl. de Physiol., 1919, iii, 315.

(28) Wourr: Arch. f. Anat., 1902, 179.

THE EFFECT OF ADRENALIN ON VENOUS BLOOD
PRESSURE

; HELENE CONNET
From the Physiological Laboratory of the Johns Hopkins University

Received for publication July 15, 1920

A REVIEW OF THE LITERATURE ON VENOUS BLOOD PRESSURE

Regulation of venous pressure by: 1. Peripheral resistance. It is usu-
ally stated that, in general, increased peripheral resistance (in the
arterioles)—other things being equal—causes a rise in arterial pressure
and a fall in venous pressure and that decreased resistance has the
opposite effect.

Bayliss and Starling (7) and Plumier (61) advance the sdk that an
increased peripheral resistance caused by vasoconstriction decreases
the capacity of the vascular system and tends to cause a rise of pres-
sure in all parts of the system. What change will occur in the venous
pressure will depend upon whether the influence of the decreased flow
from arteries to veins causing a fall, or the decreased capacity of the
system causing a rise, predominates. The opposite state of affairs
holds in case of decreased peripheral resistance. Sometimes the tend-
ency for the venous pressure to rise is exactly counter-balanced by the
tendency to fall. Bayliss and Starling (7) cite, as an illustration, the
absence of venous pressure change when vasomotor paralysis has been
induced by section of the cord just above the first thoracic segment.
Plumier (61) also illustrates this point. He finds no change in venous
pressure after weak or strong stimulation of the central stump of the
vagus, both vagi being sectioned. In the case of weak stimulation,
vasodilatation was produced while strong stimulation produced vaso-
constriction. In each case, however, the influence of the peripheral
resistance on the venous pressure was balanced by the opposite influence
of the change in the capacity of the system.

According to Bayliss and Starling (7), sometimes the venous pres-
sure-raising factor in vasoconstriction of the arterioles predominates, as
when the splanchnics are directly stimulated or the vasomotor center

96

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 97

is stimulated by asphyxia. The objections, as advanced by Hill and
Barnard (37) and Plumier (61), to attributing this rise in venous pres-
sure to decreased capacity of the vascular system will be discussed
later. | i #

2. Heart rate. ‘The authors mentioned above (Bayliss and Starling,
and Plumier) state that a rise in venous pressure is obtained when
the heart slows or stops beating because there is a tendency toward
equalization of pressure throughout the system, resulting in a fall in
arterial and a rise in venous pressure due to the elasticity of the arteries
forcing more blood into the veins. Usually in a slowly beating heart
the output per minute decreases and hence the heart does not. pump
into the aorta per unit of time as much as it did before, thus causing a
back pressure in the pulmonary veins, which eventually affects the
right heart and causes a rise in the vena cava pressure. This would
be true especially when there was an increased resistance to the blood-
flow in the arteries, as in vasoconstriction. |

Bayliss and Starling (7) consider that most of the rise of venous
pressure after peripheral stimulation of the vagus is due to the de-
creased capacity of the system which comes as a result of the anemic
stimulation of the vasomotor center following the low arterial pres-
sure brought about by such stimulation. Their proof of this is that
only: a slight rise is occasioned by stimulation of the vagus when either
the cord or the splanchnics are cut. Plumier (61) has a different ex-
planation for this. He considers the slowing of the heart of primary
importance and the vasoconstriction of secondary. He. says that
cutting the cord or splanchnics causes such a marked vasodilatation,
(arterial pressure in one of Bayliss and Starling’s experiments fell from
120 mm. to 60 mm. Hg.) that tendency toward equalization of pres-
sures cannot show the effect that it would, if the conditions of the
vascular system were normal. ‘This point is perhaps brought out more
clearly by considering what changes take place in the vascular system
under asphyxia, produced by removal of artificial respiration in an
animal whose chest has been opened. Bayliss and Starling offer an.
explanation for this rise in venous pressure, similar to that given in
connection with vagus stimulation. Plumier (61) attempts to prove
his point that vasoconstriction is of only secondary importance, by
comparing the effect of asphyxia before and after section of the vagi.
With vagi intact the arterial pressure rises only slightly but the heart
beat soon becomes very slow, and coincident with this slowing the
venous pressure in the inferior vena cava and the external jugular rises

98 . HELENE CONNET

markedly. As soon as artificial respiration is renewed and the heart.
beats faster, the venous pressure falls. In the experiment when both

vagi are cut, the arterial pressure rises, due to vasoconstriction, but

the venous pressure remains unchanged, until (100 seconds after the
beginning of asphyxia) the heart becomes paralyzed and consequently
slows, and the blood pressure falls. At this time the venous pressure
rises. Furthermore, after artificial respiration has been renewed, the
arterial pressure rises, due to continued vasoconstriction, and when
it is at its maximum the venous pressure has already fallen to normal.

The difficulty here, it seems to me, is in trying to make either heart-
slowing or vasoconstriction alone account for the rise in. venous pres-
sure. Bayliss and Starling (7) themselves, when discussing the rise of
venous pressure after splanchnic stimulation, say (p. 172): ‘‘In experi-
ment 5 one of the vagi was intact and the heart was slowed as usually
occurs when the splanchnics are stimulated. One might be inclined
to ascribe the rise of venous pressure to this slowing of the heart,
were it not that in other experiments where both vagi were divided, we
still obtained a slight rise on stimulation of the splanchniecs.” Evi-
dently, then, the slowing of the heart is responsible for the greater
part of the venous pressure rise, if not absolutely all. On the other
hand, one cannot'see how Plumier can be sure that Bayliss and Starling
are wrong in ascribing some of their rise in venous pressure, after
asphyxia for instance, to the decreased capacity of the system, since
no experiment has been performed in which that factor has been ruled
out, granting that Bayliss and Starling’s attempt to rule it out, not
only ruled it out, but introduced a new factor, that of vasodilatation
after section of the cord or splanchnics, which vitiated the comparison.
It would seen as though this point might be settled by an arrangement,
such as that used by Heard and Brooks (31), for keeping the arterial
pressure constant under varying experimental conditions. When,
after adrenalin or asphyxia with vagi intact, the change in capacity of
the system, due to vasoconstriction, was not allowed to be effective,
one could then see how much this factor has to do with the rise in
venous pressure on slowing of the heart.

3. “Respiratory pump.” According to Hill and Barnard (37) none
of the above explanations is adequate in accounting for the rise of
venous pressure obtained. as a result of various experimental proced-
ures. They attribute the rise which occurs on arrest of the heart
under peripheral vagal stimulation to the respiratory spasms produced,
and on asphyxia to strong abdominal and general muscular move-

Se ee a

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 99

ments, for in curarized animals these rises do not occur, at least in the
case of asphyxia. Their curarization was sufficient to abolish natural
respiration, but did not interfere with the heart or tone of the arterial
system. As regards vagal stimulation, they say (p. 348), “we our-
selves unfortunately have not been able to maintain arrest of. the
heart in the curarized animal for long enough time to settle this point.”’
Yet in figure 12, page 342 of this article, they show a rise in venous
pressure in the same animal on stimulation of the vagus before and
after the administration of chloroform. “In the first instance the
escape of the heart is complete, the respirations are greatly intensified
and by the powerful expiratory spasms of the abdominal muscles the
venous pressure is greatly raised. In the second case the inhibition is
complete while the respirations, weakened by the chloroform, remain
unaltered during the standstill of the heart.’’ On examination of the

- second curve one sees the venous pressure beginning to rise as soon as

the beat of the heart is stopped, but while it is rising the animal was
changed to the feet-up position. The venous pressure continues to
rise until the heart once more begins to beat. This rise of venous
pressure evidently cannot be ascribed to the activity of the respiratory
muscles since no change in respiration occurs during the standstill of
the heart, nor, probably, to the feet-up position which was assumed
during the rise of venous pressure. -

These authors object to any explanation of these phenomena which
assumes that a rise in venous pressure can be brought about by sending
more blood into the venous system, as occurs on arrest of the heart,
or hy decreasing the capacity of the vascular system by arterial vaso-
constriction. They believe that the vascular system is not filled to
distention and that since the veins can hold all the blood of the body
without distention, no increase in amount of blood in the veins or de-
crease in arterial caliber can cause a rise in venous pressure. To prove
that the veins can hold all the blood of the body at zero pressure they
cite the condition that exists in the animal after death. -Then, since it
might be thought that after death the tone of the vascular system had
passed off, they attempt to prove their point in a living animal. After
stopping the heart by vagus stimulation, they alternately placed the
dog in the vertical feet-down and horizontal positions until all the
blood had passed from the arteries into the veins, and past the venous
valves so that no reversal of flow could take place. They say (p. 346),
“In this experiment we produced a positive pressure in the veins and
no pressure in the arteries. . . . . Since the arteries are emptied

100 aE HELENE CONNET —

of blood the whole system is not filled to distention. . . .°.” Just
because the arteries: are much more elastic than the veins and can,
when the heart stops beating, empty themselves of blood and produce
a greater positive pressure than before in the veins, it. does not there-
fore follow, it seems to me, that the vascular shen while the wie
was beating, was not filled to distention.

Furthermore, in refutation of Bayliss and Starling’ s belief that anemia
of the brain in vagal arrest causes. vasoconstriction, they say (p. 348),
‘“‘In our experiments when the heart has escaped from arrest, there
has not occurred any great rise of arterial pressure which we should
expect to indicate vasoconstriction.”” Why should one expect any
great rise of arterial pressure after the heart has begun to beat again?
With the resumption of the heart beat and consequent rise of arterial

pressure toward. normal, the cause . the: apa aS anemia of

the brain, is removed. é a
‘‘ Any appreciable increase of vena cava pressure is aud either to the
reduction of the capacity of the venous system by the action of the

respiratory muscles, or to the failure of the heart in maintaining the

systolic output” (p. 350). By this, I presume, is meant the inability
of a slowly beating heart to expel per minute as much blood as it
receives per minute, thus causing a back pressure in the veins. I fail
to see how this could cause a rise in venous pressure if their contention
is correct, that the venous system is capable of holding all the blood

of the body without distention, especially since it occurs almost imme-

diately on slowing of the heart, before the arteries could have emptied
the greater part of their blood into the veins. Of course, it may. be
contended that the cava is only a part of the venous system and it:
might become distended when a large amount of blood collected in it,
but this hardly seems a valid contention in the case of the immodiaey
rise in venous pressure on arrest of the heart. |
4. Chemical mechanism. Roy and Brown (64) in expevinndhitials
with the frog’s web, tongue and mesentery, found that temporary:
anemia was followed by dilatation of arteries, capillaries and veins.
This dilatation they attribute to a “relative diminution in the lymph
of certain of the constituents of the blood, or the presence in increased

amount of certain of the products of tissue exchange, or both of these:

combined” (p. 359). This effect, they say, is independent of cerebro-
spinal vasomotor effects; it may possibly be due to action on peripheral
vasomotor ganglia, but they think it is more probably due to direct
action on vessel walls. These causes operate in other congestions and:

ap cre at amp a a

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EFFECT OF ADRENALIN’ ON VENOUS BLOOD PRESSURE FOL:

the authors feel that this automatic nena of oe oe cireulat

tion is of very great importance. » adhidet)

Henderson and Harvey (33), and Bicadex: son: 7:(82) devel the Slow of
a peripheral chemical control of venous pressure “largely through varia~:
tions in the CO, eontent of the venous blood.’’ COs, by relaxing the:
veins, they believe, removes the resistance to the flow of blood from:
capillaries to veins, and so causes an increase in venous pressure... They:
speak of such a relaxation as though it were merely the complete ‘or.
partial removal of a clip, allowing more blood to flow into a vessel’
whose caliber remains the same. But if relaxation means more than.
this; if it means an actual increase in the capacity of a portion:of a.
blood vessel, due to the lessening of vascular tone, it is difficult to'sée
how this relaxation can cause a rise in the pressure in the vessel even:
though there is an increased amount of blood present. ). teeny

4. Nervous mechanism. Ina studyof the regulationof the blood sup-
ply of the brain in 1890, Roy and Sherrington (65) found that stimu-.
lation of the peripheral stump of the vago-sympathetic nerves in dogs
produced sometimes a rise, sometimesa fall of general venous pressure. '
They think that this probably means that the vago-sympathetic trunk
contains vasomotor fibers to the veins. As discussed in the section on :
heart rate, later observers have attributed this rise which peripheral.
vagus stimulation gives, to the slowing of the heart which is occasioned:
by such stimulation. Slowing of the heart due to paralysis from.
asphyxia with both vagi cut has been shown by Plumier to give a rise”
in venous pressure comparable to the rise caused by peripheral vagus
stimulation. This perhaps does not prove that the vago-sympathetic:
nerves possess no venomotor fibers, but there seems to be perfectly
adequate explanation for the venous pressure change without assuming
the existence of such venomotor fibers. That Roy and Sherrington
sometimes found that stimulation of the peripheral stump of the vago-
sympathetic nerves produced a fall in general venous pressure, may
possibly have been due to an escape of current to the central end of the
_ nerve through the fluid medium surounding the tissues. One cannot.
‘tell what happened to the heart rate in these cases of a fall in venous
pressure for no graphs are given or statement made in regard to it.

Thompson (72) in 1893 observed constriction of the superficial veins
of the hind limb of dogs and rabbits on stimulation of the sciatic nerve
or the spinal cord. The constriction took place in short sections, the »
diameter of the vein between the segments remaining unchanged. :
Bancroft (3) confirmed these observations and extended the work. :

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO, 1

102 HELENE CONNET

He observed the appearance of the veins of the hind limb of a cat
(rabbits also were used but were not found nearly as satisfactory)
before and after section of various nerves, and thus traced out the
vasomotor supply. The cell body of the pre-ganglionic fiber lies in the
spinal gray matter, the axis-cylinder emerging in the anterior root of
the ist, 2nd, 3rd or 4th lumbar nerve, following the corresponding
white ramus into the sympathetic chain, running down it for a certain
distance. The cell body of the post-ganglionic fiber lies in the 6th or
7th lumbar sympathetic ganglion, the axis-cylinder running to the
veins in the sciatic nerve. Langley’s nicotine method was used to
determine the position of these ganglia.

The experiments of Gunn and Chavasse (30) and Crsipetionl and
Twombly (18) on the effect of epinephrin on isolated veins lend sup-
port to the belief that a venopressor nervous mechanism exists in the
veins. The details of these experiments will be given later in this
paper under the head of adrenalin. |

Hooker (41), (42) has demonstrated veno-pressor fibers in a nerve
trunk running from the inferior mesenteric ganglion to the veins of the
large intestine. A rise in venous pressure in an isolated loop of intes-
tine was induced by stimulation of: a, the nerve to the part (peripheral
mechanism); b, a sensory nerve such as the saphenous (central reflex
mechanism); c, the central mechanism by asphyxia. Section of the
peripheral nerve or destruction of the medulla destroyed the reflex.
A probable failure of this mechanism in shock is predicated by Morison
and Hooker (55).

_A contraction of the veins seems to be the cause of the increased:
capillary pressure found in the blue-handed type of irritable heart
cases as described by Briscoe (9). This is especially illustrated in
those patients whose hands were sometimes normal and sometimes
blue. The average readings of this class, when the hands were nor-
mal, were: venous pressure, 10.6 cm. H2O; capillary pressure 25.3 em.
H:O; and when blue were: venous pressure, 10.6 cm. H:O; capillary
pressure, 33.3 cm. H,O. Whether this contraction of the veins falls
under the chemical mechanism theory or the nervous mechanism
theory, the author does not state. One presumes, from the article,
that it is the latter. eg

It is not intended in this paper to review in detail the literature on
portal venous pressure, as it forms a rather specialized type of venous
pressure. A comprehensive review of the literature which has estab-
lished the presence of a vasomotor mechanism in the radicles of the

.

4
a
t
y
i

SESE er ae

Se So ae

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 103

portal vein, is to be found in Burton-Opitz’s (13) and Edmunds’ (22)

papers.

Effect on venous pressure of: 1. Adrenalin. a. Effect on isolated
veins. Dunn and Chavasse (30) have tested the effect of adrenalin
(1 to 100,000) on isolated ring preparations from various veins in the
sheep (external jugular, mesenteric, superior and inferior cava) and
find a constriction similar to that which occurs in the arteries. The
external jugular gave a greater response than the superior and inferior
cavae. The amount of response of the mesenteric vein could not be
compared with that of the other veins, for the temperature in the

_ ease of the mesenteric was 41°C., while in the other experiments it was

36°C. From this they judge that the veins probably contain veno-
constrictor nerve fibers from the thoracico-lumbar sympathetic nervous

_ system. Crawford and Twombly (18) corroborate these observations,
finding constriction of ring preparations from femoral, iliac and saphe-

nous veins of the dog. Their work on roosters is interesting in that
they find some veins that contract in adrenalin solutions, and others
that do not. Rings of the jugular vein of white Leghorn roosters,
taken from the middle of the neck, contract slowly with 1 to 60,000
adrenalin in oxygenated Ringer’s solution, but rings from the jugular
vein near the head and from the large vein of the wattles gave no
response. This argues, they feel, against a vasomotor supply to the
cephalic end of the jugular vein and the veins of the wattles, and they
suggest that perhaps the absence of vasoconstrictor fibers in wattles
may be one of the reasons why they blue so easily. ;

b. Effect of intravenous injections. Hill (36) found that intravenous
injection of suprarenal extract into a dog whose vagi had been divided
caused a rise in arterial pressure of 1170 mm. MgSO, solution, while
the vena cava pressure remained unaltered. Plumier (61) attributed
the rise in superior and inferior vena cava pressure, which he obtained
on intravenous injection of adrenalin in the intact animal (dog), to the
slowing of the heart which such an injection occasions. He feels that
one does not get as great a rise with say a 0.4 mgm. injection as the
slowing of the heart would seem to indicate, but this may be explained
by the fact that the increased force of the heart beat tends to reduce

the venous pressure. However, after cutting the vagi, unless a very

large dose is administered, there is no change or only a slight rise in
venous pressure. In both cases there was important vasoconstriction
resulting in a considerable rise in arterial pressure, but this decreasing
of the capacity of the vascular system, he points out, is not sufficient,

104; RECETS OP . HELENE CONNET

in. itself, to change the venous pressure. Capps and Matthews (16) —

find that a small dose (they do not state the amount) of adrenalin does
not affect. the venous pressure, but a large dose, such as } mgm. (2
minims) of 1 to 1000, causes a rise of from 10 to 80 mm., and that coin-
cident with this rise, the heart is markedly slowed.. ‘The rise in
venous pressure was coincident with this halting, irregular action of the
heart, and it remained high until the normal rhythm returned.. We
found likewise that, in a slow or inhibited heart-action from exciting
the vagus nerve with faradic current, the venous pressure rose. Hence
it seems probable, as Plumier (61) states, that the rise in venous pressure

after large doses of epinephrin is explained by the halting heart-action —

rather than by any venomotor stimulation’ (p. 390). Bainbridge and
Trevan (2) exclude reflex vagus inhibition in their experiments by a
small dose of atropin early in each experiment. Under these condi-
tions they find little or no change in vena cava pressure after adrenalin
injection into a portal tributary or systemic vein (hepatic artery tied
or intact), but a rise in portal pressure due, they think, either to a
swelling of the columns of the liver cells narrowing the capillary chan-
nels, or constriction of the radicles of the portal vein. Two cubic
centimeters of a 1 to 10,000 solution of adrenalin cause a rise in portal
pressure equal to 255 mm. of sodium citrate solution. Kuno (50)
explains the slight rise in venous pressure which he obtained on injec-
tion of adrenalin, with the heart beating faster, by saying, “‘the con-
traction of the blood vessels evoked by adrenaline is most distinct in the
arterial system so that ‘a large amount of blood might escape into the
veins. The pressure in the veins does not therefore fall, on the con-
trary it rises more or less during action of the adrenaline although the
heart works extremely energetically’”’ (p. 232).

As far as I know, no measurements have been made of the effect of
adrenalin on venous pressure in man. A word of caution should,
therefore, be given about transferring data concerning adrenalin from
animals to man. As has been noted above, the rise of venous pressure
in dogs has been attributed to the concomitant slowing of the heart.
In man, however, adrenalin does not slow, but quickens the heart
rate.

Clinical findings reported by Donaldson (21) and Miller (54) indicate

that in practically every person (normal or pathological) adrenalin

injection causes no change or causes an increase in pulse rate. Only
one case of a fall in pulse rate was reported (Donaldson) and this was
in a patient much collapsed from: hemorrhage. With the subcutaneous

>

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 105

injecion of 0.5 ce. of 1 to 1000 adrenalin, used in the ‘‘Goetsch”’ test
(29), (57), there is no reaction on the part of normal patients and an
increase of ten to fifty beats per minute in patients hypersensitive to
adrenalin. This result is further corroborated by the work of Wearn
and Sturgis (74) and of Tompkins, Sturgis and Wearn (73). There-
fore it would be incorrect to assume that the results obtained by Capps
and Matthews (16) on dogs, in their studies on ‘‘ venous blood-pres-
sure as influenced by the drugs employed in cardiovascular therapy,”
necessarily apply to man. Similarly, Meek and Eyster’s conclusion
(53) (based on experiments on unanesthetized dogs with good vagal
tone), that adrenalin cannot be the immediate cause of cardiac accel-

eration which follows moderate exercise, because the heart slows after
adrenalin, cannot be held good for man in whom the heart increases
its per-minute rate after adrenalin. Still another example of the
- different way in which adrenalin affects the same structure in different
animals is given by Barbour (5), who found that adrenalin caused con-
striction of human coronary arteries, but relaxation of the coronary
arteries of the calf, sheep and pig.

2. Various other influences. Since the experimental work in this paper
deals only with the effect of adrenalin on venous blood pressure, it does
not seem appropriate to review, in detail, the effect of various other
in uences on venous pressure. The following references may, how-
ever, serve to make this general review of the subject more sorapicte.
The relation of venous pressure to:

a. Gravity. Hill (85); Hill and Barnard (37); Barach and Marks (4)

b. Respiration. Jacobson (46); Wertheimer (75); Hill and Barnard
- (37); Burton-Opitz (12); Plumier (61).

c. Exercise. Burton-Opitz (11); Hooker (38); Elpers (23); Jones
(47); Henderson and Harvey (33). Krogh’s (49) recent work on the
effect of exercise in opening up new capillary beds, is of interest in
this connection.

d. High altitudes. Schneider and co-workers (66), (67), (68), (69);
Kellaway’s (48) work on the relation of anoxemia to the output of
adrenalin may throw some light on the question of the effect of high
altitudes.on venous pressure. :

e. Increased atmospheric pressure. Hill (36). :

f. Drugs, other than adrenalin. Hill and Barnard (37); Plumier (61); ;
Burton-Opitz (14); Capps and Matthews (16).:

_g. Injection of physiological solution. Bainbridge. si Kuno (50)...

106 HELENE CONNET

Venous pressure work on man. <A review of various methods for meas-
uring venous pressure in man is found in an article by Hooker and
Eyster (43), in the Johns Hopkins Hospital Bulletin for 1908. The
following is a list of articles dealing with these methods:—Oliver, 1898,
(58); Frey, 1899, (26); 1902, (27); Gaertner, 1903, (28); von Basch,
1904, (6); Sewall, 1905, (70); von Recklinghausen, 1906, (62); Oliver,
1907, (59); Moritz and von Tabora, 1910, (56); Frank and Reh, 1912,
(25); A. A. Howell, 1912, (45); Lombard, 1912 (51); Hooker and Reese,
1914, (44); Hooker, 1914, (39); Brown, 1918, (10); Wiggers, 1918, (76).
For comparison with the normal venous pressure values in man, given
in the above articles, it may be of interest to note the venous pressure
values obtained by Jacobson, 1867, (46), in sheep, and by Burton-Opitz,
1903, (12), in dogs..

Pathological values are given in the articles by Calvert (15); Hooker
and Eyster (43); A. A. Howell (45) and Clark (17). Diurnal varia-
tions are given by Oliver (60); Hooker (39) and Clark (17); the effect
of age and sex on venous pressure by Elpers (23) and Hooker (39),
(40), and the effect of temperature by Elpers (23); Hewlett (34) and
Hooker (39).

AN EXPERIMENTAL STUDY OF THE EFFECT OF ADRENALIN ON VENOUS
BLOOD PRESSURE

Introduction. It is evident from the foregoing discussion that in
studying the effect of any drug on venous blood pressure, one must
take into consideration the effect of this drug on the various mechanisms,
changes in which are known to cause changes in venous pressure. So
close is the relationship between the various parts of the circulatory,
vasomotor and respiratory systems, that any change in one part usually
causes changes in several other parts. Hence, a change in venous pres-
sure must very often be ascribed to the operation of several factors,
sometimes supplementing, sometimes antagonizing one another. Some-
times one factor is so predominant than any other is lost sight of.
This seems to have been the case in the previous study of the effect
of adrenalin on venous pressure. A greatly slowed heart causes a rise
in venous pressure. Adrenalin, injected into the blood stream of dogs
with good vagal tone, causes slowing of the heart and a rise in venous
pressure. Naturally, the conclusion was that the rise in venous pres-
sure was due to the slowing of the heart rate, especially since little
_ change in pressure occurred on administration of adrenalin to these
dogs after vagotomy.

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 107

It has been demonstrated for sheep by Gunn and Chavasse (30),
and for dogs and roosters by Crawford and Twombly (18), that adre-
nalin chloride causes contraction of isolated vein rings. The work of
Bainbridge and Trevan (2) indicates that the radicles of the portal
vein in the intact animal contract under the influence of adrenalin.
Nerves to the veins of the limb have been demonstrated by Thompson
(72) and Bancroft (3), and to the portal vein by Mall (52) and others.
The experiments of Hooker (41), (42), demonstrate the existence, in
the intestinal veins at least, of a nervous constrictor mechanism, which
ean be stimulated directly or indirectly. In view of this evidence, one
naturally wonders whether or not such a nervous mechanism is fune-
tioning when adrenalin chloride is introduced into the circulation, and
if the general venous pressure rise occasioned thereby is not, in part
at least, due to a constriction of the veins. No evidence, as far as I
know, has been found that points to such a mechanism coming into
play after adrenalin, except in the case of the portal vein.

It is with these elt in mind that the present investigation has
_been undertaken, to ascertain what factor or factors are responsible
for the general rise of venous blood pressure which follows the intra-
venous injection of a solution of adrenalin chloride.

Experimental procedures. In the course of these experiments about
fifty dogs and twenty-five cats were employed, weighing, on the aver-
age, 8 kgm. and 2.3 kgm., respectively. The experiments on dogs
were carried out under ether anesthesia. The cats were decerebrated
according to the method described by Sherrington in his recent labora- _
tory manual (71), an anesthetic, ether, being used only during the
procedures prior to the siebrebeation, The decerebrate cat prepara-
tions were always kept on an electric pad throughout the experiment.

Arterial pressure was recorded by a mercury or a Hiirthle manom-
eter, sometimes by both. Venous pressure was recorded as follows:
brass cannulae (7 cm. long by 1 to 1.5 mm. bore, for cats, and 16
em. by 2 to 2.5 mm., for dogs), connected with manometers containing
2 per cent sodium citrate, were inserted in the external jugular and
femoral veins and pushed in past the valves so that the pressures re-
corded were those in the superior and inferior cavae. Whenever there
was any doubt that the cannulae were not properly inserted, a dissee-
tion was made at the end of the experiment. The entrance of the
superior cava into the heart was taken as the zero level for pressure.
To make sure, from time to time, that no clots had formed, the pres-
sure was tied in the manometer, by means of a pressure bottle, to

108 _. | ‘HELENE: CONNET

several centimeters above the pressure existing in the vein and the
citrate solution then allowed to run into the vein. Thisit did promptly,
‘if there was no obstruction.. It was always noted on the tracing when
‘such a procedure occurred. ‘Controls were made which showed that
injections of such amounts of 2 per cent sodium citrate (} to 1 ce.)
had no visible effect on the circulatory system, and the amount of
fluid: injected was insufficient to cause any change in venous pressure.
- Simultaneous tracings of arterial pressure, respiration and time
relations were made on a smoked paper kymograph. Since the experi-
menter worked alone, it was necessary to resort to some method of
recording, so that venous pressure readings could be made and injec-
‘tions. carried out and recorded by one person, and the exact corre-
‘spondence between the various pressures be noted on the tracing.
One lever recorded respiration, another the arterial blood pressure,
and a signal magnet, which marked the base line for the arterial pres-.
sure (mercury manometer); recorded the duration of the injection and —
“was also connected with a Harvard clock recording half-minutes. In
-this circuit there was an-electric buzzer which signaled every time the
-marker was recording a half-minute, and at:the sound the level of the
venous pressure manometers was noted, the: pressures later being
‘marked at the proper place on the tracing. On the same line with
-this marker, and writing only a millimeter or two behind it, a Jacquet
:chronograph recorded seconds.

‘Parke, Davis & Company’s adrenalin chloride 1 to 1000 solution was
-used thromabant, Before injection, it was diluted with 0.7 per cent
-godium chloride to the desired strength, usually 1 to 10,000. _-

:: Special experimental procedures, not followed in all. experiments,
_are described in connection with the eDaeeeer data. obtained. from
-those procedures. 6 !

Of course, if small rises of venous pressure aré to be considered sig-
‘nificant, one must make sure that they are not caused by the introduc-
«tion of fluid, usedinthe injection. For this reason, very small amounts
tof:adrenalin solution were slowly injected (in most cases 0.5 to 3.0 ce.
-in 5 seconds. or: more), and control experiments were several times
made to:ascértain- how-:large an amount of 0.7 percent NaCl had to be
-introducéd. to cause any change in venous pressure. One-half to 3.0
dé. were without effect, .5.0. cc. caused 4 rise of 3 or 4 mm., while:20.0
-eais+a@ larger amount::than was ever employed in adrenalin chloride in-
-Jections—caused a.rise of only 10 or 15 mm. Nai citrate solution, .~

. ¢ \ * ’ . e
at ee tteers Mere peeteentye ye gt fey beey fers gy . . Ne 3 eee & 7: ¢ ee
ahs qed ee cate oe ae. ‘S2 6 ee wogrr ak X Ch 34 sOectak fied aS ore

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE -109

Experimental work on: 1. Changes in peripheral resistance and ca-
pacity of the vascular system. Under all conditions studied, adrenalin
always caused a rise in arterial pressure due to arterial constriction.
The rise was higher for the same dose when the heart rate was in-
creased than when it was decreased, but there was always arise. Such
a constriction, other things being equal, would tend to cause a fall in
venous pressure due to the obstruction to the flow from arteries to
veins but, on the other hand, the decreased capacity of the system
brought about by arterial constriction would tend to raise the pres-
sure in all parts of the circulatory system. In some cases of arterial
constriction, as shown by Bayliss and Starling (7) and discussed in the
historical review in this paper, the two factors may balance and give

no change in venous pressure. There is one factor here which seems

to have received little notice in this connection, and that is the rate at
which plasma may leave the blood stream under varying arterial pres-
sures. How far this may operate toward compensating the ‘‘ decreased
capacity of the system” factor, is not known. At all events, in the
case of adrenalin, when all other causes for change in venous pressure
are ruled out, as far as possible, these factors of peripheral resistance
and capacity of the system seem to balance, for no change in venous
pressure occurs. Chart 1 (I and II) illustrates this point. About a
quarter of an hour after the decerebrated cat (chart 1, I) was curar-
ized, according to the procedure described under ‘‘respiration,’’ 0.15
mgm. (3 cc.) of adrenalin chloride was injected into the right femoral
vein causing no change in vena cava pressure, although the arterial
pressure was raised from 34 to 167 mm. Hg.

~ Chart 1 (III and IV) shows an experiment in which the venous pres-
sure fell after adrenalin, a very unusual occurrence...It is probable
that, in this case, the increased peripheral resistance so obstructed
the flow from arteries to veins that there occurred a fall in venous
pressure. The decrease in heart rate would tend to cause a rise in
venous pressure, but this was apparently overbalanced by the-factor

or factors tending to cause a fall. In any case, it seems evident that

the usual rise in venous pressure caused by adrenalin is not brought
about, directly, by changes taking place in the arteries, though, as will
be explained later, a rise in arterial pressure may assist in causing a

"rise of venous pressure when it occurs in conjunction with a slowing of

2. Chemical regulation. It is conceivable that adrenalin may act as
a chemical stimulant to venous musculature, independent of any effect

110

HELENE CONNET

CHART. NO. |
Ir

- mz mw
0.15 MG.(3 CC.) 0.25 MG.(5CC.) O.IMG.CiCC.) O.1MG.(CI CC.)
240 aS sss ’
220 JZ A 7% ae
<- / x e- -e- -4~ e--e
a00 Ge els See es s ramet y's f
180 ra i. me | Jokes yon
160|----a bs —
kw Bb
140 ols z
i00 : -— eo “—~s—_—_@——_-»
100 fay = Ooe- eee r
30 wet NI + + 4as0r® a 0d ia ee oe?
bO| "epee poe) SSE: aad biel
—C
Chee “et
OL. 2
tS Ol Le aOR BANG! Hie Se ae
. TIME IN MIN.
Ir a CHART NO.Qm ac
O| NERVE I> VIE Oo. Jaee) \. & oalmMe-
°0 Sim : Sim ek hs '\ @ oa PED
280 . tom, 1 \ 4 , i et
260 . ‘ \ pea a!
240 : ALT ge
a20 3 --?¢— —s NY ~2 iy
180 ; eu
1604 :
14.0 ‘ Zz ‘= S.
120 “ie +n, bs bore we Ez
TT) essed: Reber ets ae = Nz.
0 N oo oPPti me ot ree
x“ “ a <x *
bO < é oe oes.
40 gS Oot rereelt® D> ore
20 3. eed CAL ol
te a Gat oa -
0 < one lu
-20 E =
4 =
-40 < re)
ie A Ss)
Dial Rel e Tegiae ) , ea umes i
= . oF IME IN MIN. : * ite t .
- CHART NOQ.3
mi Ir Ir Ww xr
0.25 MG. 0.05 MG. 0.1 MG. 4 *
220 és4 Osea BS 0. 03 MG. (0.59
200 : wt, .
ae m
40} |e %, adi. ee
120] I; : |
eo NA
‘ nn —t—— ?
80 4 ¥ ay ae J
bol Tt WS’ Ben Wot sj
+0 Ooeoes of Ye eee **teq 54, een
a0 on gee” jb “ pote = ore
ie) , errerde
S foe” Oe oe WE EDS | 3° ee eee
TIME IN MIN. = a te

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 111

through a nervous mechanism. As far as I know, no work has been
done on this subject. However, adrenalin phcdaces an effect upon
venous rings similar to that upon arterial rings, and it seems reason-
able to assume, until disproved, that in the veins, as has been shown
for the arteries, adrenalin acts by stimulating the myoneural junctions
of the sympathetic nerve fibers in the vascular musculature.

3. Respiratory and muscular factors. The contraction of muscles
against the veins, as in exercise, and changes in respiration, are known
to be very efficient in causing changes in venous pressure. Take for
example chart 2 (II), showing the result of stimulation of the saphenous
nerve before and after administration of curare. The dog was not
completely curarized, as can be seen from the fact that a slight change
in rate and atleaile of respiration was elicited by strong sensory
stimulation. The interesting thing is that the venous pressure response
seems to bear a definite relation to the amount of respiratory response.

When adrenalin chloride is injected in small amounts (usually 0.1 to

0.3 mgm.) there may be no respiratory response, or a decrease in height
and frequency of respiration occurs. This change evidently has little
or nothing to do with the rise in venous pressure, since that rise takes
place when no respiratory variations occur, and a decrease in rate
causés a fall, not a rise in venous pressure. However, to fully rule out
any change in venous pressure due to muscular contraction or respira-
tory change, curare was administered in a number of experiments,
and as soon as breathing ceased artificial respiration was given. The
air was heated to 30°C. before reaching the animal. To be sure that

other muscles besides the respiratory muscles were paralyzed, the

woeeee- pulse rate per minute.

arterial pressure in mm. Hg.
Le vanind superior cava pressure in mm. 2 per cent sodium citrate.
++++ inferior cava pressure in mm. 2 per cent sodium citrate.
respiratory rate per minute.

Wavy line, respiratory amplitude (relative only).

Large dots indicate actual determinations.

Circles indicate that venous pressure cannula was tested and found free of
clots.

Chart 1. Adrenalin chloride injections: I and II, decerebrate cat 7; III and
IV, dog 4 B.

Chart 2. I and II, dog 35 A—Stimulation of saphenous nerve before and after
partial curarization; III and IV, decerebrate cat 9—Adrenalin chloride injections,
before and after bes curarization; vagi cut.

Chart 3. Adrenalin chloride injections: I and II, dog 1 A, vagi + Gat between
I and II; III and IV, dog 4 B; V, dog 29 A.

112 HELENE CONNET

minimal nerve stimulus necessary to cause contraction of some skeletal
muscle in the fore limb was determined before curare, and then sufficient
curare was given to cause this stimulus and other stronger stimuli to

become ineffective. In such experiments adrenalin had practicallythe -

same effect on the venous pressure before and after curare: chart 2
(III and IV) is an example. Changes in the rate of the artificial res-
piration seem, also, to be ineffective. It has been noticed a number
of times that the venous pressure response to adrenalin is not as
marked immediately after the administration of curare as it is later.
In a few cases, as shown in chart 1 (I and IT), the effect persists. This
depressant action on venous pressure response is probably due to an
action on the nervous mechanism in the veins. That it usually passes

off very rapidly, fits in with the general idea that the primary depres-

sant action of curare on the circulatory system is of very short duration.

When not curarized, occasionally the act of injecting was a sufficient
sensory stimulus to cause a muscular response. This muscular re-
sponse, often seen in a stretching out of the lower limbs and conse-
quent stretching of the abdominal muscles, was not evident on the
tracing given by the usual pneumograph. Therefore, instead of re-
cording respiration by’means of a rubber bag around the chest, a small
rubber balloon was inserted through a small opening into the abdominal
cavity near the diaphragm, and the skin closed tightly around the glass
tube to which the balloon was attached. The balloon was then blown
up until it held about 5 cc. air, and connected with a recording tam-
bour and lever as the bag around the chest had been before. This has
the advantage of recording not only respiratory changes but also changes
in abdominal pressure which very markedly influence venous pressure,
as shown by Hill and Barnard (37).

4. Heart rate and output per minute. Plumier (61), in his work on
venous pressure, laid great stress on slowing of the heart rate as a very
efficient means of raising the venous pressure. Occasionally the heart
suddenly stops beating after an injection of adrenalin. The venous
pressure may then rise very high; in one case it rose five times as high
as it had after a similar dose of adrenalin when the heart rate was
increased. In dogs with good vagal tone, the heart usually slows
markedly after adrenalin and the venous pressure rises (see chart 3, I).
Even with the vagi cut the pulse rate is often slowed, due: probably
to a direct action on the heart musculature, as is shown in chart 3 (II).
A. rather uncertain venous pressure response is often seen in vagotomized
dogs when the heart rate is increased after an adrenalin injection.

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 113

Such results would lead one to attribute the rise in venous pressure
solely to: the decreased heart rate as did Plumier (61) and, later, Capps
and Matthews (16). Chart 3 (III, IV and V), however, makes one doubt
that’ heart rate is the only factor involved. Work on dogs under ether
did not prove satisfactory in clearly demonstrating whether or not a
nervous mechanism played some part in venous pressure response to
adrenalin. There are several reasons for this. In order to rule out
the slowed heart rate, the vagi had to be cut. This caused a very great
increase in the depth of respiration which brought about such a marked
rise and fall in venous pressure that it was difficult to compare such
fluctuations in pressure with the change occurring after adrenalin,
since the respiratory variations were then often absent, due to tempo-
rary cessation of respiration. Besides, even with the vagi cut, the
heart frequently slowed after adrenalin. Still more serious, perhaps,
is the possible effect of ether on the nervous mechanism. Ether de-
presses the arterial vasomotor response to adrenalin, as shown by
Berry (8) and Rous and Wilson (63), and could naturally be expected
. to affect a nervous mechanism in the veins—all the more so because,
as shown by Hooker (41), this mechanism is extremely sensitive. If
very light ether anesthesia were employed, there would then be the
difficulty of muscular and respiratory responses discussed above.
Chart 3 (III and IV) shows an unusual experiment in which the ether
anesthesia was light, and yet respiration remained constant throughout
the experiment. Experiments on decerebrated cats, which needed no
anesthetic after the operation, and whose vagal tone is not nearly so
marked as dogs’, proved to be very satisfactory. Also, it was no small
consideration, since curare is very difficult to obtain, that it took only
a small amount of curare to curarize a cat, as compared with a dog.
Since sensory stimuli have been shown to be effective after curare, it is
desirable, for humanitarian reasons, to work on a decerebrated prepa-
ration asta there can be no question of not giving enough anesthetic
during curarization to cause analgesia.

Concerning the influence of change of the heart rate on venous pres-
sure, it should be said that it is really the per-minute output of the heart,
rather than the heart rate alone, that is essential. Quoting from
Erlanger and Hooker (24), (p. 161), ‘‘If the pulse pressure is approxi-
mately dependent upon the amount of blood that escapes from the
arteries during one cardiac cycle, then it is obvious that the amount
that escapes must vary directly as the pulse rate.’”’ Therefore, pulse
pressure multiplied by pulse rate gives us an expression of the output

114 HELENE CONNET

of the heart per minute. It is possible to increase or decrease the out-

put per minute by increasing or decreasing either or both of these

factors, and to keep the output constant by varying the two factors
proportionately in the opposite directions. Hence one cannot say that
a slowed heart necessarily causes a decreased output per minute, and
hence a rise in venous pressure. It is theoretically possible that the
pulse pressure might so increase that even with a lower heart rate the
output per minute would increase. In the case of adrenalin, however,
this possibility seems never to be realized, at least when the pulse
rate is greatly reduced. For example, in one experiment, before
adrenalin, the pulse pressure was 180 mm. Hg. and the pulse rate per
minute 158, making a per-minute output of 28,440. After adrenalin,
the pulse pressure was 240 and the pulse rate 20, giving the greatly
decreased per minute output of 4800. The slowing in this case was
way out of proportion to the increased pulse pressure, and was accom-
panied by a rise in venous pressure from 25 to 120 mm. Na citrate.
Experiments were performed on dogs, as illustrated in chart 4, in
which the vagi were cooled by perfusing ice water through glass tubing
in a V around each nerve, and afterwards allowing the nerves to come
to room temperature again. The pulse rate, arterial pressure, respira-
tory rate and amplitude were-all affected but very little change in
venous pressure took place. When the perfusion of ice water around
the nerves was discontinued, the pulse rate decreased from 144 to 108,
the venous pressure decreasing also a few millimeters. When the
heart stops beating the rise in venous pressure is due to a back pres-
sure in the veins and also to the tendency for equalization of pressures
to take place throughout the vascular system. When the heart
slows, but does not stop, these factors operate to cause a rise in venous
pressure, but to a less extent. If now the arterial pressure rises as the
slowing of the heart occurs, as after adrenalin, the mean pressure of
the vascular system toward which the various pressures are tending
will, of course, be greater, and hence there is a much greater possi-
bility of a large rise in venous pressure. In one experiment, after an

adrenalin injection, the heart was slowed about as much as after the

cooling of the vagi was discontinued (chart 4, II). In the former case
the arterial pressure rose, showing that the capacity of the vascular
system was decreased, while in the latter it fell. In the case of adre-
nalin there may be a nervous constrictor mechanism in the veins at
work, but from other experiments on dogs it seems likely that this
plays little part in etherized dogs. It seems more probable that the

ee — eee

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 115

decreased capacity of the system acts in connection with the slowed
heart rate to cause a rise in venous pressure. _

5. Nervous mechanism. It can readily be seen from the foregoing
that one cannot attribute a rise in venous pressure to the activity of a
vasomotor mechanism in the veins, unless one is sure that the per-
minute output of the heart has not decreased sufficiently to explain the
rise. As we have seen,when this factor is ruled out in experiments
on dogs under ether, the evidence for the functioning of a nervous
mechanism is not very conclusive. In the experiments on decerebrate
cats, however, there is clear evidence of such a mechanism. With the
vagi intact, we may get the response which is usual in dogs—a rise in
venous and arterial pressure and a slowing of the heart, chart 5 (I
and II). When, however, the vagi are cut (chart 5, III and IV),
there is still a rise in venous pressure. The heart rate is practically
unchanged and the pulse pressure increased (as nearly always occurs
after adrenalin) so that the output per minute is now increased and the
tendency would be for a fall rather than a rise in venous pressure.
So also in this experiment, the respirations were decreased in height
and frequency which would tend to lower rather than raise venous
pressure. The rise, therefore, which one does get under these circum-
stances seems to be due to a nervous factor, rendered probably less
effective in producing a rise because of the factors tending to produce a
fall. Chart 6 shows very uniform rises in venous pressure, though in
some cases the heart rate is slowed and in some cases increased in fre-
quency. In the experiment shown in chart 7, the respiratory factor is
completely controlled by curarization. The vagi are cut in IV and V.

It is interesting to inquire whether or not the seat of this action of
adrenalin is central or peripheral. Two different types of experi-
ments were performed to investigate this question. In the first, the
cord was sectioned in the cervical region (about at the level of the
fifth or sixth cervical vertebra). As is shown in chart 8, this does not
destroy the venopressor response to adrenalin, though whether or not
any small part of the reaction is of central origin is hard to determine,
since the amount of response to a certain dose is not invariable, and so
the effect of a dose before cutting the cord is difficult to compare with
the same dose just afterwards. The response before and after is
quite similar, however.

For the second type of experiment the isolated vein preparation of
Hooker (41) was used. In the first of these experiments, Doctor
Hooker kindly demonstrated the method to the writer. An isolated loop

116

PRBUUS' ON

VAG!

gaih?

| Cle ns onapeit a

- HELENE CONNET © ©...

CHART NO. *

e-. “0-0

0.05 MG. icc.)

«
¥ e
‘6, o1rOr ae,

Oereeres Re
—
Oee- ee.

CHART NO.6
Ir

0.asSMG. GCC.)

e— -0- -4

Ps

4 rd
cee’ j $ ios Po 4 aed
Ore ers Toesreseresesee er verorre E onaibibatadcudy eum 5 faa
2 2) a
a Sat ht ty IN A ee
CHART NO. O
ae Ir IZ
0.O5MG.(ICC.) 0.05 MG. accas 0.25 ‘Me. (SCe.) hte (5CC.)
e © e--e--¢ a re o- uJ
“ns < >] MERE 0 ag anit e-- 07" Y oe s<e--
Si) aE, ig aap
a on | SS
< <
7 >
| ies
pee, es be
* . % ° ee
5 e 20. .
z F . . x Zz Nn
= ve ° NU > pee ae are
> —. hy . » a ¢
e. - ~ . “9, = . q e Fy ores
*s@es 0g oe*- Pie Oh
SIM rs "ATOR ORE IBA I OBE BOOBS 2

amr
0.5 MG. (iocec,)

ST UME IN Mine |

See Se

—

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 117

of large intestine in which the inferior mesentery artery and vein had
been cannulated, was hung up free from the surrounding intestines.
The only connection with the animal was through a nerve running from
the inferior mesenteric ganglion to the large intestine and entering
the intestine in close proximity to the inferior mesentericartery. Blood
was washed out of the vessels from artery to vein by means of warmed
(about 37°C.) Ringer’s solution under air pressure of 90 to 120 mm. Hg.
The cannulated artery was then disconnected from the air pressure appa-
ratus and allowed to hang freely from the preparation. The vein was
then connected with a venous pressure manometer containing warmed
Ringer’s solution. ‘The zero level was, in most cases, adjusted to the
level of the vein in which the pressure was being measured. The vein
was distended by a positive pressure of from 30 to 100 mm. Ringer’s
solution, by means of a pressure bottle. Any change in the caliber of the
vein would then be indicated by a rise or fall in the level in the venous
pressure manometer. Hooker (42) has shown that, in this preparation,
a rise in venous pressure may be elicited, peripherally, by stimulation
of the nerve to the part; reflexly, by sensory stimulation; and centrally,
by asphyxia. In such a dog, when adrenalin chloride was injected
into the saphenous vein in doses from 0.2 to 0.6 mgm., no rise in venous
pressure in the isolated vein occurred, but when such injections were
followed by asphyxia, produced by closing off the end of the tracheal
cannula for two or six minutes, a rise in venous pressure from 15 to
20 mm. occurred, showing that the preparation was still in good condi-
tion. This seems to show that the venopressor rise after adrenalin is
not due to any central mechanism, controlling the caliber of veins,
but to a peripheral effect on sympathetic endings, such as takes place
in ne arterioles. :

------ pulse rate per minute.
arterial pressure in mm. Hg.

Mi spreas .. superior cava pressure in mm. 2 per cent sodium citrate.
++++ inferior cava pressure in mm. 2 per cent sodium citrate.
respiratory rate per minute.

Large dots indicate actual determinations.

Circles indicate that venous pressure cannula was tested and found free from
clots.

Chart 4. Dog 17 A—Vagus nerves cooled.

Chart 5. Decerebrate cat 3—Adrenalin chloride injections, vagi cut between
II and III.

Chart 6. Decerebrate cat 6—Adrenalin chloride injections, artificial respiration.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 1

118

. 300
aso
abo
a+o
220
200
1.80
160
"140
120
100

300
280
Abd
440
eM RG
,, 200
180
160
140
(ao
100

abo
ato
aad
200
180
160
140
120
100

HELENE CONNET

CHART NO.)
am

aL Ir Ir a
O. as MG.(SCC) 0.aMG. (40c.) oF MG.(10CC) 0.2 MG. (2CcC.)
pts
5 ti een ere
o~-0} -« Pte “Treat” a a 4 Pg
e” Ym ee ame it ad éJes 4
Pe ike 8
2 z |] |
>. z
td Ww
E 5 ‘
Oy we, i 100 — cog Bet i "See 4 TPny =
tee > + Cees d . 328 bh seses ore
ete 7s z &. 2 ek Cor sere < Oressaet? *e,
er @ > 4) tvs 5. . ‘eo Oe-.e- r *+e0 waiter wa ‘3
4 ¢ -
Oo; es a Q “@ TIME "IN NIN. a. i] a [@) ] a a
I Ir mm ae
0.05* Ms. (0. 5+Cc) 0.05 MG.0.5CC.) 0.1MG.(L ee.)
a ae
ie uF Sr ae a SA IRREGULAR
RESPIRATORY RATE BW ms oe
b4 PER MIN. :
THROUGHOUT
5 = = is 4
<
S$ $2 $ >
pc «Oo da fc
Ww wo tu
r FS r -
= w - é
-_— +e ”o s rs age
ZF Zao Zoe eg fh ;
z = 5 nett re) =
= oO - fe
TTh han” tS oe
pe x mm ha gts. a iva
0.1 MS. (ICC.) G.I MG.(1CC) O.1MG.(icc. -5.M6. 0.1 MG .aM6G.(ace:
¢ im ) tae 2 ee oF acy
‘a *
a ier putt ilesida > apna

- -e-

v

/

e+ +8 of

ig. 0 a

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 119

Some experiments which were being carried out in this laboratory
suggested that histamine might have an effect on the venopressor
mechanism. Chart 9 (I, II, III) shows typical arterial and venous
responses to adrenalin in a curarized, decerebrate cat. One-half milli-
gram (1 cc.) of histamine (chart 9, IV) caused a fall in arterial pressure;
in some experiments the venous pressure also fell. A more marked
reaction was shown in another experiment, where twice the dose was
given. According to Dale and Laidlaw (19), had the cat been under
an anesthetic, these doses of histamine, from. which the unanesthet-
ized cat recovers, would have produced fatal circulatory collapse.
When histamine was followed by adrenalin in the same and larger
doses than given previously in the experiment, there was no venous
pressure response (chart 9, Vand VI). The arterial pressure rose, but
not nearly as high as before, with the same dose. A second injection,
however, gave a typical arterial pressure rise, but still no change in
venous pressure. The work of Dale and Richards (20) and Dale and
Laidlaw (19) leads them to conclude that relaxation of the capillaries
is the cause of the vasodilator effect of histamine. The fall in venous
pressure after histamine and the subsequent failure of the nervous
mechanism to respond to adrenalin, indicates that histamine has a
relaxing effect on venous tone, also, and acts in some way to depress the
activity of the venopressor mechanism.

Summary. ‘The various possible factors operating to produce the rise
in venous blood pressure which occurs in dogs and cats after intra-
venous injection of adrenalin are discussed. The two factors chiefly
responsible are the decreased heart rate bringing about decreased unit
output of the heart, and a vasoconstrictor mechanism in the veins. The

------ pulse rate per minute.
arterial pressure in mm. Hg.
avkidts superior cava pressure in mm. 2 per cent sodium citrate.
+++-+ inferior cava pressure in mm. 2 per cent sodium citrate.
respiratory rate per minute.

Large dots indicate actual determinations.

Circles indicate that venous pressure cannula was tested and found free from
clots.

Chart 7. Adrenalin chloride injections: I, II and III, decerebrate cat 8—Vagi
intact; cat curarized; artificial respiration; IV and V, decerebrate cat Gor Veg
cut; cat curarized; artificial respiration.

: Chart 8. Betensboata cat 15—Adrenalin chloride 2 a ii before rae after
section of cord; vagi cut; cat curarized; artificial respiration.

Chart 9. Deterebraks cat 17—Adrenalin chloride injections, before and after

histamine; vagi cut; cat curarized and artificial respiration begun between I and
II.

120 - HELENE CONNET

effect of the first factor is accentuated by the fact that the arterial pres-
sure is greatly raised by adrenalin in the doses here used. The first
factor has been recognized before. Reasons are given why the second
factor was previously overlooked. In dogs with good vagal tone and
under an anesthetic, the rise in venous pressure is almost entirely due
to the first factor. In cats whose vagal tone is not nearly as strong,
the second factor predominates. This nervous mechanism is shown
to be acted on peripherally by adrenalin, and to be depressed by ether,
curare and histamine, especially the last.

I may take the opportunity here to thank Dr. W. H. Howell and

Dr. D. R. Hooker of this University, and Dr. J. L. King of Goucher
College, for kindly encouragement and aid in the preparation of this
paper.

BIBLIOGRAPHY

(1) Barnprinvce: Journ. Physiol., 1915, 1, 65.
(2) BAINBRIDGE AND TREVAN: Journ. Physiol., 1917, li, 460.
(3) Bancrort: This Journal, 1898, i, 477.
(4) Baracw AND Marks: Arch. Int. Med., 1913, xi, 485.
(5) Barsour: Journ. Exper. Med., 1912, xv, 404.
(6) von Basco: Wiener med. Presse, 1904, xlv, 961.
(7) BayLiss AND Staruine: Journ. Physiol., 1894, xvi, 159.
(8) Berry: Endocrinology, 1917, i, 306.
(9) Briscor: Heart, 1918, vii, 35.
(10) Brown: Johns Hopkins Hosp. Bull., 1918, xxix, 93.
(11) Burton-Opitz: This Journal, 1903, ix, 161.
(12) Burton-Opitz: This Journal, 1903, ix, 198.
(13) Burtron-Opirz: Quart. Journ. Exper. Physiol., 1912, 329.
(14) Burron-Opitz AND Wor: Journ. Exper. Med., 1910, xii, 278; Proc. Soc.
Exper. Biol. and Med., 1910, vii, 70.
(15) Catvert: Journ. Amer. Med. Assoc. 1907, xlviii, 1168; Johns Hopkins Hosp.
Bull., 1908, xix, 44.
(16) Capps anD Matruews: Journ. Amer. Med. Assoc., 1913, Ixi, 388.
(17) CuarK: Arch. Int. Med., 1915, xvi, 587.
(18) CRAWFORD AND TwomBLy: N. Y. Med. Journ., 1913, xeviii, 327.
(19) Date anpD LarpLaw: Journ. Physiol., 1918-19, lii, 355.
(20) Date anp Ricuarps: Journ. Physiol., 1918-19, lii, 111.
(21) Donaupson: Brit. Med. Journ., 1914, i, 476.
(22) EpmMunps: Journ. Pharm. Exper. Therap., 1915, vi, 569.
(23) Exrers: (Kiel), 8°, Dortmund, 1911.
(24) ERLANGER AND Hooker: Johns Hopkins Hosp. Rept., 1904, xii, 145.
(25) FRANK AND Reu: Zeitschr. f. exper. Path. u. Therap., 1912, x, 241.
(26) Frey: Illustr. Monatschr. d. arztl. Polytech., 1899, xxi, 68.
(27) Frey: Deutsch. Arch. f. klin. Med., 1902, Ixxiii, 511.
(28) GAERTNER: Miinchen med. Wochenschr., 1903, 1, 2038.

ee ee

EFFECT OF ADRENALIN ON VENOUS BLOOD PRESSURE 121

(29) Gortscn: N. Y. State Journ. Med., 1918, xviii, 259.

(30) Gunn anv Cuavassz: Proc. Roy. Soc. ,1913, Ixxxvi B, 192.

(31) Hearp AnD Brooks: Journ. Pharm. Exper. Therap., 1915, vi, 605.

(32) Henperson: This Journal, 1916-17, xlii, 589.

(33) HENDERSON AND Harvey: This Journal, 1918, xlvi, 533.

(84) Hewxierr: Amer. Journ. Med. Sci., 1913, cxlv, 656.

(35) Hixu: Journ. Physiol., 1895, xviii, 15.

(86) Hitu: Proc. Roy. Soc., 1900, Ixvi, 478.

(37) Hi~u AnD Barnarp: Journ. Physiol., 1897, xxi, 323.

(88) Hooxrr: This Journal, 1909, xxv, 24 (Proc.) (preliminary report); 1911,
XXVlii, 235.

(39) Hooxer: This Journal, 1914, xxxv, 73.

(40) Hooxrr: This Journal, 1916, xl, 43.

(41) Hooxer: This Journal, 1918, a 543.

(42) Hooxer: This Journal, 1918, xlvi, 591.

_ (43) Hooxrr anp Eyster: Tohins Hopkins Hosp. Bull., 1908, xix, 274.

(44) Hooker anp Resse: This Journal, 1914, xxxiii, 27 (Proc. 1,

(45) Hower ..: Arch. Int. Med., 1912, he 148.

(46) Jacopson: Arch. f. Anat. u. Physiol., 1867, 226.

(47) Jones: Lancet, 1917, i, 574.

(48) Kexuaway: Journ. Physiol., 1918-19, lii, 63 (Proc.)..

(49) Kroeu: Journ. Physiol., 1918-19, lii, 457.

(50) Kuno: Journ. Physiol., 1917, li, 221.

(51) LomBarp: This Journal, 1912, xxix, 335.

(52) Matt: Arch. f. Physiol., Suppl., 1890, 57.

(53) Merx anp Eyster: This Journal, 1915, xxxviii, 62.

(54) MitueR: Lancet, 1914, ii, 158.

(55) Morison anp Hooker: This Journal, 1915, xxxvii, 86.

(56) Moritz anp von Tapora: Deutsch. Arch. f. klin. Med., 1910, xeviii, 475.
(57) NicHouson anp GortscH: Amer. Rev. Tuber., 1919, iii, 109.

(58) Oxiver: Journ. Physiol., 1898, xxiii, 5 (Proc. ).

(59) OLIVER: Quart. Journ. Med., 1907-08, i, 59.

(60) OxtveR: Blood and blood pressure, London, 1901.

(61) Puumier: Arch. internat. d. physiol., 1909, viii, 1.

(62) von ReckLINGHAUSEN: Arch. f. exper. Path. u. Pharm., 1906, lv, 463.
(63) Rous anp Wiuson: Journ. Exper. Med., 1919, xxix, 173.

(64) Roy anp Brown: Journ. Physiol., 1879-80, ii, 323.

(65) Roy anp SHERRINGTON: Journ. Physiol., 1890, xi, 85.

(66) ScunEIDER: This Journal, 1920, li, 180.

(67) ScHNEIDER AND Sisco: This Journal, 1914, xxxiv, 1.

(68) ScHNEIDER AND Sisco: This Journal, 1914, xxxiv, 29.

(69) ScHnEIDER, assisted by Curtny anv Sisco: This Journal, 1916, xl, 380.
(70) Sewauu: Colorado Med., 1905, ii, 219.

(71) SHeRRINeToN: Mammalian physiology; a course of practical exercises, 1919.
(72) Tuomrson: Arch. f. Physiol., 1893, 102.

(73) Tompxins, SturGis AND WEARN: Arch. Int. Med., 1919, xxiv, 269.
(74) WeaRn AND Srurais: Arch. Int. Med., 1919, xxiv, 247.

(75) WertHermer: Arch. d. physiol. norm. et path., 1895, vii, 107.

(76) Wiccers: Journ. Amer. Med. Assoc., 1918, lxx, 508.

STUDIES ON THE VISCERAL SENSORY NERVOUS SYSTEM

II. Luna AUTOMATISM AND LUNG REFLEXES. IN THE SALAMANDERS
(NrectuRus, AXOLOTL)

A. B. LUCKHARDT anp A. J. CARLSON
From the Hull Physiological Laboratory of the University of Chicago

Received for publication July 17, 1920

In a previous article (5) we showed that the lung of the frog contracts
immediately following the external respiratory act; that the lung mus-
culature can be induced to contract reflexly by the stimulation of any
visceral or cutaneous nerve with practically no exceptions; that the
lungs of this animal possess a peripheral automatic mechanism which
at times shows rhythm but which under normal conditions is kept in
a state of inhibition by tonic central inhibitory impulses via the vagi;
that these nerves, furthermore, contain not a few motor fibers which
apparently exert no marked tonic activity; and that the cervical sym-
pathetic nerves contain but few motor and no inhibitory fibers for the
lungs. In addition, we made a brief study of the action of certain
drugs (atropine, adrenalin and histamine) on the lungs besides using
nicotine extensively in differentiating between the motor and inhibitory
nerves contained in the vagus fibers to the lungs.

On completion of this work it seemed quite important to us to in-
vestigate the neuro-muscular apparatus and physiology of the nerves
of the lungs of other available amphibia to determine whether or not
the physiological mechanism discovered in frogs is the same or similar
in this class of animals.

METHODS

The only amphibia available for this study were the axolotl and
necturus. The general methods of study were those reported in our
first article, modified to meet the anatomical peculiarities of the type
of animal which we were studying.

In every instance the cord was pithed below the medal As in
previous work on the frog we cannulated the tips of the lungs exposed

122 —

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VISCERAL SENSORY NERVOUS SYSTEM 123

by a ventral incision and connected each cannula with either a water
manometer or delicate tambour. After recording the normal respira-
tions or respiratory attempts of the animal, we attempted to elicit
reflex lung contractions as the result of mechanical and electrical stimu-
lations applied to the animal anterior to the spinal transection. We
subsequently closed the glottis with a small mosquito forceps (in
axolotl only) and pithed the brain (specifically the medulla) to note
any tonic inhibitory influence the vagi nerves might have on the neuro-
musculature apparatus of the lungs. Stimulation of the same nerves
to the lungs next engaged our attention. This was followed by the
effect of the intravenous injection of drugs alone or in combination.
Because of the poor or imperfect circulation in necturus, as a result of
the preparation of the animal for experimentation, for certain purposes

‘we injected the drug deep muscularly, 15 to 20 minutes, before prepar-

ing the animal in the manner described above; for we found that even
injection of the drug in Ringer’s solution by way of the bulbus arterio-
sus or into the pulmonary artery failed to reach the posterior end i
the lung of this animal.

The lungs of necturus are paired sacs, extending from the level’ of
the heart almost to the anal region. There are no alveoli or septa in
these lungs. According to Miller (9) the course of the smooth muscle
fibers in the lung walls is circular, except at the apex. Both lung artery
and lung vein lie superficial to the muscle layer. fia! a a

Miller has described medullated and non-medullated nerve fibers
and nerve nets in the necturus lung. The pulmonary fibers of the vagi
enter the base of the lungs in many branches, not along the pulmonary
blood vessels, as in the frog, but between the blood vessels. There

- are many ganglion cells (bipolar and multipolar) in the main nerve

trunks and in the nerve plexuses. According to Miller, the non-medul-

lated fibers connect with the ganglion cells. Similar ganglionated nerve

plexuses have been described in the lungs of other tailed amphibians,
especially by Stirling (11) in the case of the newt. The older anatomical
literature is reviewed in detail by Oppel (10). Miller’s description of
the ganglionated nerve plexuses in the necturus lung is practically
identical with the local nervous system of the necturus heart, as wren
by one of us (6).

In necturus the glottis or opening from the pharynx into the trachea
is exceedingly small, and certainly not suited for rapid filling or emptying
the lung sacs with air. We have repeatedly seen these animals attempt
to swallow air, the air escaping by the gill slits and in no instance

124 A. B. LUCKHARDT AND A. J. CARLSON

entering the lungs. There can be no doubt that in necturus the gaseous
exchange is carried out by the gills, supplemented by the skin, and the
role of the lung sacs in respiration is an open question. The lung sae
of necturus has the same histogenesis as the lung of other vertebrates,
although it remains most primitive as regards differentiation. Should
these structures be called lungs, especially if they serve mainly or
exclusively as ‘hydrostatic organs?”’

In our review of the anatomical and zodélogical literature on amphibian

respiration, we came across the surprising fact, apparently well known.

to zodlogists though not to physiologists, that many species of tailed
amphibians have neither lungs nor gills, and in other species without
gills the lungs appear to be too rudimentary to function in respiration.
The normal condition of these species is that of the frog with the lungs

extirpated, that is, the gaseous exchange is carried out entirely by the’

skin. But several zodlogists (Wilder (12), Lénnberg (8), Camerano
(4), Bethge (8) and others) have concluded mainly on anatomical
grounds (great vascularity of the mucous membrane of the buccal
cavity and upper end of the esophagus), that the pharyngeal cavity
serves lung functions, especially in those species having neither lungs
nor gills. It appears to be a fact that the so-called buccal respiratory
movements (including that of the external nares) are carried out even
in those species in which the lungs are absent. But Babdék and Kiihnova
(1) have shown that the brain centers controlling these movements are
different from those governing the filling and emptying of the lungs,
the latter are, the former are not influenced. by asphyxia, which fact
seems to throw doubt on the respiratory character of these buccal
movements. Lapique and Petetin (7) have also shown, in the case
of one species of salamanders devoid of lungs and gills, that the main
respiratory organ is in the skin.

None of these questions can be settled shins by direct physiological
experiments, but we may regard the necturus lungs, provisionally at
least, as lungs on the basis of organogenesis. Their motor control
also places them in the same category with the lung of the frog and of
the axolotl.

Since the entire respiratory apparatus (including the lung) of the
necturus is strikingly more primitive than that of the other salamander
used in this study, we shall describe the results obtained from necturus
first and end with a description of those obtained from the axolotl. -

Necturus maculatus. External respiration in this animal is essentially
performed by the large gills which under usual conditions are kept in

VISCERAL. SENSORY NERVOUS SYSTEM 125

more or less constant motion. Occasionally the animals come to the
surface for air which is promptly expelled by an act of swallowing
through the gill slits. The lungs of this animal consist essentially of
two thin elongated muscular sacs which are well supplied with blood
vessels. With but one exception we found them collapsed. In an
inflated and atonic condition their diameter is about 1 cm. at their
greatest circumference. At their ends they taper off into blunt tips,
at their base they communicate with a tracheal sac similar to that
possessed by the frog. This sac communicates with the oral cavity
through a glottis: situated far down in the pharyngeal region. The
glottis is exceedingly primitive. It consists essentially -of a slit which
is quite easily overlooked on direct inspection. We agree with the
description of Oppel that the glottis is exceedingly delicate. Taking
everything into account we feel that the lungs may serve an excretory
function (elimination of CO.) but are rarely if ever filled with air during
any act of external respiration. The lungs receive their innervation
through pulmonary fibers carried by the vagi. It was found impracti-
cable to isolate these latter nerves in the neck for direct electrical stimu-

lation. They were isolated at their exit from the skull by a dissection

to right and left of the median line after a sagittal section of the skull.

Because of the inaccessibility of the glottis for closure by hemostat
as practised in the frog, we prevented communication of the lungs
through the glottis with the mouth by dorso-lateral traction and fix-
ation in that position of the fore legs after pinning the animal on its

back. A wad of cotton wedged under the neck of the animal at the

level of the tracheal sac or put over the tracheal sac and held firmly
in that position by the constriction of a rubber band was an additional
measure employed in not only closing off the glottis but in preventing
intercommunication between the lungs through the tracheal sac.
Axolotl. The lungs of this genus of amphibia are certainly more
complex than in necturus. On opening the abdominal cavity one is
at first glance struck with the resemblance of the lungs of this animal
with the reptilian lung. The upper portions of both lungs are sub-
divided into alveolar sacs by septa; the lower appendages which extend
down the abdominal cavity for a considerable distance are more saclike
in their texture. As in the frog and necturus the lungs communicate
at their base with the tracheal sac which in turn communicates with
the mouth cavity through a well-developed glottis. In the specimens
which we used gills were present and functioning. It was equally
apparent, especially on opening the abdominal cavity, that the animals

126 A. B. LUCKHARDT AND A. J. CARLSON

could and did make use of the lungs for gaseous exchange. In. this
animal the vagi could be isolated in the neck as in the frog. The
result of our investigation on this form is confined to a study of less
than a dozen animals. Although most of the animals were in good
condition at the time of experimentation, they deteriorated more
quickly than the frog and the necturus similarly prepared. We are
confident, however, that our results are characteristic of this form
especially since they fit in well with what we observed in the frog and
necturus.

All the tracings reproduced with this report were taken with the
same speed of the kymograph. A single time tracing showing 5 second
intervals will be found at the bottom of figure 1. It can be used in
a study of the time relations in all other tracings should the reader care
to do so. |

RESULTS

1. Necturus maculatus: a. The central inhibitory control of the lungs
’ through the vagi. As mentioned above it was found impossible for -
anatomical reasons to isolate the vagus nerve or its pulmonary branches’
in the neck to note the effect of ligation and sections of these nerves
on the tonic activity of the lung musculature. Since destruction of
the medullary centers in the frog effected the same result, we adopted
this expedient in necturus. Having closed the glottis and prevented
intercommunication of the lungs as described above we connected the
lungs each with a water manometer and destroyed the brain entirely
by rapidly pithing it. Figure 1, A, shows the effect of such a procedure.
The destruction at a of the cerebral lobes and midbrain was followed
by a temporary escape of the lungs from inhibitory control. The
subsequent destruction at 6 of the medullary centers was followed by
permanent hypertonic state of the lung as in the frog. In another
animal whose cord was not pithed at all but which lay quietly on its
back as a result of a rubber band placed tightly about the front legs,'
ligation of the base of one lung was followed by an immediate. escape
_ of this lung from tonic inhibitory control in a manner identical with
destruction of the medulla (fig. 1, B). |

1 Both in the frog and in the necturus pressing the front legs tightly together
with a rubber band renders the animals quiescent, and they will lie quietly on
their backs for long periods without attempting to turn to a normal position.
This procedure depresses also some of the skeletal reflexes.

VISCERAL SENSORY NERVOUS SYSTEM Ize

It should be noted that in this animal the entire central nervous
system was intact and that there was the minimum trauma produced
in exposing the tip and base of one lung. Nevertheless, on severing
this lung from its physiological connection with the central nervous
system, the typical lung tetanus was produced. The results of this
type of experiment strengthen our position that the striking peripheral
automatism of the amphibian lung is a normal physiological state and
not induced by the trauma rendered necessary by the experimental
procedures.

From these experiments it would seem to follow that in this animal
the lung is kept in tonic inhibition by central vagal control. If now
one proceeds further and stimulates the peripheral end of the exposed ©
vagus nerves at the base of the skull as was done in many animals, one

obtains temporary escape of the lung from the hypertonic state as is

shown in figure 2. This animal suffered at a an exposure of the anterior
end of the brain by resection of the end of the upper mandible. This
operative procedure was followed possibly by a slight inhibition of the
lung tonus. The medulla was rapidly pithed at 6 resulting in perma-
nent hypertonus of the lung. Stimulation of the peripheral end of the

left and the right vagus at c and d respectively was in each instance

followed by a temporary inhibition of the lung with an unusually rapid
return to its hypertonic state. If the peripheral vagus stimulation is
of sufficient strength the inhibition of the lung tonus usually puts the
lung back temporarily to the identical tonus state prior to the destruc-
tion of the medulla. This is additional evidence that the state of the
lung tonus in the intact animal is governed by the inhibitory control
through the vagi. :
Since ligation of the base of the lung effects the same result as de-
struction of the medulla, namely, an escape of the lung from its state
of inhibitory control, it might be expected that electrical stimulation
of the base of the lung would cause an inhibition comparable if not
identical with stimulation of the peripheral end of the vagus. Figure
3 records the result of such an experiment. The lungs being in a state
of hypertonus as a result of destruction of the medulla by pithing, stimu-
lation of the vagus of the right lung at a@ caused an inhibition which is
virtually duplicated by stimulation of the base of the left lung at 6.
It appears to us from these observations that there is no escape from
the conclusion that the vagi nerves possess inhibitory fibers for the
lung which under normal conditions exercise a maximum inhibitory
control over the lungs by tonic impulses from the medullary centers.

ZBL

128

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VISCERAL. SENSORY NERVOUS SYSTEM 129

b. Peripheral lung rhythm. In two instances the lungs of necturus
showed a type of tonus'rhythm seen occasionally in the frog. Figure
4, A and B, are records taken from these animals. In figure 4, A,
this tonus rhythm appeared on pithing the medulla at a. Here a fast
and later a slow tonus rhythm is written on the curve indicating release
of the lung from central vagus inhibition. In figure 4, B, the tonus
rhythm followed a decidedly rapid inhibition resulting from faradiza-
tion of the base of the lung at a with a strong tetanizing current.

The interpretation of these results is based entirely upon speculation.
It is possible that the rhythm which appeared as a result of the destruc-
tion of the brain (fig. 4, A) arose from occasional inhibitory impulses
reaching the lungs through the vagi from more or less intact portions
of the medulla which escaped complete destruction during the pithing
of the latter. The tetanization with a strong current of the base of
the lung in figure 4, B, at a might have effected changes in the physi-
ological state of the automatic tissue present in the lungs which initiated
an occasional and recurring refractory state of this peripheral automatic

Fig. 1. Water manometer tracings of the intrapulmonic pressure in necturus.

A: Spinal cord cut and destroyed below medulla, cannula in tip of lungs.
Glottis closed by dorsal traction on front legs. Lungs isolated by ventral median
incision; a, destruction of cerebral lobes and midbrain; 6, destruction of the
medulla. Showing permanent lung hypertonus on destruction of medulla.

B: Animal rendered quiet by tying rubber band around front. legs, placed
on dorsal side, no restraint, with water running over gills. Abdominal incision
at base and tip of lung; z, ligation of base of lung. Showing permanent lung
hypertonus identical with that following destruction of medulla. Time tracing: —
5 second intervals.

Fig. 2. Water manometer tracings of the intrapulmonic pressure in necturus.
Upper tracing, left lung; lower, right lung. Spinal cord cut and destroyed
below medulla; cannulae in tips of lungs. Lungs isolated by a ventral median
incision. Glottis (trachea) closed by pressure exerted by dorsal traction on
front legs.

a, Transverse section of upper mandible exposing anterior end of brain.

b, Pithing brain.

c, Stimulation of left vagus at base of skull.

d, Ditto, right vagus.

Showing hypertonus of lungs induced by destruction of the medulla, and
inhibition of this tonus by vago stimulation.

Fig. 3. Water manometer tracings of the intrapulmonic pressure in necturus.
Cannulae in tips of lungs. Spinal cord and brain destroyed. Lungs in hyper-
tonus. Upper tracing, left lung; lower, right lung; a, stimulation of right vagus
at base of skull; 6, stimulation of base of left lung.

Showing identical inhibitions of the lung tonus by vagus and by direct lung
(base) stimulation.

130 A. B. LUCKHARDT AND A. J. CARLSON

Fig. 4. Water manometer tracings of the intrapulmonic pressure in necturus.
Cannula in tip of lung. Spinal cord cut and destroyed below medulla, lungs
isolated by ventral median incision.

A: A pithing of the medulla showing a tonus rhythm of the lungs following
destruction of the brain.

B: Lung in hypertonus from destruction of the brain; a, direct stimulation
of the base of the lung with a weak tetanizing current.

Showing primary inhibition of lung tonus followed by a tonus rhythm.

Fig. 5. Water manometer tracings of the intrapulmonic pressure in necturus.
Cannula in tip of lung. Brain and spinal cord destroyed, giving the lungs per-
manent hypertonus.

A: a, injection 0.5 ec. adrenalin; b, 0.6 ec. of (1:1000) adrenalin in5 ce. Ringer’s
into heart. Showing inhibition of lung tonus by adrenalin.

B: a, injection of 3 mgm. nicotine into heart, showing inhibition of lung
tonus by nicotine.

C: a, injection of 0.6 cc. histamine (1—1000) into heart after partial recovery
of preparation from a previous injection of nicotine. Showing direct stimulating
action of histamine on lungs after paralysis of inhibitory nerve mechanism by
nicotine.

Re arageai

PRS ey ISS

VISCERAL SENSORY NERVOUS SYSTEM 131

mechanism, the inhibition of the hypertonic state of the lung giving
rise to the appearance of a rhythm.

c. Reflex lung contractions as a result of cutaneous stimulation. We
have been unable to effect reflex contractions of the lungs of the necturus
by electrical or mechanical stimulation of cutaneous nerves anterior
to the spinal transection.

d. The action of pituitrin, histamine, nicotine and adrenalin on the
hypertonus of lung. In most of the work on drugs a cannula was tied
into bulbus arteriosus. The drug was diluted with Ringer’s and the
injections made under moderate pressure through the bulbus. The
method was poor even under favorable conditions. Direct intravenous
injection was out of the question because active circulation through

_ the lungs was absent in every animal although heart appeared in good

condition. Even when the drug was injected under pressure we never
felt certain that it reached all parts of the lung. This was due partly
to the peculiarity of the pulmonary circulation in this animal and partly
to the fact that a good deal of the fluid containing the drug escaped
from the vessels ruptured in destroying the brain and isolating the
vagi.

Adrenalin. The injection of adrenalin caused a marked inhibition
of the hypertonic lung as is recorded in figure 5, tracing A, at a and b.

Pituitrin. According to the commonly accepted view, pituitrin is
a direct stimulant of smooth muscle tissue. We were somewhat sur-
prised to find that this drug in small or large doses causes a prompt
and marked inhibition. Figure 6, A, is a record taken from an animal
which suffered destruction of the medulla at a followed by a hypertonic
state which was markedly reduced-by the injection of pituitrin at b.
The slow recovery is also recorded. No attempt was made to deter-

mine the point of action of pituitrin. Pituitrin not only caused a

relaxation of the lung; but when weak solutions of this drug were used
to irrigate the intestines all intestinal movements ceased after a short
latent period. It would seem, therefore, that in these animals pituitrin
does not act as a direct: sieccute stimulant but rather as a stimulant
of the inhibitory mechanism.

Histamine. Figure 6, B, illustrates at c the typical inhibitory effect
of this drug in an Saemal whol lungs were in hypertonus as a result
of destruction of the medulla at 6. Both lungs showed pronounced
relaxation with very slow recovery. In all types of animals other than
necturus this drug causes a more or less powerful contraction of the ©
lung musculature (turtle, frog). Necturus proved to be an exception

A. B. LUCKHARDT AND A. J. CARLSON

132

VISCERAL SENSORY NERVOUS SYSTEM 133
to the generally aecepted view that’ the drug supposedly acts: asa
direct muscular'stimulant. The results obtained:suggest the possibility

that the drug acts primarily on the inhibitory mechanism. We there=

fore’ attempted to eliminate the latter by means of nicotine. As a
matter of fact we found that the injection of histamine after previous
nicotinization effected a contraction of the sp in xg ir instance’ as is

_ recorded graphically in figure 5, Cat a.

~ Nicotine. It will be recalled that this drug shed on the me s eth
in a manner similar to section of both vagi. We furthermore showed
that in this animal the drug acted by paralyzing the inhibitory center
in the medulla as well as the vagus inhibitory terminations in the lungs;
for we found that after nicotinization stimulation of the peripheral
end of the vagus gives rise to a lung contraction instead of the inhibition
seen before giving this drug.

The subcutaneous or deep muscular injections of large (5 to 10 mgm.)
doses into the normal intact necturus is in 15 minutes followed by

- general clonic convulsions with an increase in the rate and amplitude

of the gill movements. Tetanus of the gills supervenes and active
external respiration comes to an end some time before the general

Fig. 6. Water manometer tracings of the intrapulmonic pressure in necturus. .
Cannula in tip of each lung. Spinal cord cut and destroyed. below medulla.
Lungs isolated by ventral median incision. Glottis and tracheal sac closed by
mechanical pressure through dorsal traction on front legs.

A: Upper record, left lung; lower, right lung; a, pithing of brain; b, injection
of 0.5 cc. pituitrin in 5 cc. of Ringer’s solution into heart. Showing inhibition
of lung hypertonus by pituitrin.

B: Upper tracing, left lung; lower, right lung; a, section of upper mandible ©
exposing anterior end of brain; b, pithing of brain; c, injection of 0.7 cc. hista-
mine (1-1000) in 5 cc. Ringer’s solution into heart. Showing inhibition of lung
hypertonus by histamine. ;

Fig. 7. Axolotl., Water manometer tracings of the intrapulmonic pressure.
Spinal cord pithed and destroyed below level of innervation of front legs. Lungs
isolated by abdominal incision; cannula in tip of lungs. Glottis open. Spon-
taneous respiration (quick movements of lever) except where indicated.

* A: a, Gentle mechanical stimulation of gills.
b, Gentle mechanical stimulation of skin of front legs.
_e, Gentle mechanical stimulation of skin of mandibles.

d, Strong mechanical stimulation (pressure) of toes of front leg.

x, strong attempt at respiration (swallowing).

B: a, Moderate mechanical stimulation of the gills.

b, Gentle stroking of skin of front leg.

c, Strong mechanical stimulation of the gills.

Showing lung contractions following spontaneous respiratory movements and,
on stimulation of various sensory nerves.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

134 A. B. LUCKHARDT AND A. J. CARLSON

convulsions cease. The lungs of such animals were found contracted.

Pithing the brain, stimulation of the vagi themselves, or stimulation of

the lung itself at cts base were without effect.

If the dose of nicotine injected subcutaneously is odiie still further
(2.5 to 1 mgm.) the same symptoms appear in 10 to 15 minutes but
are less severe. Pithing of the brain or stimulation of the vagi is again
without effect. Direct stimulation of the lung at its base with a strong
tetanizing current gives rise to an inhibition of the lung wie any
or with but feeble return.

From these results it would appear that a, the lungs of this amphibian
is supplied only with inhibitory fibers through the vagi; 6, that nicotine
paralyzes the respiratory center for the lungs and also the junction
between the preganglionic fiber endings and postganglionic cell body,
since external respiration ceases with the lungs in hypertonus and since
stimulation of the base of the lungs will still yield inhibition when
stimulation of the vagus nerve itself gives nothing; c, the vagus nerve

contains no motor fibers for the pulmonary musculature. Without —

commenting at this time on the possible significance of this fact, we
call attention to the fact that in this most primitive lung which we
have studied, the lung is solely under the tonic influence of inhibitory
fibers carried by the vagi and motor fibers are apparently absent. We
have been unable to elicit a motor response of the lung either by stimu-
lation of the vagi or the lung itself in any normal or nicotinized. animal.

The apparent resistance of the peripheral lung mechanism to this

drug compared with that of frog may possibly: be due to the rather

sluggish and imperfect circulation through the lungs.

2. Axolotl. Tarlier in this paper we called attention to the fact that
the axolotls with which we worked, although possessing gill remnants,
were essentially air-breathing animals; and that the lungs were decidedly
better developed for that purpose than the lungs of the necturus.

a. Lung contractions at the end of normal respiration. Records were
taken of the changes in the intrapulmonic pressure occurring during
normal respiration with the glottis open. It can be seen from figure
7, B, that every spontaneous respiratory effort (gulp) as indicated in
the tracing of the quick movement of the lever is followed by con-
traction of the lung. These lung contractions compare favorably with
those obtained under similar experimental conditions from the frog.

b. Reflex lung contractions. Gentle mechanical stimulation applied
to the skin of the mandible, gills or front legs induced lung contractions
of reflex origin as is seen in figure 7, A, at a, b and c. In these three

VISCERAL SENSORY NERVOUS SYSTEM 135

instances the lung contraction appeared without a preceding attempt
at respiration. Subsequent attempts at respiration were followed in
each instance by lung contractions in every way similar to those of
reflex origin induced by gentle stimulation. It is obvious that the con-
tractions are not.the result of changes in the intrapulmonic pressure
due to movements of the animal during external respiration; for they
occur in the absence of all visible movement of the head region.

Fig. 8. Axolotl. Water manometer tracings of the intrapulmonic pressure.
Spinal cord transected below medulla and pithed posteriorly. Cannula in tip
of lungs, lungs isolated by median abdominal incision. Glottis closed by forceps.
All injections intravenously (in 2 cc. Ringer solution).

A: Lower tracing, right lung; upper, left lung.

a, Transection of upper mandible exposing anterior end of brain.

b, Pithed brain.

c, Injection of 1 ec. 1:10,000 adrenalin.

B: a, Pithed brain.

b, Injection of 0.01 ce. 1:1000 histamine.

c, Injection of 2 mgm. nicotine.

Showing the inhibitory action of these drugs on the lung hypertonus follow-
ing destruction of the brain, the latter being equivalent to section of the vagi
nerves.

Strong mechanical stimulation of the fore legs or gills may lead to
partial or complete opening of the glottis with partial or complete
collapse of the lungs as is illustrated in figure 7, A, at d, and figure 7, B,
ata,bandc. Strong attempts at respiration after marked stimulation
of pain fibers as at xx in figure 7, A, were not followed by the filling
of the lungs until considerably later. It might be assumed that the
strong stimulation interfered temporarily with the central control of
the mechanism normally at the service of the animal in filling its lungs

136 A. B. LUCKHARDT AND A:: J. CARLSON

with air or depresses the! central inhibitory. control over the lungs.:
‘If the latter is the correct. interpretation the animal failed to fill its:
lungs in spite-of violent respiratory attempts because of their hypertonic:
state... Although we -have no direct evidence on this matter ss latter:
ipherprejating séems to be the more probable one.

_c..Fhe central inhibitory control of; the:lungs through heed vagt: It i-
coctin that in the axolotl-the medullary.centers exert, under normal
conditions, a tonic inhibitory control over the lungs as iis the frog and
necturus; for as a result of the destruction of the medulla which is_
- equivalent to section of both vagi (as in fig. 8, A, at b) both lungs go
into a state of hypertonus.

d. Action of certain drugs on the hypertonic lungs. Adrenalin. This ~
drug temporarily inhibits the hypertonus of the lungs resulting from
destruction of the medulla as seen in figure 8, A, at c.

Histamine. In axolotl the intravenous Sileatiel of 1 mgm. of his-
tamine-HCl caused inhibition of the lung musculature not unlike the
action of this drug in the hypertonic lung of necturus (fig. 8, B, at b).

Nicotine. The invariable effect of nicotine on the hypertonic lung
of the axolotl is inhibition (fig. 8, B, atc).

As previously noted we were not only restricted to a few animals in
study of this form but found that the physiological state of the animals
rapidly declined as the result of our operative procedures. The axolotl
furnishes a less hardy physiological preparation than the frog or necturus.
As far as our results go they check with those obtained from the frog
and necturus. |

SUMMARY

1. The vagus center exerts a tonic inhibitory control over the lungs.
Destruction of the’ medulla releases the lung from this control. As
a result, the lungs assume a state of more or less permanent hypertonus
(necturus and axolotl).

2. Electrical stimulation of the peripheral end of the vagus nerves
causes a temporary inhibition of the hypertonic state of the lungs,
only on the side of stimulation, during and for some time after the stimu-
lation (necturus and axolotl). |

3. The efferent vagi fibers to the lungs are solely of the inhibitory
type. We have never seen the slightest indication of a motor response
on stimulation of these nerves. Unlike the frog, the vagi of these forms
of amphibian life possess few if any motor fibers for the lungs (necturus
and axolotl).

VISCERAL SENSORY NERVOUS SYSTEM i3¢

4, Electrical stimulation of the base of the lung yields the same results
as stimulation of the peripheral end of the vagus (necturus and axolotl).

5. We have been able to elicit reflex lung contractions from gentle
mechanical stimulation of cutaneous nerves only in the axolotl. In-
tense stimulation of sensory nerves (pain) probably prevents filling of
the lungs during attempts at respiration because of a hypertonic con-
dition of the lungs due to an inhibition of the inhibitory center.

6. There may .appear as a result of destruction of the medulla or
strong faradization of the base of the lung a tonus rhythm in the dener-
vated lungs of the necturus. This rhythm has not been seen in axolotl.

7. Adrenalin, pituitrin, histamine and nicotine cause a marked
inhibition of the hypertonic condition of the lungs resulting from the
destruction at the medulla. Histamine injected into the animal after

nicotine causes a contraction of the lung. In axolotl these drugs act
in the same direction. Pituitrin was not used in this form; nor was
histamine injected after nicotinization of the animal.

BIBLIOGRAPHY

(1) BaBix anp Kiunova: Arch. f. d. gesammt. Physiol., 1909, cxxx, 444.
(2) Barrows: Anat. Anz., 1900, xviii, 461.
(3) Betuce: Zeitschr. f. Wissensch. Zodl., 1898, lxiii, 680.

(4) Camerano: Anat. Anz., 1894, ix, 676; Arch. ital. d. Biol., 1896, xxv, 219.
(5) CARLSON AND LuckHarpT: This Journal, 1920, liv, 55.

(6) Caruson: Arch. f. d. Physiol., 1905, cix, 51.

(7) Lapique AND Pretetin: Compt. Rend. Soc. Biol., 1910, lxix, 84.

(8) L6nnper@: Anat. Anz., 1899, xxii, 545.

(9) MituerR: Bull. Univ. Wisconsin, Science Series, II, 1900, 203.
(10). Oppeu: Lehrb. d. Vergl. Mikr. Anat., 1905, vi, 277.
(11) Strruine: Journ. Anat. and Physiol., 1882, xvi, 90.

' (12) WitpEr: Amer. Naturalist, 1901, xxxv, 183.

CHANGES IN ACID AND ALKALI TOLERANCE WITH AGE
IN PLANARIANS

Wirth A Notre oN CATALASE CONTENT

JOHN W. MacARTHUR

From the Hull Zoélogical Laboratory, University of Chicago, and the Department
of Biology, University of Toronto

Received for publication July 17, 1920

During a study of the effects of certain typical acids, bases and salts
upon living planarians (Planaria dorotocephala, P. maculata and P.
velata), observations were made which are here reported on account of
the wider interest of their bearing upon tolerance of H+ and OH7— ions,
upon the relative efficiency of the mechanism for regulation of neutrality
in young and old individuals, and upon the problem of acidosis in
general.

Methods. A closely graded series of concentration of the acids (hy-
drochloric chiefly, also sulfuric and acetic) or alkali (sodium hydroxide)
is made up from standardized N or 0.1 N solutions by dilution with
aerated well water or other (Lake Michigan or Lake Ontario) water in
which the worms live and thrive. The well water, which was used
chiefly, has a pH value of 7.5 to 7.6 and an ion and gas content to be
published with the larger study above mentioned. Both distilled water
and strongly chlorinated tap-water are in themselves injurious to these °
worms and hence could not well be used for the purposes of these ex-
periments; but similar tests are now being made with P. maculata in
distilled water, this species being but little affected by distilled water
in the period of time required.

Into the series of dilutions of an acid or alkali in 500 cc. or 1000 ee.
Erlenmeyer flasks, filled and ready to be plugged with rubber stoppers,
are introduced the flatworms, usually ten larger (18 to 20 mm.) and
ten smaller (8 to 12 mm.) specimens together, all selected sound from
established well-fed cultures. In some cases a similar group of three
easily distinguishable sizes was used (22+ mm., 15-+ mm. and 8+
mm.). In control flasks all such individuals live practically indefinitely.

138

ACID AND ALKALI TOLERANCE IN PLANARIANS 139

The hydrogen ion concentrations, already in a graded series from the
“method of diluting the normal solution, are measured and corrected at
critical points by the colorimetric method with appropriate indicators:
thymol-blue, brom-phenol-blue, methyl-red, brom-cresol-purple, phe-
nol-red and phenol-phthalein, and the Hynson, Westcott and Dunning
apparatus (1). Certain difficulties were encountered from the fact that
glassware requires particularly thorough cleaning after each usage, and
because strong acids added to water containing so much carbonate as
do these naturally generate CO, and such solutions tend to return grad-
ually toward neutrality. But these facts have no great significance here
except as they render impracticable a precisely accurate determination
of the actual limits of tolerance, as may be made in distilled water and
by electrometry. In all cases it was the relative rather than the abso-
lute susceptibility that was sought and that is here emphasized. i‘
Extensive physiological studies of planarians have been made by Child
(2), (3), who showed for P. dorotocephala by various means that the small-
est worms (up to about 6 or 7 mm.) consist of but one zodid, while the
medium-sized ones (12 to 14 mm.) usually possess a second zoéid region,
and larger specimens (20 to 25 mm. or more) exhibit at the posterior
end a third zoéid or group of very small zodéids, constituting what is
essentially a “growing tip.” At least the chief, the second and the
third of these zodids were demonstrable physiologically by both “ direct’’
and ‘‘indirect’’ methods in cyanides, but only after fission do the typical
head structures of the zodids become visibly differentiated morphologi-
cally. The growing tip is evidently involved repeatedly in the reproduc-
tive process; hence as this region grows its zodids become more independ-
ent and acquire a higher rate of metabolic reaction and, like young indi-
viduals, are more susceptible to high concentrations and less susceptible
_ to low concentrations of lethal agents (KNC, anesthetics, etc.).
Experimental. The chief observations are recorded and summarized
graphically in the accompanying figure. The time records are averages
from repeated tests made of each dilution; such averages for separate
tests show but small deviations from the general average, but individ-
ual deviations are often large and overlapping wherever the curves lie
close together. Dr. C. M. Child, to whom the writer is indebted for
many opportunities and suggestions in this work, has recently used
these experiments as part of a class course at the University of Chicago.
Acids. Immersed in HCl solutions that kill almost instantly (e.g.,
in acidities greater than about pH =-2) all worms are fixed and _ pre-
served intact (range of preservation). In lower concentrations, from

140 ' . JOHN W. MACARTHUR

pH = 2 down to about pH = 4.5, all individuals are killed and caused
to disintegrate, the older somewhat later and more slowly than the
younger (range of direct susceptibility and inhibition). In this disin-
tegration all regions of the body are not equally and simultaneously
involved, but usually the posterior tip and the head are first attacked

hours : ' oien
‘ | ! i
17 ( 7 417
'
16 416
15 ‘ his
H
13} ' 413
12 ‘ 42
mi t di
10 i dio
‘,
8 49
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ef : 18
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6} $ 16
. St i a 5
4 +
3 3
x
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| aoe |’
oH 8... ake 45, DA ec oe
iii? tae ane aca ae pees a. 4 «oe 3

Fig. 1. Time in hours of distinct initial disintegration of young worms
---0---0--- and old worms ——x——x—— in solutions of different pH
values (abscissae) up to pH = 10. Small numbers below abscissae indicate
volumes of well water added to one of N /10 HCl (left) or NaOH (right).

Below pH = 2+ is the range of preservation or fixation. Then follows .
with increasing dilution up to pH 4.4 + the range of distinct direct susceptibility
which grades off by a transition range into the range of acclimation or indirect

susceptibility.
With NaOH the range of preservation is evidently pPecnt, direct susceptibility

differences between young and old much more marked, and the indirect a
bility differences less marked than with acids.

and then the regions behind the head in order from in front backward;
and, as might be expected from the small differences in direct suscepti-
bility between young and old, the anterior end of a second zodéid is not
distinguished. As the acidity approaches pH = 4.5 or pH = 4.6, the
age difference in survival time decreases more and more and finally

,
S,

ACID AND ALKALI TOLERANCE IN PLANARIANS 141

becomes nil, and disintegration does not discriminate between the small
and the large. In still more dilute solutions, however (pH = 4.7 to
4.9), where death is delayed for several hours and a certain amount of
recovery from the initial inhibitory effects of the.agent is possible, the
relative susceptibility of young and old individuals is reversed (range of
indirect susceptibility and acclimation), and smaller worms disintegrate
last or not at all, while larger ones either disintegrate entirely or lose
their head region. Posterior zodids are left intact, and the posterior
part of the first zodid was never seen to disintegrate before the anterior

end. Often the young worms live for days or indefinitely after the old

are partially or wholly gone. Recovery tests, made by returning young
and old alike to fresh water after various periods of exposure to the
agent, are even more delicate in their indication of the sites and degrees
of injury, and were used to extend and confirm the results obtained by
leaving the animals in the agent up to the end of the experiment.

A more or less definite sequence of changes leads up to the final dis-
integration with acids. After the initial stimulation, during which the
flatworm assumes for a time a slender form, moves rapidly and secretes
some mucus, it gradually loses its power of adherence to the glass walls
of the container and becomes shortened, cylindrical and swollen. This
state is soon followed by discoloration or whitening, the loss of color
occurring, as noted, first at the sensory tip, margins and ventral sur-
face of the head and gradually extending backwards, often more rap-
idly on the ventral surface, and in larger individuals beginning early
also at the growing tip. The whitening appears to indicate that semi-
permeability or some similar property of the surface layer has been
abolished at the approach of death, for following close upon the loss of
pigment occur disintegrative changes of a characteristic kind: as the
parts become sticky and adherent to glass the regularity of the external
contour is interrupted by small breaks in the continuity of the surface,
the protoplasmic granules swell and mass into small clumps or liquid
spheres and scatter out into the medium until finally little remains of
the old body but a soft white shreddy outline composed of the more
resistant connective and supporting tissues quite stripped of all the
relatively susceptible epithelial parts.

Results differ in no essential way if sulfuric acid in slightly higher
concentrations be substituted for the hydrochloric, or if a considerably
greater strength of acetic acid be used—the difference probably being
necessary to compensate for the lesser dissociation of the organic acid.

142 JOHN W. MACARTHUR

A change of response occurs upon addition of acid to the normal
medium. The planarians then exhibit a fairly strong negative geo-
tropism, climbing always up the walls of the container, whether in doing
so they approach or attain a surface or not. Since strong acids cause a
release of COsz into the solution, the response may perhaps be consid-
ered generally appropriate and adaptive, inasmuch as ordinarily an
increase of COzis doubtless associated with an insufficiency of oxygen (to
which a similar response is made) and both could doubtless be avoided
by rising to a better aerated surface layer.

Alkalz. In alkaline solutions the same general results are obtained
with certain more or less significant modifications. In the hydroxide
(NaOH) stimulation is evidently more marked than in acids, and both
whole worms and surviving parts of any size are more active both spon-
taneously and upon mechanical stimulation up to the very point of
death. Alkalies also cause the secretion of a very excessive amount of
mucus, which collects, as often as removed, in the bottom of the vessel.

Even quickly killing concentrations do not produce a definite fixation
and preservation. Disintegration, if rapid, occurs, by a rather violent
process of splitting and bursting of the dorsal surface in darkened lines;
if slow, it begins at the margins and dorsal surfaces of the head and the ~
posterior tip, and in larger specimens may also appear at what is pre-
sumably the anterior end of the second zodéid.

Results with NaOH differ from those with HCl and resemble more
nearly those with KNC in one respect—in the slowly acting concen-
trations, allowing partial acclimation of the larger animals, death some-
times begins at the posterior end of the first zodid and proceeds forward,
while the head region of the first zodid and all of the posterior zodids —
remain intact for some time or indefinitely. In short the details of
disintegration with this alkaline agent resemble those with most ‘acid
dyes,’ while “basic vital dyes’’ rather resemble acids in their effect .
(unpublished work).

By the method of direct susceptibility there is much greater differ-
ence in survival time of young and old with NaOH than with HCl, the
old surviving about twice as long as the young. The effective range of
concentrations for indirect susceptibility, on the other hen is less
extended than with acids.

It will be noted that the range of critical concentrations, within —
which young animals and young parts only are able to regulate slight
H* ion alterations, is a comparatively limited one and lies just beyond
the limits resisted by all alike. Thus increase of H+ ion up to pH =

tee ee Oe ae ne
= ip - S

ACID AND ALKALI TOLERANCE IN PLANARIANS | 143

4.9 on the one side or of OH~ ion to about pH = 9.1 on the other come
within the normal range for all members of the species; slight additional
changes (from pH = 4.9 to pH = 4.8 or 4.7 and from pH = 9.1 to
pH = 9.2 or 9.3) can be met by the young individuals and parts alone;
still greater changes are beyond the powers of acclimation of any,
though the old resist the longer. |

The greater tolerance by younger planarians and the posterior zoéid
region of such dilute acid and alkaline solutions is almost certainly only
another example of the greater power of acclimation to mildly depress-
ing conditions associated so generally with more active metabolism (3).
In fact the general principle underlying the indirect susceptibility
method is founded on the discovery that organisms or parts of organ-
isMs possessing an intenser metabolism can acclimate or acquire toler-
ance more quickly and more completely than less active organisms or

parts to low concentrations of cyanides, narcotics, ete. Child also

showed later that the anterior, ventral and median regions (the regions
of high direct susceptibility and presumably of most rapid metabolism)

4 in Kchinoderm and Annelid embryos, developing in low concentrations

of NaOH, alcohol or HCl in sea-water, acclimated or acquired tolerance,
or after temporary exposure recovered most quickly and underwent a
proportionately accelerated and increased development in the larvae (4). —

The explanation of this power of acclimation is not known, but may
be in some way associated, as regards acids and alkalies, with differences
in protoplasmic conditions, such as the higher percentage water content
of metabolically active parts or individuals. If this water carries, as
seems probable, at least an equal proportional and a greater total salt
content, such inorganic salts of these as are buffer-acting substances
(carbonates, phosphates, etc.) would act here much as in mammalian
blood, to increase resistance to additional H+ and OH™ ions in the
medium. Or, if a greater proportion of mid-products of protein metab-
olism, or more ionized protein, be present during rapid metabolism, then
these amphoteric substances may serve as acids or bases according as
there are excess bases or acids in the medium. Naturally such buffers
and metabolites would be protective only against slightly and slowly
injurious concentrations; with higher concentrations the projective
action is quickly overcome and the agent may diffuse and act most
rapidly in the parts with greatest water content.

The acid or alkali effects may of course be produced through injury
to some enzyme or enzymes essential to continuance of metabolic proc-
esses. Inasmuch as the almost ‘universally occurring enzyme, cata-

144 ; JOHN W. MacARTHUR

lase, may eventually be shown to play some réle in metabolism generally
and in oxidation in particular (5), the writer wishes to record here the
results obtained from numerous experiments to determine the catalase
content or activity of planarians of different ages. It was found that
equal weights of crushed young worms (8 to 15 mm.), maturer worms
(18 to 20 mm.), and of very old worms (25 to 30 mm.) liberated in 15
minutes at 22°C. from 13 per cent unneutralized hydrogen peroxide the
following quantities of oxygen respectively per gram weight of tissue:
653.3 cc., 460.4 cc. and 317.6 cc. Without a single exception, in many
repetitions of the experiment, the rule was found to hold that the larger
(older) the worm the lower is the catalase content. It is of interest to note
that the oxygen consumption of young planarians has been found to
be from 15 per cent to 100 per cent greater than that of old ones (6),
showing a higher basal metabolism, just as the Benedict method does
for man.

Some significance should be attached to the fact that though there |

are such large differences in direct susceptibility of young and old with
alkalies, these differences are small with acids; while, on the contrary,
though the differences by indirect susceptibility are small with alkalies,
they are larger with acids. The young are evidently comparatively and
absolutely less resistant to alkalies, but relatively more resistant to acids.
With advancing age there would appear to be a decreased relative resist-
ance to acids and an increased relative resistance to alkalies—a set of
changes such as would result from a gradual onset of a state of acidosis
and the more or less incomplete oxidation of the larger-acid products
left from a state of lowered metabolism. MacNider (7) has shown
that as age advances the acid-base equilibrium of mammals is more and

more easily disturbed or overtaxed; that uranium nitrate, for instance, —

is more toxic to the old than to the young and produces sooner in the
old a condition of true acidosis (8), characterized by a depletion of re-
serve carbonates in the blood, etc.; and that the aged, after uranium
treatment, received intravenously without injury considerably more
alkali than did the young. :

For all the species used by Child (4) in controlling form and propor-

tions of developing embryos, he found that “‘the agents which are most

effective in producing the differentially inhibited type of form are least
effective in producing the types of form characteristic of differential
acclimation, and vice versa,” his series being effective in producing ac-
climation types in the order: HCl, aleohol, NaOH, NH,OH, KNC.
Thus acclimation was rapid in acids and alcohol, slow in NaOH and

a

ACID AND ALKALI TOLERANCE‘ IN" PLANARIANS 145

NH,OH, and exceedingly slow in KNC.: A similar contrasting physi-
ological effect between HCl and NaOH is here shown PY other means
for fresh-water planarians.

Comparing allied species with P. Duletotanhils it may he said. hast
in general P. maculata has a slightly. wider range of normal tolerance,

while P. velata is distinctly less resistant to’ acids and more resistant to
alkalies. ~

SUMMARY AND CONCLUSIONS

1. Planaria dorotocephala of all ages used tolerate HCl up to about
_ pH 4.9 and NaOH up to about pH 9.2 in the well water (pH = 7.5 to
_ 7.6) in which they live, i.e., they Seip a range of pH from 4.9+ to
about 9.2+.

2. Smaller, physiologically younger, individuals are on the average
tolerant of a slightly wider range of hydrogen ion concentration (from
pH = 4.7 to pH = 9.3) than are larger, physiologically older individu-
als, this difference of susceptibility being apparently somewhat greater
on the acid than on the alkaline side of neutrality. The young possess
a greater power of neutrality-regulation than do the old, explanatory
suggestions for which are offered.

3. In concentrations of alkali which kill within a few hours suscep-
tibility is reversed in relation to age, the young being very much more
susceptible than the old. In similar concentrations of the acids young
specimens are likewise on the average more susceptible, but only slightly
more so. In other words, high concentrations of OH~ tend to increase
and high concentrations of H+ tend to diminish differences in direct
susceptibility between young and old individuals. This suggests a
possible increasing average acidity with senescence and decreasing
metabolism.

‘4. Young planarians have about double the catalase content of old
planarians per gram weight of tissue.

5. In acid solutions liberating CO2, in which worms live for some time,
they commonly assume, as in conditions of oxygen deficiency, a nega-
tive geotropism of an obviously ad apie nature as a normal means of
escape from excess CO..

6. These facts indicate the necessity of taking into account the factors
of age, size and metabolism in defining range of tolerance to agents and
conditions.

146 JOHN W. MacARTHUR

BIBLIOGRAPHY

(1) CuarK anpD Luss: Journ. Bact., 1917, ii.

(2) Cuitp: Journ. Exper. Zoél., 1913, xiv. :

(3) Cuiup: Senescence and rejuvenescence, 1915, Chicago.

(4) Cuitp: Journ. Morph., 1916. xxviii, 65; 1917, xxx, 1

(5) Buras: Science, 1918, xlviii, 327. Also literature of ALVAREZ AND STARK-
WEATHER, This Journal, 1918, bey 186, and AppLeMAN: Amer. Journ
Bot., 1918, v. 207.

(6) Hyman: Biol. Bull., 1919, xxxvii, 388.

(7) MacNIpER: Selonibe, 1917, xlvi, 643.

(8) HenpEerson: Science, 1917, xlvi, 73.

Se ee er Oe ee Pen eee

Se

STUDIES ON THE ALKALINE RESERVE OF THE BLOOD OF
THE INSANE

NOBUHARU SUITSU
From the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania

Received for publication July 19, 1920

These studies were undertaken to ascertain whether or not the
alkaline reserve of the blood of the insane shows any significant differ-
ences from that found in the blood of normal individuals, and whether
or not the blood of the various types of insanity studied vary one from
the other in their carbon dioxide combining capacity. During the
course of the investigation certain other material was obtained which
will be briefly discussed.

The subjects of these studies were patients ‘ss the Pennsylvania
Hospital, Department for Mental and Nervous Diseases,: Philadelphia.

Since Van Slyke, Stillman and Cullen (1) have shown that a slight
rise in plasma carbon dioxide tension usually follows eating, the samples
of blood to be ana!'yzed were taken at the uniform hour of eleven o’clock
in the morning, three and a half hours after breakfast, unless otherwise
noted. The blood specimens were drawn from an arm vein with a
Record syringe containing a small amount of potassium oxalate. Care
was taken to avoid the sucking in of air. After filling the syringe the
point of the needle was plunged under paraffin oil contained in a small
test tube and 2 cc. of blood expressed and centrifuged. The plasma
was then pipetted off and the carbon dioxide capacity determined as
described by Van Slyke (2), and Van Slyke and Cullen (3). The deter-
mination was carried out two or three times on one and the same sample
and the average adopted as the result.

A general comparison of the findings obtained from excited and depressed
cases. It is well known that not only mechanical but also psychic
activities have great influence upon respiratory and circulatory func-
tions (1), and that the alveolar carbon dioxide tension under ideal
normal conditions indicates the level of the blood bicarbonate, but
since the alveolar carbon dioxide tension is altered by numerous factors,
psychic, physiological and pathological (4), it is not a reliable measure

147

148 © NOBUHARU SUITSU

of the blood bicarbonate except when it is certain that both the mechan-
ical and nervous factors controlling respiration are normal.

With these facts in view an attempt was made to determine whether
or not any relation existed between the plasma carbonate and conditions
of excitement and depression. The figures in table-1 give the plasma.
CO, capacity in twelve cases classed as depressed and ten individuals
exhibiting excitement.

It will be seen that in general terms all the values fall within normal
limits and that no significant differences can be observed between the
two groups. The mere fact that no differences occur in these two groups

TABLE 1

The dtholene reserve of the blood of the excited and depressed insane

Cubic centimeters of CQ:-reduced to 0°, 760 mm. bound. as bicarbonate in
100 cc. plasma

EXCITED CASES DEPRESSED CASES
64.42 72.08
64.42 ° 69.14
63 .95 66 .36
63 .57 66 .36
63 .48 66 .36
62.08 63.54
60 .20 59.74
54.38 59.68
51.24 57.39
45 .04 55.94

52.06
51.18
Average........ 59.28 - 61.65

of patients permits the supposition that in long-continued excitations

a compensatory reaction occurs sufficient to preserve the normal level

of the alkaline reserve of the blood, which one would naturally suppose
to be lowered by consequence of the increased activity.

The alkaline reserve of the blood during individual changes in mental
condition. A confirmation of the general findings that no evident

differences are to be found in the alkaline reserve of the blood of excited’

or depressed insane patients is afforded by the figures given in table 2
These results represent the analyses of the bloods from single individuals
at frequent intervals over periods of several weeks during which there
occurred a marked change in mental condition with respect to excite-

RE ROR IE Ee Re yO nee ete ee NL

Soe, Foe aareeter

ALKALINE RESERVE OF BLOOD OF INSANE 149

ment and relative depression. It is seen that in these cases at least
there failed to occur any consistent change in the alkaline reserve of
the blood accompanying the change from excitement to depression or
vice versa. The four individuals studied were all diagnosed as dementia
precox cases. |

The variability of the alkaline reserve of the blood of the insane from week
to week. During the course of these investigations opportunity was
afforded to determine the degree to which the alkaline reserve of the
blood varies in the individual from week to week in cases where the
general trend of mental condition was uniform.

The results are given in table 3. The variability is given He, the value

for the average deviation calculated for each individual. An inspection
of the table shows that although the absolute amounts all fall within
normal limits and no significant differences occur, yet there are differ-
ences in the degree of variability to be observed in different individuals.
These differences, however, cannot be evaluated since the data are
insufficient.
- Table 4 is a compilation of the analyses cbtaiiea from 112 bloods
from 51 individuals arranged in the order of their descending value.
The figures represent the cubic centimeters of CO, reduced to 0°, 760
mm. bound as bicarbonate by 100 cc. plasma. It is seen that the
absolute amounts do not exceed the limits usually attributed to normal
bloods save at the extremes. The main fact of interest lies in the
relatively low variability of this blood factor which is comparable with
that obtained for the average deviation of the blood creatinine nitrogen
in similar patients (5).

In table 5 there have been arranged the values representing the -
average amounts, the average deviation, and the range of fluctuation
of the alkaline reserve of the blood according to diagnosis. From the
data it is evident that no significant differences obtain in the absolute
amounts of the alkaline reserve of the blood in the different types here
studied. It should be noted, however, that there is a tendency for
the variability of this blood factor to be greater in the mentally dis-
turbed than in normal individuals.

The alkaline reserve of the blood taken three and a half and fourteen
hours after eating. Since it is often inconvenient to take samples of
blood early in the morning and before breakfast a comparison was made
of the alkaline reserve of the blood taken three and a half hours after
breakfast, and fourteen hours after the night meal. The data are given
in table 3. : %

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

150

NOBUHARU SUITSU

TABLE 2

The alkaline reserve of the blood of four dementia precox patients during states of

excitement and states of depression

CO2 REDUCED TO 9°
760 MM. BOUND AS

NUMBER CONDITION BICARBONATE IN 100 _
Cc. OF PLASMA
cc.
1 CIGG i si Bo dois thin 50k SABC eb UL LN ou, pias eae ee 50.24
Bixoited 4.535 ¢ 4 i. «hs 2a ea ck a ee 64.77
PUROS GIG 6 soars 5 vases Saeeeeee es Ca Oars 4 pace ane eae 63 .30
2, ea PI genie ARS ok MMR Balle nn el 59.68
Depressed. 2.) OPO a US CL 60.56
Bixoited .. 0:30 sia wes ohh Bee ted asia erie 63 .48
2 Excited..... ob hh aa tole chad a eS Cla te ee « weet Vat 60.70»
Wexcited. ..2. oo cas. coeeaey RE ROLOS as ces dae 63.43
TIROPORSOG Ae. co cos vas bee ES Cee eee pare 68.51
Wxelted.\ 6... a 1 OR OI OD, SF 61.07
EXC boi5 coos 3 oes SER OAS ORME len « Sale ee 74.80
3 FOS OIEOG oo s orn. gh 03.e & 5.0 hk abe ed La be 63 .30
OUI OG, acs 4 agi ccie-a 5-400 5 OST ER Ru RRC cr ach ae 60.19
GIUIOT, suc fe bo oo ns 0b oS nd PEO Oe Ree Meee one nee 57.78
4 PXoitiad fi.) 6. oi 0 Hes ees es Was eas ea 56.71
Cb ao. h-2r3 sh nae ns =. ee Rs ar ke 54.22
Quite. ach « sn9 25:05.) sare ace 6 SE CRORES ie ics ace 52.49
TABLE 3

: The” individual variability of the alkaline reserve of the blood of the insane as

observed from week to week

Cubic centimeters of CO2 reduced to 0°, 760 mm. bound as bicarbonate in 100

ec. of plasma .

seT1 | ser2]| sev 3| sev4|ser5| ser 6| set7| seT8]} seT9

Average...

set 10

set ll

SET 12

58 .46 |55 .34/53 .21/70 .07|64 .30/65 .47|62 60/64 . 12/74 .90|59 80
58.15 |62 :79/63 .48/72 .00/63 .42|64 .38/63 .51/58 .90|73 .26)65 .46
66 .73 |62 .40/62 56/75 .38/65 .30/57 .74/65 50/59 .80/70 . 10/62 .58
65 .66 |53 .92/71 .02/75 .31/63 .36|67 .81/61 .54/62 .56/70 . 17/63 .30

63.42 |66 31/63 .66/74 .85
63 .44 |64.34/63 .42)/73 .57
69.14 |70.41

63.45 |62 .22/62 .89)73 .53 64 . 10/63 .85/63 .29 61.35/72 .11,62.79
1 4.8) 1.

69.20
62.49
66.79
63 .30

2.8 |2.3| 4.4

65.4716

- Variability. 4.0. | 7.0 | 5.3 | 2.3

ee ee ee ee eS a

ALKALINE RESERVE OF BLOOD OF INSANE

151

TABLE 5

reserve of the blood of the insane according to diagnosis

TABLE 4
The carbon dioxide combining capacity of 112 bloods from 51 insane and normal
Individuals
yom ial DIAGNOSIS paar pang DIAGNOSIS gap oe DIAGNOSIS poe Fa hy DIAGNOSIS
75.38 | M. D. 66.36 | M. D. 63.42 |.M. D. 58.90: 1 DP.
40.04. | M. 1). 66.36 | M. D. 63.42 | N. 58.62. | D,'P,
74.90 | D. P. 66.31 -| M. D. 63 .42 I. M. 58.46 | M. D.
74.85 | M.D. 65.66 | M. D. 63.36 )N. | 58.40 | D. P.
74.80 | M.D. 65.50 | D. P. 63.30 | D. P. 58.15 | M. D.
7o.o¢ | M. D. 65.47 D. P. 63.30 | D. P. 57.78 1: P;
7a.20 | D.P. 65.38 | N. 68:47 F DP: 57.74 | D. P.
72.08 | M. D. 65.30 | N. 63.09 | D. P. ‘67-39 | M.D.
72.00 | M.D. 64.77 | D. P. 62.79 -| M. D. 56:72 tsD: Pi
71.04 | N. 64.42 | M. D. 62.60 | D. P. 55.94 | M. D.
71.02 | I. M. 64.42 |S. D. 62:56 °° | DeP. 55.52 DP:
»A0.4i |) M. D. 64.38 | D. P. 62.56 bik M. 55.34 | M: D.
70.17 Si ial 64.34 M. D. 62 .44 M. D. 55.10 i.
7U.40 1) DD: P. 64.30 N. 62:40 | M. D. 54.388 | M. D.
70.07 | M. D. 64.12 | D. P. 62.08 | M. D. 64°22 4:D. P.
69.53. .1°.D..P. 63.95 | M.D. 61:54). |. Do BP 53.92 | M.D.
69.14 | D. P. 53.66. | I. M. 61.52 pS Tale ogi 63.21 Ry.
69.14 | M.D. 63.61 N. 61.33 | N. & .O7''"|, Bat,
oo.4c. | D. P. 63.57 | N. 61.07 | M. D. 52.75 | M.D.
69.12 N. 63.57 «| M.D: 60.70 | M. D. 52.65 ?
68.60 | D. P. 63 . 54 ? 60.56 Dik: a2:46 0 BoP,
58.51 M. D. 63 .54 DP. 60.20 | M. D. 52.06 | D. P.
68 .32 DP. 63 .54 N. 60.19 | D. P. 51:74 | D..P.
67.88 | D. P. 63 .51 os Be 60.03 | N. 51.18 ?
67.81 | D. P. 63.48 | M.D. 59.80 | D. P. 50.24 |'D. P.
67.22 | N. 63.48 | D. P. 59.74 | 1I.M. 50.20 | N.
66.73 | M. D. 63.48 | I. M. 59.68 | D. P. 47:76 |}. D. P.
' 66 .36 M. D: 63 .43 M. D. 59.68 Der. 47.61 ?
i Average, 62.99; Variability, 7.35; Range, 73.38-47.61.

M. D., Manic depressive; D. P., Dementia precox; I. M., Involutional mel-
ancholia; 8. I., Senile involution; N., Normal.

The range of fluctuation, average amounts and average deviations of the alkaline

Fe ae ee OF EN eg ON RR Se Petpet a ee

oka prAeNosis sonata Wy ore
ce. ce. per cent
15 MRS a Ne Tinlad ba viele wi cautee aioe 47 .61-71.04 | 62.60 6.6
39 Manic depressive........63-s.2hes0s, 52.75-75.38 | 64.42 7.3
i 8 Involuntary melancholy............. 51.18-71.02 | 61.03 mY g
47 SEMOINGNEIG DTCOCOX, 0s. . 5s obs Kone c eats 47 .76-74.90 | 61.84 ‘8.2

Sea see Ee ea ee

152 NOBUHARU SUITSU

Sets number 7-8-9 are the values obtained after the shorter fast, and
sets 10-11-12 those found after the longer period of abstinence in the
same individuals respectively.. The investigation extended over eight
weeks, the first four weeks of which being the period when the blood
specimens were taken once a week after breakfast, and the second four
weeks being the period when the samples were taken before breakfast.

It is evident that there are no valid or consistent differences in the
bloods taken at these times.

SUMMARY

The results of the studies here reported indicate that:
1. The alkaline reserve of the blood of the insane appears to fall
_ within the limits considered normal for healthy persons.

2. There are no demonstrable differences in the absolute amounts of
the alkaline reserve of the bloods from excited or depressed patients
here studied.

3. The variability of the plasma carbon dioxide combining capacity
seems to be higher in the i insane than in the small group of normals
here studied.

4. No noteworthy differences obtain in the alkaline reserve of the :
blood taken three and a half and fourteen hours after eating.

I take this occasion to express my appreciation of the courtesy of
Dr. Owen Copp in affording me the facilities of the Pennsylvania
Hospital, Department of Nervous and Mental Diseases. The work
was carried on under the direction of Dr. Frederick 8. Hammett, for
whose help and advice I am deeply grateful. ;

BIBLIOGRAPHY

(1) Van Styxn, StizitMANn, AND CuLLEeN: Journ: Biol. Chem., 1917, xxx, 401.
(2) Van Stryke: Journ. Biol. Chem., 1917, xxx, 347.

(3) Van SLYKE AND CULLEN: Journ. Biol. Chem., 1917, xxx, 289.

(4) Hiaains: This Journal, 1914, xxxiv, 114.

(5) Hammett: Journ. Biol. Chem., 1920, xli, 599.

ee

5 ny ae ee Ee it

GASTRIC TONUS OF THE EMPTY STOMACH OF THE FROG

CoMPARATIVE StupiEs IV!

T. L. PATTERSON

From the Hull Physiological Laboratory, The University of Chicago and the
Physiological Laboratory, Queen’s University

Received for publication July 23, 1920

Sherrington (1) in 1915 called our attention to the reflex postural
activity of muscle and nerve as being the main outcome of the function-
ing of the proprioceptive part of the nervous system for at least the
skeletal muscle. He pointed out that the muscle fiber possessed the
property of exhibiting different lengths while exhibiting one and the
same degree of tension, and that it was not to be regarded as an elastic
band. Furthermore, he believes that unstriated muscle, like skeletal
muscle, possesses the same properties as is shown by the ease with
which the hollow visceral organs, like the bladder and stomach, adapt
their size to the volume of their contents and with very little alteration
in their intravesical pressure. Under these conditions, visceral tonus
is therefore postural configuration. In confirmation of this Hurst
(2) found that the relaxation of the rectum was analogous to what

Sherrington described as the “lengthening reaction” of the “postural

tone’’ in the skeletal muscles and in the bladder, and which he at an

‘earlier date had described in connection with the stomach and intestine,

although he had not actually used the expression “visceral tone.”’ In
case of the skeletal muscle the reflex postural action depends normally
upon the afferent nerve of the posturing muscle itself, while in the un-
striated muscle it is far less dependent on the central nervous system
for its adjustment and maintenance.

More recently Grey (3) has shown by slowly filling the empty viscus
with warm physiological saline solution and recording the fluctuations
in the intragastric pressure that the norma] stomach in rabbits and

1 A preliminary report of this work was made before the 1919 meeting of the
American Physiological Society at Baltimore, a brief abstract of which was
published in the Proceedings of that society.

153

154 T. L. PATTERSON ©

cats is capable of adapting its size to the volume of its contents with
very small changes in the intragastric pressure. According to this
investigator, the mechanism involved in the postural configuration of
the stomach is situated in the wall of the viscus itself and concerns
solely its musculature together with its intrinsic nervous mechanism,
while the extrinsic nerves exhibit no direct influence, but serve rather
to regulate the tension of the stomach wall.

The experiments summarized in this report were undertaken with
the view of securing further data on the gastric tonus (postural activity
—Sherrington, Hurst, Grey) of the neuro-muscular apparatus as ap- —
plied to the empty stomach. While the term “postural activity” is
very applicable to the skeletal musculature it appears to me that it
is not well suited for the unstriated musculature which makes up the
larger portion of the walls of the hollow visceral organs, therefore the
older and simpler terminology of gastric tonus will be used throughout
this paper. The results tend to show that the extrinsic nerves exert
a partial influence on the tonal activity of the stomach viscus, as well
as serving to modify and regulate the gastric activity at least in the
frog. This animal is particularly adapted for such a study for it has
been shown in a previous paper (4) of this series that the gastric hunger
contractions show no periodicity and no appreciable change in gastric
tonus, both features of which are present in the higher animals. In
contradistinction to the higher animals, the contractions are practically
continuous with scarcely any distinction between the digestive peri-
stalsis and the hunger movements. :

Among the first to make observations upon the internal pressure of
the hollow visceral organs were Mosso and Pellacani (5) who investi-
gated the bladder in man and in the dog. These authors found that
the bladder is capable of adjusting its cavity-volume to different quan-
tities of content, which it enfolds with about the same light tension
of grasp whether the viscus is nearly empty or well filled. . Somewhat
similar observations have been made upon the fundic portion of the
stomach. Kelling (6) found that within certain limits the intragastric
pressure remained unaffected by the quantity of fluid within the viscus
and that the intra-abdominal pressure altered very little in the dog
before and after the taking of a copious meal, although the intake of
the volume of food might amount to 50 per cent of the total contents
of the abdomen in the fasting condition. He infers from these latter
observations that the additional volume of contents must be accom-
modated for by a reflex adjustment of the postural contraction of the

2 RS agg PS

GASTRIC HUNGER CONTRACTIONS 155

abdominal muscles. Pike and Coombs (7) in confirmation of the above
have reported that the introduction of fluid into the stomach or into
the peritoneal cavity of cats causes lengthening of the rectus abdominis
muscle while the flow of fluid out of the stomach causes a shortening
of the same muscle. These changes in the length of the muscle are
small and do not occur if the posterior roots of the spinal nerves supply-
ing the muscle have been cut, or if the spinal cord has been transected
at the level of the lower cervical roots. The section of both vagi has
no marked effect on the response of the muscle. The authors regard
the change in the length of the muscle corresponding to the increase
or decrease in volume of the contents of the abdominal cavity as a

reflex process dependent upon afferent impulses ‘which falls into line

with other known instances of postural activity of muscle and nerve.
The observations of Sick and Tedesko (8) and others have shown that

the gradual filling of a cat’s stomach is not accompanied by a rise in

intragastric pressure and that the excised stomach, kept alive in a bath
of warm oxygenated Ringer’s solution also exhibits the same phenom-
enon to an unmistakable extent.

Cannon and Lieb (9) have also brought forth evidence that each
passing of the cardia by swallowed food is accompanied by a rapid
small dilatation of the fundus, and that this dilatation is a reflex oper- |
ated through the vagus. Rogers (10) has reported that central stimu-
lation of one vagus nerve with the opposite nerve intact in the
decerebrate dog and after complete splanchnic section leads to reflex
spasmodic contractions of the entire stomach and increased gastric tone.
Therefore it would appear that the adaptability of the normal stomach
at all times is a form of receptive expression brought about by changes
in the intragastric pressure as the volume of its contents slowly increases
or decreases. ‘. ;

Experimental procedure. 'The same general method of experimenta-
tion was used in the following experiments on the bullfrog (Rana
catesbiana) as that described in the preceding paper (11) of this series,
with the exception that the gastric balloon was inflated with a known
quantity of air by means of a graduated glass syringe sufficient to
maintain a constant pressure of 2 cm. in the water manometer and
the number of cubic centimeters of air necessary in the different
experiments to produce this constant pressure was recorded. Normal
contractions of the empty stomach were obtained from each ani-
mal, extending over a period of several days, and then these animals
were either vagotomized, splanchnetomized or vago-splanchnetomized

156 T. L. PATTERSON

(section of both sets of nerves) and the respective observations repeated
over a period of from two to three weeks and compared with the normal.
The recorded tracings were taken on a slowly moving drum making a
revolution in fifty to sixty minutes. ;

The changes in volume capacity of the stomach as influenced by partial
and complete isolation from the central nervous system. ‘The influence of
the vagi and splanchnic nerves on the activity of the empty stomach
of the frog has been reported in a previous paper (11). According to
these results, double vagotomy leads to a sympathicotonie condition
of the stomach followed with nearly the normal type of hunger con-
tractions with the exception that they appear to be of a somewhat slower
rate and slightly weaker. On the other hand, section of the splanchnic
nerves leads to a hypertonic stomach with shallow contractions, showing
an increased rate and tending to run into incomplete tetanus, while
complete isolation of the stomach from the central nervous system
leads to a hypotonic stomach with about the normal type of gastric
hunger contractions. Somewhat similar changes have been described
by Cannon (12) on cats for the digestive movements and by Carlson
(13) on dogs for the movements of the empty stomach.

Although the sectioning of these nerves in various animals has led
to certain changes in gastric tonus, as arising from the influence exerted
through the extrinsic nerves supplying the stomach, no attempt has
‘been made to analyze the question quantitatively. In order to study
the changes in volume capacity of the empty stomach as influenced by
partial and complete isolation from the central nervous system, twenty-
‘one animals were used for the various observations recorded herein, as
follows,—seven were vagotomized, seven splanchnetomized and seven
-vago-splanchnetomized. In addition, fifteen other animals were used
but as the length of the duration of these experiments was more or less
brief due to parasitization or other causes leading to an early death,
the data from these were excluded. However, in none of these experi-
ments in which results were obtained were they contradictory to the
typical results as tabulated. There were also a few animals of this

number excluded because of incomplete nerve section. The following —

tables have been prepared as showing typical results of the experiments.

The animals used for the observations in the preceding experiments
were twelve to thirteen inches in length, extended, and it was found,
without exception in this size of animal, that 10 cc. of air introduced
by a syringe into the gastric balloon was sufficient to maintain a con-
stant manometric pressure of 2 cm. in the stomach of the normal ani-

Se a

a eee SS en ea

ry ene
aie

&o eae

GASTRIC HUNGER CONTRACTIONS 157

mal. Larger animals in proportion to size require greater quantities
of air to obtain this constant manometric pressure, and vice versa.
In one case, a very large frog measuring sixteen inches, the only one
used in the series of experiments, 15 cc. of air were necessary to produce

SS
~~

TABLE 1
Effect of section of the vagus nerves on volume capacity of stomach and contractions*
DATE 1918 CONDITIONS + EEA fetdrorveygh intent REMARKS
ce. cm.
- August 7 | Stomostomized
August 10 Normal 10 6.5
August 11 Normal 10 6.0
August 12 Normal 10 6.5
August 13 Vagotomized Operation O.K.
August 16 Vagotomized 15 6.5
August 17 Vagotomized 15 8.0
August 18 Vagotomized 15 6.8
August 19 Vagotomized 15 7.0
August 20 Vagotomized 15 8.0
August 21 Vagotomized 15. 6.5
August 22 Vagotomized 13 6.5
August 23 Vagotomized 10 6.0
August 24 Vagotomized 10 4.5
August 25 Vagotomized 10 5.5
August26 Vagotomized 10 5.8
August 27 Vagotomized 10 6.2
August 28 Vagotomized 10 5.0
_ August 29 Vagotomized 10 4.0
. August 30 Vagotomized 10 3.5
August 31 Vagotoniized 10 Very weak
September 1 Vagotomized 10 Very weak
September 2 Vagotomized Animal died. Au-
topsy showed
both vagi cut

* Work now in progress on lung tonus of the frog shows that double vagotomy

leads to practically the same effects as extirpation of the lungs, and this may
shorten the life of the animals.

the constant pressure of 2 cm. in the water manometer. The question
of the elasticity of the rubber balloon may arise here for, as Osborne
(14) pointed out, in thin-walled rubber bags the extensibility of the
elastic material is great and its dimensions, including its thickness,
alter much under the stretch imposed. Furthermore, a subspherical

158. T. L. PATTERSON

bag may change in general figure as its size is altered, or changes in the
physical consistence of the rubber membrane may occur as inflation
and deflation proceeds, all of which would lead to serious complication
for the analysis of results. However, in the case of the gastric balloon ~
used 10 or even 15 ce. of air do not fill the rubber balloon, so that the
tension of the bag’s elasticity complicates the stomach tonus.

TABLE 2
' Effect of section of the splanchnic nerves on volume capacity of stomach and
contractions
DATE 1918 CONDITIONS yoga Bed cra) ries? REMARKS

cc. cm.
August 7 | Stomostomized
August 10 Normal 10 6.0
August 11 | Normal 10 6.7
August 12. | Normal 10 6.5 .
August 13 | Splanchnetomized Operation O.K.
August 16 | Splanchnetomized 4 0.5
August 17 | Splanchnetomized 4 1.0
August 18 | Splanchnetomized 4 0.8
August 19 | Splanchnetomized 4 0.6
August 20 | Splanchnetomized 4 0:5
August 21 Splanchnetomized 4 1.4
August 22 | Splanchnetomized 6 0.5
August 23 | Splanchnetomized 10 0.3 '
August 24 | Splanchnetomized 10 0.4
August 25 | Splanchnetomized 10 0.3
August 26 | Splanchnetomized 10 0.3
August 27. | Splanchnetomized 10 0.2
August 28 | Splanchnetomized 10 Very weak
August 29 | Splanchnetomized 10 Very weak
August 30 | Splanchnetomized 10 Very weak
August 31 | Splanchnetomized : Animal died. Au-

topsy showed
splanchnics cut

In testing out the amount of air necessary to produce the constant

manometric pressure it was found without exception in all the animals
that a much smaller amount than 10 cc. of air would produce changes
in the manometer amounting to 2 or more centimeters but the length
of its duration was very short and the pressure soon fell to the zero
level or closely approximated it depending on the quantity of air intro-
duced. This is indicative of the ease with which the stomach adapts
its size to the volume of its contents.

GASTRIC HUNGER CONTRACTIONS

159

In female animals filled with large egg masses the number of cubic
centimeters of air necessary to produce the constant pressure showed
no variations from that of the non-egg-carrying female and the male,
although the abdomen was much enlarged. This condition in the egg-

TABLE 3

and contractions

’ Effect of section of the vagi and splanchnic nerves on volume capacity of stomach

AIR IN

STRENGTH OF

DATE 1918 CONDITIONS srt abiven neriont REMARKS
ce. cm.
October 22 Stomostomized —
October 25 Normal 10 6.5
October 26 Normal 10 6.5
October 27 Normal 10 6.9.
October 28 Normal 10 6.7
October 28 Vagi and splanchnics cut Operation O.K.
‘November 2 | Vagi and splanchnics cut | 15 6.7
November 3 | Vagi and splanchnics cut 15 7.0
November 4 | Vagi and splanchnics cut | 15 6.9
November 5 | Vagi and splanchnics cut | 15 4.2
November 6 | Vagiand splanchnics cut | 15 6.6
November 7 | Vagi and splanchnics cut | 15 8.0
November 8 | Vagi and splanchnics cut 15 6.5
November 9 | Vagi and splanchnics cut | 15 5.8 Morning
November 9 | Vagi and splanchnics cut 13 6.0 Night
November 10 | Vagi and splanchnics cut | 13 5.0 }
November 11 | Vagi and splanchnics cut | 13 4.4
November 12 | Vagi and splanchnics cut | 13 3.5:
November 13 | Vagi and splanchnics cut |. 13 3.4
November 14 | Vagi and splanchnics cut | 13 3.5
November 15 | Vagi and splanchnics cut 13 | Very weak
November 16 | Vagi and splanchnics cut 13 | Very weak
November 17 | Vagi and splanchnics cut | 13 | Very weak
November 18 | Vagi and splanchnics cut Animal died.
Autopsy
showed both
vagi and
-splanchnics
cut

carrying female is doubtless accounted for,

at least in part, by a reflex

mechanism leading to a relaxation of the abdominal muscles, an adap-

‘tation similar to the reflex relaxation of the rectus abdominis muscle .

in increased volume contents of the stomach as has been described by

160 T, L. PATTERSON

Pike and Coombs (7). The animals with very. few exceptions were run
continuously as soon as recovery was complete after the its and
the fast commenced immediately.

Section of both vagi or the vago-sympathetic nerves (11) i in the neck
of the frog increases the volume capacity of the stomach temporarily,

as shown in table 1, from the normal of 10 cc. to 15 cc. of air. This

condition invariably lasts from eight to nine days. Usually on the
ninth day following the cutting of the nerves there is a decrease in the

Bie

|
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Fig. 1. The spaces left to right indicate the number of days experiment ran.
Vertical spaces above and below the heavy line A B, representing the normal
pressure of 10 cc. of air necessary to maintain a constant pressure of 2 cm. in
the water manometer, indicates the positive or negative changes from the con-
stant in the volume capacity of the stomach as influenced by the extrinsic nerves.

Curve C C, shows effect of sectioning both vagi on stomach. Curve DD, effect

of splanchnic section. Curve EF E, combined effect of section of both vagi and
splanchnic nerves. Note complete recovery of gastric tonus in first two cases,
while in the latter there is only a partial recovery. Heavy line A B also indi-

* cates negative effect of decerebration on stomach. Figures at left indicate

number of cubic centimeters of air in balloon.

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GASTRIC HUNGER CONTRACTIONS 161

intragastric pressure to about 13 cc. of air and on the next day it drops
again to the normal or 10 cc. level, and remains there. In other words,
the normal tone of the stomach has been reéstablished (fig. 1), and this
condition as it exists in the frog may be comparable to the temporary
loss of tonus as described by Cannon (12) in cats. The contractions

- of the empty stomach tend to approach the normal, but on the whole

they are of a slightly slower rate and more irregular. The amplitude
of the individual contractions may even appear greater than normal
and this may be because the contractions start rather suddenly and
without any marked preliminary increase in tonus in the fundic end
of the stomach. , |

Section of the splanchnic nerves in the frog markedly decreases the
volume capacity of the stomach temporarily, as shown in table 2, from
the normal of 10 ce. to 4 cc. of air. This marked diminution in size
like the increase after double vagotomy invariably lasts from eight to
nine days. Usually on the ninth day following the cutting of the
nerves there is an increase in the intragastric pressure to about 6 cc.
of air, while on the next day it reaches again the normal or 10 ce. level
and remains there. Here again the stomach has reéstablished its gastric

-tonus (fig. 1). This condition in the frog is much more marked than

Carlson (13) found it to be in dogs for just as the number of cardio-
inhibitory fibers vary in the vagus of the cat and the dog, so also may
not the number of motor fibers in the vagi destined for the stomach
vary in different animals? The contractions of the empty stomach
are small, showing an increased rate and a tendency to approach in-
complete tetanus. This is especially true.during the temporary period
of high tonal activity when only 4 cc. of air are required to maintain
the constant manometric pressure and in one animal 3 cc. of air were
found to be sufficient. In afew such animals I have found in the morn-
ing following the removal of the balloon the night before such strong
gastric and esophageal contraction that it was impossible to introduce

the balloon through the short esophagus into the stomach without first

introducing a small glass seeker and stretching it. I have even had
difficulty in introducing the seeker the first time on one or two occasions
because of such marked contraction. This would seem to uphold the
views of Cannon (12) and Kelling (6) that the gastric fibers of the vagi
function to make the gastric muscles exert a tension.

Section of the vagi and splanchnic nerves in the frog increases the
volume capacity of the stomach permanently, as shown in table 3,
from the normal of 10 cc. to 15 ce. of air, but in this case there is not

162 T, L. PATTERSON

a complete recovery. After this complete isolation of the frog’s stomach
from the central nervous system, the 15 cc. stomach invariably lasts
from twelve to thirteen days, which is a longer period than in either
the vagotomized or splanchnetomized stomach. Usually on the thir-

teenth day following the sectioning of these nerves which is accom-—

plished at one operation there is a fall in the intragastric pressure to a
13 cc. level, where there are no further changes (fig. 1). This new and
partial readjustment of the hypotonic stomach is evidently determined
by the intrinsic local gastric motor mechanism of the stomach wall for
the gastric hunger contractions: persist after its isolation from the
central nervous system. ‘The appearance of the individual contractions
is much the same as when the vagi alone are cut. These contractions
may exhibit a greater or lesser amplitude and show a tendency toward
irregularity. All the animals in the different groups were autopsied
to verify more especially the sectioning of the respective nerves. In
a few of these animals in which the heart was still beating regularly
the effect of vagal stimulation on the stomach was determined. This
resulted usually in a phase of inhibition followed: by a stronger phase
of excitation immediately upon the removal of the stimulus and is in

confirmation with the findings of Hopf. (15) on frogs. Stimulation of —

the sectioned splanchnic usually resulted in a relaxation of the body
of the stomach, if any change at all occurred, and if the stimulation
was repeated several times in succession it seemed to bring about a
constriction of the pyloric sphincter and perhaps also that of the cardiac
sphincter, a condition which would seem to indicate that the splanchnics
might possess a few fibers of the motor type. The stimulation of these
two nerves does show, however, that the fibers of neither have degenerated,
and since the rate of nerve degeneration differs in different animals and
in frogs requires from thirty to one hundred and forty days, depending
upon the season of the year (16), there is no possibility of the regener-
ation of these nerves. The normal functioning of the two sets of nerves

to the stomach is indicated by the results of sectioning, as well as by’

the results of stimulation. In the case of the isolation of the stomach
from the influence of the vagi with the splanchnics intact, or vice versa,

there is a perfect physiological readjustment of the normal tonus of the ~

gastric musculature. On the other hand, after complete isolation of
the stomach (vagi and splanchnics severed) from the central nervous
system there is only a partial physiological readjustment of the gastric
musculature. This indicates that the extrinsic nerves play a prominent
part in the maintenance of gastric tonus, at least in the frog. When

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the splanchnics are sectioned it must be the motor fibers of the vagi
that produce the high and temporary gastric hypertonicity, i.e., hyper-
tonic stomach. When the vagi are cut and the splanchnics permitted
to exert their full influence on the gastric musculature it is reasonable

to believe that these nerves must possess motor fibers probably to the

sphincter muscles and that these areas then act as tonic rings which,
in connection with the intrinsic or local reflex mechanism of the gastric
wall, are capable of producing a perfect physiological readjustment.
Whereas, in the case of the stomach completely isolated from the central
nervous system, this intrinsic or local gastric mechanism is incapable
of bringing about a complete readjustment and in consequence of this
it creates a new level of gastric tonus. Thus, every reflex is in its own
measure an integral reaction, and is purposive in that it bears some
biological purport for its organism. This physiological readjustment
occurred regularly in all the animals, that is, it could be looked for

-after a lapse of a certain number of days following the sectioning of

the nerves. For example, in the case of the vagotomized stomach when
this readjustment started I have seen in a few instances the pressure
in the manometer increase from the constant level of 2 cm. to 5 or 6
em., but I have never observed it in the stomach of the normal animal.
In the splanchnetomized stomach of the course the first readjustment
stage is marked by a fall in the manometric pressure to zero.

The changes in gastric tonus observed throughout this series of
experiments are so slight in the normal frog that they are practically
unmeasurable. However, tonus is the prime condition for that tension
which must be developed before contraction can result and if the tension
persists the contraction recurs (17). Furthermore, the importance of

_ the tonic state in the normal functioning stomach is reinforced by the

fact that when all the extrinsic nerves are cut the stomach develops in
time within itself a tonic state, while the adaptability of the abdominal
cavity to the volume of its contents is left to the postural reflex.

The effect of decerebration on the tonus of the stomach. It has been
shown by King and Connet (18) that the rate of the gastric contractions
is increased in decerebrate guinea pigs and that the stomach becomes
hypertonic. According to Rogers (19) the hyperactivity of the crop
of the decerebrate pigeon is inhibited by food and water as in the normal
bird, while the writer (4) has reported no change in the type of the
contractions from the empty stomach of the normal and the decerebrate

_ frog. In order to study the effect of decerebration on the volume ca-

pacity of the stomach, observations were made on six decerebrate frogs.
The following table has been prepared from a typical experiment.

164 T. L. PATTERSON

Decerebration in the frog has no effect on either the volume capacity
of the stomach or the amplitude of the individual contractions, as is
shown in table 4. There is also no change in the type of contractions,
which confirms the work of the previous paper (4). The negative

findings in these experiments show that the higher cerebral centers in

the frog play no appreciable part in either the maintenance of gastric
activity or the tonic state. Since section of the vagi leaves the stomach
in a temporary hypotonic condition (15 cc. stomach) while the decere-
bration effects are negative we may infer that impulses from centers

TABLE 4

Effect of decerebration on volume capacity of stomach and contractions

is oe ane STRENGTH
DATE 1918 CONDITIONS nainon OF CON- REMARKS
TRACTIONS

cc. cm.

July 26 Stomostomized

July 31 Normal 10 6.0

August 1 Normal : 10 6.5

August 2 Normal 10 6.0

August 2 | Decerebrated 4:15 p.m. Operation O.K.

August 2 | Decerebrated 10 6.0 Contractions
started again at

: 5:35 p.m.

August 3 | Decerebrated 10 6.3

August 4 | Decerebrated 10 6.0

August 5 | Decerebrated 10 —~§.2

August 6 | Decerebrated Animal died. Au-

topsy showed
complete _—re-
moval of cere-
bral hemi-
spheres |

in the mid-brain and medulla exercise the controlling influence and
produce after section of the splanchnics the temporary hypertonic
stomach, i.e., high gastric tonus. It may be further implied that there
is a dynamic readjustment in the central nervous system which leads
to an actual diminution in the inhibitory impulses through the splanch-
nics after vagal section or an inverse motor condition existing through
the vagi after splanchnic section, or else the stomach may bring about
its physiological readjustment by an increased resistance or tolerance
of the splanchnic or motor impulses over the respective nerves to the
gastric mechanism.

GASTRIC HUNGER CONTRACTIONS 165

CONCLUSIONS

1. The normal stomach of the frog possesses a marked capacity for
adapting itself to the volume of its contents with only minimal changes
in the intragastric pressure. This is in confirmation with the work of
Grey and others. : noe Aa ie

_ 2. Both the intrinsic and extrinsic nerves take part in the mainte-
nance of gastric tonus as is shown by partial and complete isolation of
the stomach from the central nervous system. |

3. Section of the vago-sympathetic nerves (double vagotomy) with
the splanchnics intact increases the volume capacity of the stomach
temporarily, but there is later a complete readjustment. |

4. Section of the splanchnic nerves with the vagi intact decreases
the volume capacity of the stomach temporarily, but there is one a
complete readjustment as above.

5. Section of both the vagi and splanchnic nerves (complete isolation
from the central nervous system) increases the volume capacity of the
stomach permanently, and in this case there is only a partial readjust-
ment, at least for a period extending over three weeks. and the tonus
of the stomach is established upon a new level from that of thenormal.

6. Decerebration affects neither the volume capacity of the stomach
nor the type of the contractions. :

The writer desires to acknowledge his indebtedness to Doctor Carlson
for his kindly and valuable criticism.

BIBLIOGRAPHY

(1) SHERRINGTON: Brain, 1915, xxxviii, 191.

(2) Hurst: Seale Hayne Neur. Studies, London, 1919, i, no. 4, 208.

(3) Grey: This Journal, 1918, xlv, 272.

(4) Parrerson: This Journal, 1916, xlii, 56.

(5) Mosso anp Peuuacant: Arch. ital: d. Biol., 1882, i, 96.

(6) Kewuina: Zeitschr. f. Biol., 1903, xliv, 161.

(7) Prke AND Coomss: This ‘Journal. 1917, xlii, 395.

(8) Stick anp TepeEsko: Deutsch. Arch. f. klin: Med., 1908, xcii, 146.

(9) CanNON AND Ligs: This Journal, 1912, xxix, 267.

(10) Rogers: This Journal, 1917, xlii, 605.

(11) Parrerson: This Journal, 1920, liii, 293.

(12) Cannon: This Journal, 1906, xvii, 429; 1911, xxix, 250.

(13) Caruson: This Journal, 1913, xxxii, 369.

(14) OsporNneE: Proc. Roy. Soc., 1909, B, lxxxi, 485.

(15) Horr: Zeitschr. f. Biol., 1910, lv, 409.

(16) Berne: Allgemeine daatokois und ErrBlO Waa des Nervensystems, Leipzig,
1903, 158.

(17) Cannon: Arch. Int. Med., 1911, viii, 417.

(18) Kine anp Connet: This Journal, 1915, xxxix, 123.

(19) Rogers: This Journal, 1916, xli, 555

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

STUDIES ON THE SUBMAXILLARY GLAND

VI. ON THE DEPENDENCE or TissuE ActTiviry UPON VOLUME-FLOW
oF BLoop AND oN THE MercHANISM CONTROLLING
THIS VOLUME-FLOW OF BLOOD

ROBERT GESELL

From the Departments of Physiology of Washington University Medical School and
of the University of California

Received for publication July 27, 1920

INTRODUCTION

This paper has to do with two problems: one the dependence of
tissue activity upon volume-flow of blood, and the other the mechanism
by which the volume-flow of blood is controlled. While these problems
may be considered as distinct from each other, yet they have a certain
interdependence which may warrant their discussion in common.

I have previously reported results bearing upon both problems (1),
(2). Although no definite conclusions were reached concerning the
mechanism of volume-flow control, it was shown that in hemorrhage
the organism as a whole suffers from a reduced flow of blood as is
indicated by the reduced alkaline reserve of the plasma of the blood.

But despite the fact that the organism suffers from a reduced flow of —

blood we know from the work of others (3), (4) that a tissue may be
stimulated to great activity even though the flow of blood may be very
low or even absent. This apparent independence of volume-flow and

tissue activity is shown in figures 1 and 3, where secretion of saliva is —

used as the index of tissue activity.

DEPENDENCE OF SECRETION UPON VOLUME-FLOW OF BLOOD

To show the dependence of tissue activity upon volume-flow of blood,
using secretion as the index to activity, special methods must be em-
ployed. The greatest care must be taken that change in volume-flow
of blood be the only variable. When the gland is activated by stimu-
lation of the chorda tympani the periods and strength of stimulation

166

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VOLUME-FLOW OF BLOOD 167

must be equal and periods of rest must be chosen which will avoid .the
augmenting effect of previous stimulation. Results obtained under
such conditions can be compared with results obtained when the blood
supply is modified. Such results are shown in figures 1, 2 and 3, in
which blood pressure, volume-flow of blood, secretion, electrical deflec-
tions, time in seconds and moment of'stimulation of the chorda tympani
are recorded. The volume-flow of blood was measured with the blood-
less method previously described.

Figure 1, A and B, shows the effects of occluding the carotid artery
during a short period of stimulation lasting 14 seconds. In the first
record, the artery was unoccluded and stimulation of the chorda tym-
pani produced the usual rapid acceleration of blood flow. In the second,
where the artery was occluded, the flow of blood during the period of
stimulation was slow but the amount of saliva secreted was not dimin-
ished. The results might be taken to indicate that even large fluctu-
ations in volume-flow of blood need not affect the metabolic processes
of the gland, were it not for the change in the contour of the electrical
deflections which suggests that the glandular processes were modified.
On the other hand, the absence of an after-flow of blood following
de-occlusion indicate that the gland was not overtaxed by the ~ a
ary reduction in the blood-flow.

Figure 1, C and D, shows the effect of occlusion of the artery during
greater activation. The secretion of saliva during occlusion was not
reduced, yet the electrical deflection was modified again as in 1, B.
The difference between the results shown in 1, A and B, and in 1, C
and D, is that de-occlusion in the latter observations was followed by
a markedly accelerated after-flow of blood indicative of an overstrain
of the tissue resulting from activation without sufficient blood supply.

Figure 2, A, B and C, shows the effect of more prolonged occlusion
of the artery lasting through a period of stimulation of 30 seconds.
Even this longer period of occlusion has little effect upon the amount
of saliva obtained—16.2 drops during the period of occlusion as com-
pared with 18.2 and 19.2 drops during the preceding and subsequent
periods of free flow of blood. In figure 3, A, B, C and D, where the
periods of stimulation and occlusion were still longer, the reduced flow
of blood again had relatively little effect. In fact (C) during occlusion
18.4 drops of saliva were secreted compared with 19.0, 19.6 and 20.3
in the preceding and subsequent periods.

Upon the whole, the results indicate that relatively short periods
of occlusion of the carotid artery affect little the amount of saliva

168 ROBERT GESELL

elicited by stimulation of the chorda tympani. This absence of marked
effects may be apparent only and may be due to the fact that the gland
at the moment of stimulation has recovered from previous activation
and readily liberates its stored material and energy regardless of the
momentary decrease in flow of blood during the period of activation.
If so, it would be better to study’ the dependence of tissue metabolism

Fig. 1. Effect of occlusion of the carotid artery on the response of the submax-
illary gland to stimulation of the chorda tympani. 1A, normal; 1B, artery oc-
cluded; 1C, normal; 1D, artery occluded; B.P., blood pressure; V.F., volume-flow
of blood; S., salivary secretion; E., electrical deflection; 7'., time in seconds and
moment of stimulation of the chorda tympani.

169

VOLUME-FLOW OF BLOOD

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VOLUME-FLOW OF BLOOD 171

upon tissue which has not fully recovered from previous activation;
for this purpose the gland might be activated over a longer period of
time, and the blood supply modified during this period of activation,
that is while activity and recuperation are going on hand in hand.

A series of such experiments is shown in figure 4, A, B, C, D and E.
In figure 4, A, B and C the gland was activated by the injection of
pilocarpin and the flow of blood was restricted in three ways: in A by
obstruction of the carotid artery; in B by injection of adrenin; and in
C by stimulation of the vago-sympathetic. In every instance the slow-
ing of the flow of blood affected the response of the gland to the stimu-
lation of pilocarpin. The objection might be raised that we not only
interfered with the blood supply but also with the supply of pilocarpin
which stimulates the gland. This objection can not hold in theexperi-
ment represented in figure 4, D and C, where the gland was activated
by stimulating the chorda tympani. In 4; D, the flow of blood was
decreased during stimulation by occluding the artery. The volume-
flow of blood was not recorded but the moments of occlusion and de-
occlusion are evident in the blood pressure tracing. It will be noted
that the effect of occlusion became progressively greater as occlusion
continued, suggesting that stored up saliva may be easily liberated,
whereas the storage and liberation of new saliva required a greater
flow of blood. The slowing of the secretion is undoubtedly due to the ©
slowing of the blood stream and not to fatigue of the gland from pro-
longed stimulation, as is indicated by the subsequent acceleration of
secretion upon de-occlusion of the artery. In this record secretion
continued for some time after cessation of stimulation. Occlusion of
the artery during this after-secretion again retarded secretion. The
effects of injection of adrenin during prolonged stimulation of the
chorda tympani were quite as striking—see figure 4, E.

The gland from which figure 5 was obtained was extremely sensitive
to changes in volume-flow of blood. The period of stimulation of the
chorda tympani lasted from A to B. The close parallelism between
secretion and volume-flow of blood is very evident. Short irregular
_ fluctuations which were not due to occlusion of the artery also are to
be seen. |
Figure 6 is taken from the same experiment as figure.5. It shows

again the effect of volume-flow of blood upon the threshold of stimula-
tion for secretion. Stimulation of the chorda.tympani elicited an
increased volume-flow of: blood and though no visible secretion occurred
it excited the sec ‘as is evidenced by the electrical deflection. ‘That

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during prolonged activity. In A, B and C, the gland was activated by the injec-
tion of pilocarpin and the blood flow interfered with by arterial occlusion, injec-
tion of adrenin and stimulation of the vagosympathetic respectively. In Dand £
the gland is activated by stimulation of the chorda tympani and the flow of blood
interfered with by arterial occlusion and the injection of adrenin.

172

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174 ROBERT GESELL

occlusion of the carotid artery during excitation affected the processes
in the gland is indicated by another change in the electrical deflection.
When the artery was de-occluded about 20 seconds after the cessation
of stimulation a rapid flow of blood occurred. This accelerated blood
flow was accompanied by a copious secretion. The fact that an ac-
celerated blood flow was associated with secretion some time after the
effects of stimulation had wholly or at least partially worn off, seems
to indicate that secretion in this instance occurred in two definite stages.
The results are in line with the observation that the elicitation of visible
secretion requires a stronger stimulus than does the elicitation of an

Fig. 6

electrical deflection which is an index to glandular activity. There
may, however, be another explanation of the results shown in figure 6.
We know from previous work that adrenin, provided it does not inter-
fere with the flow of blood through the gland, may occasionally augment
secretion elicited by either stimulation of the chorda tympani or in-
jection of pilocarpin. It is possible that the short period of arterial
occlusion led to an asphyxial discharge of adrenin into the circulation
(5) sufficient to augment secretion. Unfortunately results similar to
those seen in figure 6 were not obtained frequently enough to make
possible the determination of the factor underlying the accelerated
secretion.

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VOLUME-FLOW OF BLOOD 175

THE MECHANISM CONTROLLING THE VOLUME-FLOW OF BLOOD

It is well known that after atropinization of an animal stimulation
of the chorda tympani may accelerate the volume-flow of blood through
the submaxillary gland without eliciting visible secretion. This obser-
vation is used as evidence of the presence of vasodilator fibers in the
chorda tympani. But Barcroft (6) pointed out that even though there
be no visible secretion resulting from stimulation of the chorda tympani,
oxidations in the gland may be increased. The accelerated flow of
blood may, therefore, be due to liberation of dilator metabolites rather
than to stimulation of dilator fibers.

The fact that stimulation of the chorda tympani, too weak to produce
visible secretion, may elicit an increased volume-flow of blood is likewise
cited as evidence for the presence of dilator fibers in that nerve. But
the fact that such an increase in volume-flow of blood is accompanied
by an electrical deflection also makes possible the explanation of dila-
tation through dilator metabolites (1).

Since these methods, which indicated the existence of dilator fibers,
fail to yield crucial data concerning the mechanism of volume-flow
control, I attempted in a previous research to throw further light on the
problem by a variety of indirect methods (1). The results obtained
were summarized as follows:

As to the existence of vasodilator nerves the question which initiated this

_research nothing definite can be said. We have no proof that such nerves do not
exist, neither have we proof that metabolites can not adequately control the

volume-flow of blood. All that can be said is, that if the dilator fibers do control

_ the volume-fiow of blood, this flow may be augmented still more by an accumula-
tion of metabolites. Many observations might apply to both theories, some how-

ever point more strongly to the metabolite control.

It was shown in that research that with conditions constant the

flow of blood is very finely adjusted to the activity of the gland. By
’ plotting the superbasal flow of blood on the ordinates against progres-

sively increasing amounts of salivary secretion onthe abscissas we found
superbasal flow of blood to be a linear function of superbasal metabolism.
Such accelerated flow accompanying tissue activity is undoubtedly a

purposive reaction to make good the excess of liberated energy, but

does not help toward determining the mechanism of volume-flow

control. This reaction of accelerated flow of blood is studied to advan-
tage by plotting continuous curves of glandular activity and volume-
: flow elicited by stimulation of the chorda tympani. This method not

176 ROBERT GESELL

only brings out in another way the parallelism of the two phenomena
but in addition shows the time relation of the two processes.

Figure 7 shows the effect of stimulation of the chorda tympani of
12 seconds duration in 7, A, and of 60 seconds in 7, B. -The activity of
the gland (secretion) is plotted on the ordinates in solid black against
time on the abscissas. Volume-flow of blood is plotted as a line. The
portion of the curve of volume-flow of blood preceding stimulation of
the chorda tympani represents the basal flow of blood or the flow of
rest; the curve above that level represents the superbasal flow. Though
in each instance the increased flow of blood followed stimulation

7A

Fig. 7. Effect of stimulation of the chorda tympani on secretion and volume-
flow of blood. Volume-flow of blood and salivary secretion are plotted on the
ordinates against time in minutes on the abscissas. The duration of stimulation
of the chorda tympani is shown.

promptly, there was considerable Jag in the flow as indicated by a com-
parison of the crests of the curves of secretion and volume-flow of
blood. This lag or after-flow of blood, which continues throughout
the experiment, is suggestive of a recuperative process following activa-
tion, and is in agreement with the findings of Barcroft and Hill on
oxidation and heat formation associated with tissue activation.

Figure 8 shows results obtained in other experiments. The graphs
were obtained from four different animals. Graphs A and B show the
relation of volume-flow of blood to prolonged activity of the gland
elicited by prolonged excitation of the chorda tympani. The usual
effect of such stimulation upon secretion is a rapid acceleration followed

eee ee, eS a

VOLUME-FLOW OF BLOOD 177

by a decrease which in turn gives way to a secondary increase. The
parallelism between secretion and volume-flow of blood is striking.
Graph 8, C and D, shows the same relation, but the fluctuations in
secretion differ from those in graph 8, A and B,in that they are elicited
by short periods of stimulation. Secretion elicited by stimulation of

Fig. 8. Effect of stimulation of the chorda tympani on secretion and volume-
flow of blood. Volume-flow of blood and salivary secretion are plotted on the
ordinates against time in minutes on the abscissas. A and B show the relation
of volume-flow of blood to prolonged secretion elicited by prolonged stimulation
of the chordatympani. C and D show therelation of volume-flow of blood to pro-
longed secretion elicited by a short period of stimulation of the chorda tympani.

178 ROBERT GESELL

the chorda tympani as a rule stops promptly upon cessation of stimu-
lation, but not infrequently, the secretion slows only to accelerate before
ultimately decreasing. Such results are represented in graph 8, C and
D. The volume-flow of blood here, too, is closely adjusted to the activ- —
ity of the gland. It is interesting to consider the significance of these
results in relation to the mechanism of vasodilatation. The after-
secretion following cessation of stimulation may last from 1 to 15°
minutes. The cause of this secretion we do not know. It may possibly
be due to a prolonged after-discharge of the vasomotor post-ganglionic
cells.. A like discharge of the vasomotor post-ganglionic cells would
explain in a similar manner the after-flow of blood. If both after-flow
‘of blood and after-flow of secretion are due to this after-discharge of
ganglionic cells it is extremely interesting that the after-discharge of
the two sets of cells should be so nearly equal and so exactly timed.
Numerous experiments similar to those represented in figures 7 and
8 show that under constant conditions and regardless of the duration
of stimulation, the volume-flow of blood is nicely adjusted to the needs
of the tissues. Although such results need not favor either theory
of vasomotor control, the lag of accelerated volume-flow following
activation might suggest at least the codperation of metabolite control.
If a lack of parallelism could be demonstrated between volume-flow
of blood and activity of the gland resulting from stimulation of the
chorda tympani, the presence of dilator fibers in the chorda tympani
would be suggested, only, however, assuming the absence of vasocon-
strictor fibers. But it appears to be a difficult matter to demonstrate
satisfactorily such a lack of parallelism. Perhaps graph 8, D, could be
looked upon as representing a slight lack in the perfection of adjustment
between volume-flow and tissue activity, in that the second increase of
secretion is associated with a relatively smaller increasein volume-flow
than that associated with the first phase of secretion. In a few experi-
ments the lack of parallelism has been far more striking in that the
volume-flow of blood actually decreased during a period of copious
secretion. i
As to the significance of these apparent exceptions, it is obvious that.
the presence of a variable number of constrictor fibers in the chorda
tympani might change the relation between glandular activity and
volume-flow of blood elicited by stimulation of the nerve. Fréhlich
and Loewi (7), believe that such fibers exist. They obtained a decreased
flow of blood, such as is described above, when the chorda tympani was
stimulated. In their, experiments nitrites were administered for the

7
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.
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:
,

- YOLUME-FLOW OF BLOOD 179

purpose of producing a maximum dilatation, thereby permitting effective
stimulation of the constrictor fibers running in the chorda tympani.
Bayliss (8) failed to obtain the decreased flow of blood under the con-
ditions given by Frohlich and Loewi and furthermore failed to confirm
the results by stimulation of the cervical sympathetic nerve of the cat,
which is known to contain constrictor fibers.

I attempted to determine the presence of constrictor fibers running
in the chorda tympani by selective stimulation of these fibers produced
by hemorrhage. I recorded the volume-flow of blood from both sub-
maxillary glands during progressive hemorrhage. On one side the
chorda tympani and the vago-sympathetic were cut and on the other
side only the vago-sympathetic. Since one gland was connected with
the central nervous system through the chorda tympani and the other
gland was completely isolated, if constrictor fibers are present in the
chorda tympani we might expect a difference in the curves of basal
flow of the two glands (basal flow of blood plotted on the ordinates
against mean blood pressure upon the abscissas). A comparison of the
curves of basal flow of blood failed to indicate the presence of constrictor
fibers in the chorda tympani.

The results shown in figure 9 are more helpful in explaining the differ- .
ence between the results of Frohlich and Loewi and of Bayliss. In this
figure the curve of secretion and of basal-flow of blood and of salivary

_ sécretion elicited by short periods of stimulation of the chorda tympani

at various levels of blood pressure during progressive hemorrhage are
plotted. The period of stimulation in each case lasted about 20 seconds.
The curve beginning on the abscissas is the curve of secretion. The
other is the curve of blood flow. The horizontal portion preceding
stimulation of the chorda tympani represents the flow of rest or basal-
flow and the remaining portion the secretory or super-basal flow of
blood. Record A was obtained during normal blood pressure. In
record D the pressure had fallen to about 40 mm. Hg.

From the work of Barcroft we know that during secretion water is
abstracted from the blood flowing through the gland. As the basal-
flow of blood diminishes with decreasing pressure the basal and super-

‘ basal-flow of blood decrease and the configuration of the curves changes

in a way indicative of this abstraction of water, for it will be noted that
in the final stages of hemorrhage the flow of blood during secretion is

actually less than the flow of rest. The irregularities of the curves _

of blood flow in graphs B and C apparently are the result of abstraction
of water from the blood, but only when the blood pressure is too low

180 ROBERT GESELL

to take sufficient advantage of the dilatation which presumably occurs,
does the abstraction of water reduce the flow below that of rest. This
reduction gives the appearance of constriction.

The exceptions to the proportionality of tissue activity and volume-
flow of blood, therefore, can not be said to be real, but figure 10 shows
results which may possibly be of significance. This figure shows the
effect of reducing in steps the strength of a prolonged and continuous
stimulation of the chorda tympani. The period of stimulation lasted

8 D
Fig. 9. Relation of volume-flow of blood to secretion during progressive
hemorrhage.

approximately 7 minutes. Figure 10, A, shows the usual results of
such a procedure and figure 10, B, the unusual results. In figure 10, A,
it will be noted that at the points B, C and D, where the strength of
current was decreased, volume-flow of blood and secretion showed
proportional changes. Not so in figure 10, B. For some reason the
response of the gland to the same procedure was strikingly different.
The chorda tympani was stimulated at A and the strength of stimulation
kept constant up to B. During this period the usual relations between

VOLUME-FLOW OF BLOOD 181

volume-flow of blood and secretion obtain. At B the strength of
stimulation was suddenly decreased and, barring the smaller variations

loA

k
7
4

Fig. 10. Effect of suddenly reducing in steps the strength of a prolonged stimu-
lation of the chorda tympani. ;

in rate of secretion, there was little if any change of rate. On the other
hand the volume-flow of blood was enormously reduced—almost to
the basal flow of blood. ‘Though the strength of stimulation and rate

182 ROBERT GESELL

of secretion remained constant up to point C, the flow of blood remained
reduced for a short period only, coming back again to the original super-
basal flow. Another sudden reduction in the strength of otinnulaas
at point C produced the same results.

It is exceedingly interesting that the reduced volume-flow of blood
should suddenly accelerate though the strength of stimulation remained
constant between the points of sudden diminution of strength of stimu-
lation, to reach again the level obtaining between A and B where the
stimulation is considerably stronger. From the fact that the volume-
flow of blood diminished though the secretion remained constant, it
would appear, assuming the presence of dilator fibers, that the strength
of stimulation was reduced below the threshold of these fibers. Whether
the subsequent acceleration of volume-flow of blood is due to an auto-
matic lowering of the threshold of stimulation of the dilator fibers

resulting from accumulation of metabolites or whether it is due to the

direct action of the metabolities on the vessels, the results do not defi-
nitely indicate. They do show a lack of parallelism between metabo-
lism and volume-flow and accepting the absence of constrictor fibers
in the chorda tympani they point to the existence of dilator fibers.
The value to be placed on these findings depends upon the significance
we can attach to an isolated exception of this kind. It should be
mentioned in this connection that although this result was obtained on
only one animal it was obtained repeatedly.

SUMMARY

The dependence of tissue activity on volume-flow of blood was studied
on the submaxillary gland of the dog—

a, by comparing the amount of secretion obtained during periods of
normal and reduced flow of blood;

b, by noting the effect of decreased flow of blood upon the electrical
detlecGohs:

c, by studyike the after-flow of blood following de-occlusion of the
artery immediately following tissue activation. (An exaggerated after-
flow of blood was used as an index to overstrain of the tissue).

Reduction of the volume-flow of blood during a short period of stimu-
lation of the chorda tympani, from 10 to 30 seconds, did not decrease
the amount of secretion.

The glandular processes, however, were affected by such procedure,
for the electrical deflections were invariably altered.

=r
a ca

- a eo
je PL ee. ee

VOLUME-FLOW OF BLOOD 183

Reduction of the volume-flow of blood during a period of more intense
stimulation, but also of short duration, although it did not reduce the
amount of secretion elicited, resulted in a more prolonged flow of blood
as well as an altered electrical deflection.

With more prolonged stimulation of the chorda tympani the various
glands responded differently to arterial occlusion. On some: glands
occlusion of the artery for a period of about 1 minute was without
effect upon secretion, while in others a noticeable reduction in secretion
occurred.

The temporary independence of tissue activity of volume-flow of

blood as evidenced by secretion is probably apparent only and is due
to the recovery of the gland between periods of stimulation and to the
relative independence of the process of liberation of secretion on volume-
flow of blood.
_ The dependence of tissue activity upon flow of blood is better shown
by reducing the flow through a tissue which has already been activated
for some minutes, that is, in a tissue in which recuperation and activity
are going on hand in hand.

Such methods showed a very close dependence of tissue activity -
upon volume-flow of blood. The results substantiate the views previ-
ously published on the significance of hemorrhage and reduced flow
of blood from other causes in the onset and sustentation of the condition
of traumatic shock.

Prolonged stimulation of the chorda tympani usually produced fluc-
tuations in secretion during the period of stimulation: first a rapid secre-
tion followed by a decrease, then another acceleration giving way to a
final decrease at the end of stimulation. Similar fluctuations were
produced by a short period of stimulation lasting only a small part of
the period of secretion. Whether the stimulation was long or short,
the volume-flow of blood and the secretion ran parallel with each other.
The significance of the findings is discussed.

The close parallelism between tissue activity and volume-flow of
blood offered difficulties in demonstrating definitely the existence of
dilator fibers.

One experiment showing a lack of this parallelism indicates the
presence of dilator fibers in the chorda tympani.

Data are presented indicative of chemical regulation of blood flow.

Experiments are cited pointing to the absence of constrictor fibers
in the chorda tympani.

6). Taucanee: The iraerncatas
Press, OO Sa ; ;
@ Frouricu AND Lonw:: mes!

ince i A ee SE Sa ei ea a a ae

STUDIES ON THE SUBMAXILLARY GLAND

VIL. On THE Errects oF INCREASED SALIVARY PRESSURE ON THE
ELECTRICAL DEFLECTIONS, THE VOLUME-FLOW OF BLOOD AND
THE SECRETION OF THE SUBMAXILLARY GLAND OF THE DoG

ROBERT GESELL

From the Depariments of Physiology of Washington University Medical School and
the University of California

Received for publication July 27, 1920

In studying the electrical deflections of the submaxillary gland of the
dog I noted that stimulation of the chorda tympani produced a greater
and more prolonged after-flow of blood when the salivary duct was
obstructed than when unobstructed (see figs. 4, 5 and 8). In so far
as these observations aid in elucidating the mechanism of the control
of volume-flow of blood they pertain to the problem of papers II, III,
IV, VI and VIII of this series. The observations, however, will be
considered from a broader point of view, namely, in their relation to
the general problem of the physiology of the salivary glands.
The data will be discussed under the following heads:

1. Effects of increased salivary pressure upon the electrical deflections of the
gland.
a. Occlusion of the aalfciry duct during secretion elicited by the injection of
pilocarpin.
b. Occlusion of the duct synchronous with secretion elicited by the stimulation
of the chorda tympani.
c. Backward injection into the salivary duct of the resting Bata.
2. Effects of increased salivary pressure upon the volume-flow of blood through
the gland.
a. Occlusion of the salivary duct during secretion elicited by the injection of
pilocarpin.
b. Occlusion of the duct synchronous with secretion elicited by the stimulation
of the chorda tympani. |
c. Backward injection into the salivary duct of the resting gland.
3. Effect of increased salivary pressure upon secretion.
a. Occlusion of the duct during secretion elicited by the injection of pilocarpin.
b. Occlusion of the duct synchronous with secretion elicited by stimulation of
the chorda tympani.
185

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

186 | ' ROBERT GESELL

c. Backward injection into the salivary duct of the resting gland.

d. Effect of occlusion of the duct during secretion elicited by the stimulation
of the chorda tympani upon secretion elicited by subsequent stimulation of the
chorda tympani and vago-sympathetic.

_ e. Effect of backward injection in the resting gland upon subsequent secretion
elicited by stimulation of the chorda tympani or vago-sympathetic before and
after atropinization.

Le EFFECTS OF INCREASED SALIVARY PRESSURE UPON THE ELECTRICAL
DEFLECTIONS OF THE GLAND

a. Occlusion of the duct during secretion elicited by the injection of
pilocarpin. When the submaxillary gland is activated by the injection
of pilocarpin a definite electrical deflection occurs and when the secretion
approaches a constant rate the two electrodes tend again to assume a
constant difference of potential as is evidenced by the horizontal direc-
‘tion of the recorded electrical deflection. If the salivary duct is now
occluded a typical disturbance of this equilibrium occurs which is
shown in figure 1, A, B,C, Dand E. The first effect of occlusion was
an upward PeAneticn which gave way in a few seconds to a downward
deflection. De-occlusion produced the reverse effect. The downward
deflection accompanying occlusion was suddenly accelerated and
changed as suddenly into an upward deflection. The contour of the
deflection differed considerably from time to time and from animal to
animal as is obvious from figure 1, A, B, C, D and E, yet the four phases
were present in all. The electrical deflection elicited by occlusion and
de-occlusion of the duct may therefore be looked upon as more or less
accurately indicating the sequence of certain glandular po set
up by these procedures.
b. Occlusion of the salivary duct synchronous with secretion elicited
by the stimulation of the chorda tympani. If the chorda tympani is
stimulated at regular intervals with stimuli of equal strength and dura-
tion equal amounts of saliva may be elicited and electrical deflections
of the same contour may be obtained, provided, the period of rest inter-
vening between stimulations is of such duration as to prevent the aug-
menting effect of previous excitation. When such constant results
were obtained it was found that occlusion of the salivary duct along
with stimulation of the chorda tympani produced a definite change
in the electrical deflection as is seen in figures 2 and 3. These figures
show five sets of observations—2, A, B and C, and 3, A and B. In
any single set of observations the chorda tympani was stimulated at

EFFECTS OF INCREASED. SALIVARY PRESSURE 187

equal intervals of time with equal strength of stimulation and the duct
occluded equal lengths of time. But in the different sets of observations
the periods and strength of stimulation, the periods of rest and of
occlusion of the duct were variable. This variability must in part

WHEE tbe

PEM WILLE PEEUELT ELD EP EE

ees es ee ULU LLL WITT TT TP tet

Fig. 1

account for the differences of the deflections. Bearing the variability
of the conditions of the experiments in mind it is quite remarkable
that the changes in contour produced by occlusion of the duct should
be as uniform as they are.

‘188 ROBERT GESELL

Fig. 2

EFFECTS OF INCREASED SALIVARY PRESSURE 189

3A

Fig. 3

190 -ROBERT GESELL

Prolongation of the electrical disturbance is a point more or less
common to the observations in which occlusion of the duct occurred.
This prolongation is associated with a prolongation of the normal period
of secretion. Other characteristics common to many of the modified
deflections are the notch and the elevation of the base line. To be
sure there are some dissimilarities in the deflections of different groups
of observations, yet in any single set of observations repeated occlusion

produced changes in the deflections so nearly alike that here again the |

deflections seem to indicate with considerable exactness the sequence
of glandular processes. | |

In some instances de-occlusion of the duct produced a downward
deflection similar to that associated with de-occlusion during secretion
from the injection of pilocarpin. But it is obvious that the deflection
as a whole showing the effects of occlusion and de-occlusion cannot be
compared to advantage with the deflections obtained by occlusion and
de-occlusion during secretion from the injection of pilocarpin, for in
one instance the deflection is a result of a change in secretion already
in progress and in the other it is a resultant of the activation of the
resting gland and the obstruction of the secretion formed.

c. Backward injection into the salivary duct of the resting gland. ‘The
backward injection of saline or gum-saline produced deflections com-
parable to those elicited by occlusion of the duct during active secretion
elicited by the injection of pilocarpin. Such deflections are shown in
figure 1, F, G and H. The injection produced an upward deflection
followed by a downward deflection. Cessation of the backward in-
jection was associated with a downward deflection followed by an up-
ward deflection. The general contour of the deflection, to be sure,
differs from that produced by occlusion during active secretion, yet
- the four phases are present. It is of interest to note here that atropini-
zation apparently does not influence the effects of backward injection
upon electrical deflections.

2. EFFECTS OF INCREASED SALIVARY PRESSURE UPON THE VOLUME-FLOW
OF BLOOD THROUGH THE GLAND

a. Occlusion of the salivary duct during secretion elicited by the in-
jection of pilocarpin. Occlusion of the salivary duct during secretion
resulting from the injection of pilocarpin as a rule retarded the flow
of blood during the period of occlusion, as is shown in figure 5, A. On
de-occlusion the flow accelerated again to reach or surpass the flow

——- =

AG

Seen ee

i ee ee

—

EFFECTS OF INCREASED SALIVARY PRESSURE 191

preceding occlusion. The de-occlusion flow of blood in figure 5, A,
only approximated the pre-occlusion flow, but in consideration of the
fact that the volume-flow of blood as a rule is proportional to the
activity of the gland and of the fact that the de-occlusion secretion
was considerably slower than the pre-occlusion secretion, the results
suggest that even in this observation occlusion in reality produced an
accelerated after-flow of blood. |

Fig. 4

b. Occlusion of the duct synchronous with secretion elicited by stimu-
lation of the chorda tympani. The above results are substantiated by
the universally accelerated after-flow of blood noted on de-occlusion
of the duct after occlusion synchronous with secretion elicited by stimu-
lation of the chorda tympani (see figs. 4 and 6). A comparison of
record B of figure 6, in which occlusion of the duct occurs, with records
A -and C, in which there was no occlusion of the duct, shows the ac-
celerated flow of blood. The results of two such experiments are
plotted in figure 4 in which secretion (solid black) and volume-flow
of blood (single line) are plotted on the ordinates against time in minutes

ROBERT GESELL

192

Leo teat ee

4

y

EFFECTS OF INCREASED SALIVARY PRESSURE 193

on the abscissas. The duration of stimulation of the chorda tympani
is marked by the rectangle near the abscissas at the beginning of the
record. The extra-flow of blood elicited when the duct was occluded
greatly exceeded the extra-flow occurring when the duct was not oc-
cluded. Though the two experiments represented in this figure show
little reduction of the flow of blood during the occlusion of the duct, such
slowing not infrequently occurred, in agreement with the results pro-
duced by occlusion of the duct during secretion elicited by the injection
of pilocarpin.

c. Backward injection into the salivary duct of the resting gland. Back-
ward injection into the salivary duct of the resting gland produced
comparable results to those obtained by increasing the salivary pressure
in the two ways discussed above (see fig. 5, B and C). During the
period of increased salivary pressure the flow of blood was greatly
reduced. On cessation of injection, which is indicated by the salivary
record, the volume-flow of blood accelerated to remain accelerated for -
several minutes above the basal-flow of blood preceding injection.

3. EFFECT OF INCREASED SALIVARY PRESSURE UPON SECRETION

a. Occlusion of the duct during secretion elicited by the injection of
pilocarpin. WDe-occlusion of the salivary duct after occlusion during
secretion elicited by the injection of pilocarpin was followed by a
momentary rapid acceleration of secretion which within a few seconds
usually gave way to a rate of secretion below that obtaining before
occlusion (see fig. 5, A). How much of the momentary accelerated
secretion was due to the emptying of distended ducts, to the passage
of saliva which escaped into the tissue spaces back into the ducts, to
the liberation of secretion accumulated in the cells themselves due to
failure to overcome the increased salivary pressure in the ducts, the
experiments do not show. The retarded secretion following de-occlu-
sion suggests some form of tissue damage resulting, possibly, from back-
ward filtration. The effect which occlusion of the duct has'upon the
rate and amount of secretion following de-occlusion depends largely
upon the duration of occlusion, the rate of the pre-occlusion secretion
and the variability common to the glands themselves. Some saliva is
always unaccounted for in the compensatory secretion. |

b. Occlusion of the duct synchronous with secretion elicited by stimu-
lation of the chorda tympani. Occlusion of the salivary duct during
stimulation of the chorda tympani followed by subsequent de-occlusion

194 ROBERT GESELL

produced variable results, depending again upon the rate of secretion
elicited by stimulation, the duration of occlusion of the duct and the
peculiar reaction of the gland itself. Various results.from several
animals are shown in figures 2 and 3. The records in each case are
arranged in their proper sequence. Figure 2, A and B, which is com-
piled from two different animals, shows the effects of prolonged occlusion
of a copious secretion and a momentary occlusion of a scant secretion.
In figure 2, A the chorda tympani was stimulated at regular intervals
of 4 minutes with a constant strength of stimulation lasting 16 seconds.
With the duct unobstructed each stimulation elicited 16 drops of secre-
tion as is shown in the first record of that series. In the following two
records where the duct was occluded for 2.5 minutes 13 drops of saliva
were secreted following de-occlusion, that is, only 3 drops of secretion
were lost. In the final control when the duct was unoccluded 16 drops
were again elicited. In the following record, 2, B, a scant secretion of
only 3 drops of secretion was elicited by a relatively weak stimulus

lasting 5 seconds. The duct was occluded only 10 seconds. It will

be noted that occlusion of such scant secretion for only 10 seconds
resulting in a loss of about 13 per cent of the secretion and producing
a definite change in the electrical deflection, stands in striking contrast
to the loss of only 3 drops out of a larger total of 16 drops of secretion
obstructed for 2.5 minutes. The results in figure 2, B indicate that
the storage capacity of the ducts of a gland weighing approximately
7 gm. may be little over 2 drops and that if an amount greater than
2 drops is obstructed, enough back pressure may be developed to inter-
fere with further secretion. If that is true, where were the 13 drops of
the experiment represented in 2, A stored?

c. Backward injection into the salivary duct of the resting gland. An
index to the capacity of the ducts as it pertains to this problem may
be indicated by the after-flow of secretion following backward injection
of gum-saline in the resting gland. When a small amount of fluid
was injected and then retained by occlusion of the duct, subsequent
de-occlusion within 40 to 80 seconds was usually followed by several
drops of after-flow. Not infrequently there was no after-flow what-

ever; but the usual flow of 2 to 4 drops suggests again that the ducts.

may accommodate, for a short time at least, approximately 3 drops
of saliva. : |

d. Effect of occlusion of the duct during secretion elicited by the stimu-
lation of the chorda tympani upon secretion elicited by subsequent stimu-
lation of the chorda tympani and the vago sympathetic. The effects of

*
— se eS a

SE Sn

EFFECTS OF INCREASED SALIVARY PRESSURE 195

occlusion of the duct during secretion elicited by the stimulation of the
chorda tympani upon secretion elicited by subsequent stimulation of
the chorda tympani and vago sympathetic are shown in figure 6. This
figure gives the results of two sets of observations, A-D and W-Z.
The record of the first set shows secretion and volume-flow of blood;
the record of the second set electrical deflections as well. The chorda
tympani was stimulated at regular intervals with stimuli of equal
strength and duration. Equal amounts of secretion were elicited with
each stimulation under normal conditions. During one such period
of stimulation the salivary duct was occluded and the effect upon the
subsequent stimulation noted. In record A a total of 12 drops of
saliva was secreted. Since 3 of these drops were secreted slowly after
the cessation of stimulation, only 7 drops were rapidly secreted during
the period of stimulation. In record B the duct was occluded during
stimulation of the chorda tympani and several minutes after stimulation
the duct was de-occluded whereupon only one drop of saliva fell from
the cannula. Next, record C, the chorda tympani was stimulated with
the duct de-occluded; 13 drops of saliva were secreted, of which 11
drops appeared within the 10-second interval of stimulation. In record

_D, the final control, 6 drops of saliva were secreted rapidly. It follows

from these results that occlusion of the duct during secretion increased
the subsequent secretion by 4 or 5 drops.

The augmenting effect of occlusion upon secretion is shown still
better in observations W, X, Y and Z, due to the fact that after-secretion
was absent. In record W stimulation of the chorda tympani for a

‘period of 10 seconds elicited 4 drops of saliva. In record X the duct
was occluded during stimulation. A-rapid flow of blood occurred. —

There was no flow of saliva on de-occlusion of the duct. In the follow-
ing record, Y, stimulation elicited 8 drops of saliva or double the amount
in record W. In the final control approximately 3 drops were again
secreted. Note the electrical deflections.

The effects of occlusion of the salivary duct during secretion elicited
by stimulation of the chorda tympani on subsequent secretion elicited
by stimulation of the vago sympathetic appear to be identical with
those just described (see fig. 7). The lower broken line on which time
is marked in seconds indicates when the chorda tympani was stimulated
and the upper line when the vago-sympathetic was stimulated. The

results of alternate stimulation of the chorda tympani and vago-sympa-

thetic without occlusion of the duct are shown in the upper tracing;
with occlusion, in the lower tracings. Without previous occlusion of

196 ROBERT GESELL

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198 ROBERT GESELL

the duct stimulation of the vago-sympathetic elicited a slow scanty
secretion of only 3 drops, but with previous occlusion a secretion of
8 drops.

—_——$—

SERRE pt

Fig. 8

e. Effect of backward injection in the resting gland upon subsequent
secretion elicited by the stimulation of the chorda tympani or vago-sympa-
thetic before and after atropinization. When equal amounts of saliva
were elicited at regular intervals by equal stimulation and gum-saline
was then momentarily injected into the duct, it was found that stimu-

RS ar PSR: ie: ; +535 Seah

EFFECTS OF INCREASED SALIVARY PRESSURE 199

lation of the chorda tympani 1 to 5 minutes following the injection

elicited a greater amount of secretion and that the latent period of

secretion was reduced by. several seconds. The same reduction in
latent period was noted when stimulation of the chorda tympani was
preceded by occlusion of the duct during secretion elicited by stimulation
of the chorda tympani.

The experiment represented in figure 8 shows the effect of atropin
upon the results described above. In record A, the first stimulation
of the chorda tympani elicited 3 drops of saliva. Following that stimu-
lation 1 mgm. of atropin was injected intravenously. This effectively
paralyzed the chorda tympani for the prevailing stimulus, as is shown
by the absence of secretion with the second stimulation. Gum-saline
was then injected backwards through the duct into the gland and the
duct de-occluded within a minute. The next stimulation elicited 2

drops of secretion though the subsequent stimulation was ineffective.

In record B, a continuation of the experiment, the chorda tympani and
vago-sympathetic were simultaneously stimulated producing 2 drops
of secretion. The accelerated volume-flow of blood shortly following
was a result of the injection of 11 mgm. of atropin. Stimulation of the
vago-sympathetic following that injection in record C was ineffective
so far as secretion is concerned, but reduced the flow of blood. In
record D, gum-saline was injected into the duct between the breaks on
the time record. One drop left the cannula on de-occlusion. Subse-
quent stimulation of the vago-sympathetic yielded 4 drops and final
stimulation none. Similar results have been obtained after the intra-
venous injection of 350 mgm. of atropin.

DISCUSSION

From the work of Langley we know that during rest some of the
constituents of salivary secretion are elaborated and stored ready for
subsequent secretion. It appears that even after the glandular cells
are well stocked with zymogen granules, secretion may yet occur in
two definite stages, final elaboration followed by ultimate liberation.
Various observations point in that direction, e.g., the observation of
Langley on the augmenting effect of stimulation of the chorda tympani
upon the secretion elicited by subsequent stimulation of the sympathetic
fibers. Stimulation of the chorda tympani too weak to elicit secretion
may produce a definite electrical deflection. In the preceding paper
it was noted that a gland subjected to a subnormal flow of blood failed

200 ROBERT GESELL

to secrete during the period of stimulation of the chorda tympani,
whereas it secreted 20 to 30 seconds after cessation of stimulation when
the flow of blood through the gland was suddenly accelerated. The
observation in the present paper, that a gland may continue to secrete
for a period of 4 or 5 minutes after the cessation of stimulation, as is
shown in the de-occlusion experiments, is likewise of significance; as
is also the fact that injection of atropin in amounts sufficient under
usual experimental conditions to paralyze the secretory endings of the
chorda tympani, fails to prevent a discharge from the gland on stimu-
lation of the chorda tympani after a Prenieue injection of gum-saline
into the duct of the gland.

Unfortunately we do not understand the reaction of the gland to
increased salivary pressure. The fact that a storage of more than 2
drops of saliva in the ducts embarrasses the gland and that 13 drops
of saliva may be secreted after the duct is occluded for a period of 2.5
minutes indicates that at least not all of the after-secretion following
de-occlusion is due to an expression of saliva contained within the dis-
tended ducts. The slowness of the secretion would also speak against
this. Yet we have some evidence pointing to the contractility of the
salivary ducts; for example, when the gland is secreting at an even rate
as a result of an injection of pilocarpin, a short stimulation of the chorda
tympani results in a momentary acceleration of secretion followed by
a compensatory slowing which in turn gives way to a rate approaching
the initial rate. ,

We know that when secretion occurs against a high resistance much
of the saliva is filtered backward into the interspaces of the gland.
The extreme slowness of secretion in certain instances following de-
occlusion of the duct suggests the passage of this saliva back into the
ducts. So far as I know we have no evidence that an increased salivary
pressure results in filtration of saliva back into the secreting cells them-
selves or prevents the liberation of saliva which is already in these cells.

Since ‘‘secretion’”’ may be elicited following backward injection after —

atropinization sufficient to paralyze secretion, there is the possibility
that under more normal conditions fluid is filtered backward into the
cells, this fluid being later liberated. A part of the excess fluid within
the cells may be liberated by a passive mechanism on the reduction
of the salivary pressure accompanying de-occlusion, but the passive
mechanism may not be sufficient to liberate all of the excess fluid or
secretion and this may then be actively liberated upon stimulation of
the chorda tympani. It seems not improbable that this active libera-

- igi SS

EFFECTS OF INCREASED SALIVARY PRESSURE 201

tion may be the result of active contraction of certain constituents
of the secreting cells themselves. This problem needs further investi-
gation and will be reported upon later. The results so far indicate
only that atropin in certain stages of atropinization paralyzes primarily
the function of elaboration of secretion leaving the function of liberation
more or less intact. :

As to the nature of the changes in volume-flow of blood produced by
increased salivary pressure we can say that they are not of a central
reflex origin if all the vasomotor nerves to the gland run in the chorda
tympani and vago-sympathetic, for these changes in flow occurred after
section of both nerves. The decreased flow obtaining during the period
of increased pressure may well be a mechanical effect of compression
of the capillaries and venules, for it obtains during stimulation of the
chorda tympani when the factors normally producing dilatation are
at work, as well as when the gland is at rest. On de-occlusion of the
duct the compressing pressure vanishes and the flow of blood not only
returns to normal.but greatly exceeds the normal. When this acceler-
ated flow was first noticed with occlusion accompanying stimulation
of the chorda tympani it was thought that possibly more energy was
required for secretion against a high pressure and that the accelerated
flow of blood supplied this needed energy. The accelerated flow result-
ing from backward injection into the duct of the resting gland is con-
tradictory to this view. The accelerated flow does not seem to be due
to a specific chemical irritation of the saliva itself for it occurs after
injection of inert solution, such as saline and gum-saline. Whether a
backward filtration into the secreting cells would bring about increased.
volume flow of blood we do not know. The only suggestion we have
at present to account for the accelerated flow is the tissue damage
resulting from backward filtration into the tissue spaces and Asai
into the cells themselves.

SUMMARY

Occlusion of the duct of the submaxillary gland of the dog during
secretion elicited by the injection of pilocarpin produced characteristic
electrical deflections.

Injection of gum-saline into the duct produced somewhat comparable
deflections.

Occlusion of the duct along with stimulation of the chorda tympani
modified in a more or less typical way the usual electrical deflection
obtained by stimulation when the duct was not occluded.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 1

202 ROBERT GESELL

Occlusion of the salivary duct during secretion elicited by the injec-
tion of pilocarpin retarded the flow of blood. The after-flow of blood
was at times accelerated.

Backward injection of gum-saline into the salivary duct of the resting
gland retarded the flow of blood during the period of increased pressure.
Release of that pressure resulted in an accelerated flow of blood at
times lasting many minutes.

Occlusion of the duct during secretion elicited by stimulation of the
chorda tympani frequently retarded the flow of blood. De-occlusion
was followed by a markedly accelerated after-flow of blood.

De-occlusion of the salivary duct during secretion elicited by the
injection of pilocarpin was followed. by a short period of accelerated
secretion. This accelerated secretion never compensated fully the
absence of secretion during the period of occlusion.

De-occlusion of the duct several minutes subsequent to injection of
gum-saline into the duct was followed by an after-flow from the duct
of 0 to 4 drops.

Occlusion of the salivary duct along with stimulation of the chorda
tympani was followed upon de-occlusion by a secretion less in amount
than that normally elicited with the duct unoccluded. The amount
of saliva unaccounted for varied considerably with the animal, the
duration of occlusion and the amount of secretion obstructed.

De-occlusion of the duct after obstructing for several minutes a
temporary secretion elicited by stimulation of the chorda tympani may
be followed by a slow secretion lasting 4 to 5 minutes.

Occlusion of the duct during secretion elicited by the stimulation
of the chorda tympani increased the amount of saliva elicited by subse-
quent stimulation of the chorda tympani or vago-sympathetic. The
latent period of secretion was also reduced by several seconds. je

Baekward injection of gum-saline into the resting gland increased
the amount of secretion el cited by subsequent stimulation of the chorda
tympani or vago-sympathetic and reduced the latent period of secretion.

Injection of atropin sufficient to check secretion elicited by the stimu-
lation of the chorda tympani or vago-sympathetic may fail to prevent
secretion with similar stimulation after backward injection into the
salivary duct.

If all the nerve fibers reaching the sabre aesllacsy gland run in the
chorda lingual and vago-sympathetic the changes in volume-flow of
blood resulting from increased salivary pressure are not of a central
reflex origin.

EFFECTS OF INCREASED SALIVARY PRESSURE 203

The decreased volume-flow of blood during the period of increased
salivary pressure is probably due to mechanical occlusion of the capil-
laries or venules.

It is suggested that the accelerated flow of blood following de-occlu- |
sion is a result of tissue damage from backward filtration into the tissue
spaces, and possibly into the cells themselves.

The effects of occlusion of the duct upon secretion elicited by stimu-
lation of the chorda tympani suggests that secretion may occur in two
definite stages. The effects of atropin on augmented secretion follow-
ing backward injection of gum-saline into the duct suggests the same
inference. :

“STUDIES ON THE SUBMAXILLARY GLAND

VIII. On THe Errects oF ATROPIN UPON VOLUME-FLOW OF Boop,
ELECTRICAL. DEFLECTIONS AND OXIDATIONS OF THE
SUBMAXILLARY GLAND

ROBERT GESELL

From the Departments of Physiology of the Washington University Medical School,
St. Louis, and the University of California, Berkeley, California

Received for publication July 27, 1920

When the chorda tympani is stimulated definite effects are produced
in the submaxillary gland among which are: secretion of saliva, an
augmented flow of blood, a change in the electrical condition of the
gland and an accelerated oxidation. This paper considers briefly some
of the effects of the intravenous injection of atropin upon these processes.

The work is a continuation of the study of electrical deflections of
the submaxillary gland and has a bearing upon the problems of vaso-
motor control. In paper II of this series (1) the nature of the electrical
deflections elicited by stimulation of the chorda tympani was discussed. _
It was pointed out that such factors as the type of lead, the strength
and duration of stimulation, the period of rest, etc., all influence the
kind of deflection obtained. It is essential then to keep these factors
in mind in connection with the effects which atropin produces upon
electrical deflections elicited by stimulation of the chorda tympani.

We know from the work of others the effect which atropin has upon
secretion of saliva. These effects, when noted in this work will, there-
fore, be discussed only in their connection with the changes in volume-
flow of blood, the electrical condition of the gland and oxidations.

Atropin influences the electrical response of the gland profoundly
‘ as was shown by Bayliss and Bradford (2). I have obtained like results.

Figure 1 shows the effect of the injection of 1.5 mgm. of atropin.
Record A, shows the effects of stimulation of the chorda tympani with
both vago-sympathetics intact before the injection of atropin. Record
D, shows the effects of stimulation after double vago-section and atro-
- pinization. Records B and C, show the effects of section of the left
and right vago-sympathetics respectively. Atropinization abolished

204

EFFECTS OF ATROPIN 205

1 emake

CIM MM Mn A MMM MMMM MRT

206 ROBERT GESELL

both the secretion and the electrical deflection elicited by stimulation
of the chorda tympani, and from records B and C, it is apparent that —

PPP EPEC POURING

PoE ED VIM

a

QF EEE EU UU
Co sre wor

Le ic Mi

I Se LE md leds sna
Ce

SET TLE RRR ee
Fao = ~

TTT Law
ieee ag 26
if

MU UL TUTTE ee

Fig. 2

volume-flow of blood per se was of little if any significance in the de-
velopment of electrical changes. In record A, where the volume-flow
of blood was least affected the largest electrical deflection occurred, and

EFFECTS OF ATROPIN 207

in records C and D, where the flow was enormously accelerated, both
passively and actively, there was practically no change in the electrical
deflection. .

Figure 2 shows the effects of repeated injections of small amounts of
atropin, each injection occurring several minutes before stimulation
of the chorda tympani. The amounts injected are noted on the record
—the upper figure represents the amount injected prior to stimulation
and the lower figure the total amount injected at that moment. In
record A, before the injection of any atropin, stimulation of the chorda
tympani elicited a copious secretion, a copious flow of blood and a
large electrical deflection. In record B, after the injection of 1 mgm.
of atropin, similar stimulation elicited approximately the same. flow
of blood but the secretion was reduced to only 4 drop and the electrical
deflection was nearly abolished. The injection of another milligram
of atropin before record C, although exerting no further visible effect
upon the volume-flow of blood, paralyzed secretion and abolished the
electrical deflection. The subsequent records followed the injections
of 4, 7, 9 and 50 mgm. of atropin respectively. The electrical response
of the gland continued to be absent, at least the deflections which oc-
curred during the period of stimulation of the chorda tympani were
no greater than those which occurred during the periods of rest.’ At
times the accelerated flow of blood resulting from stimulation of the
chorda tympani was reduced by the injection of atropin as is apparent
from a careful comparison of recordsA and B of figure 3. It is of interest
to note in connection with figure 2 that although the accelerated super-
basal flow of blood was slightly reduced, the after-flow was greatly
prolonged so that the sum total of the effects was a greater superbasal
flow for equal stimulation after the administration of atropin. Note
that in the lower records the basal flow of blood is considerably ac-
celerated. The acceleration was not due to a rise of blood pressure.
It occurred after section of the chorda tympani and the vago-sympa-
thetic and therefore was peripheral in origin.

Figures 3, 4 and 5 show results of other experiments. In figure 3,
the administration of 0.08 mgm. of atropin nearly paralyzed secretion
for the prevailing stimulus; only 1 drop was elicited as compared with
6 drops before atropinization. It likewise reduced the superbasal flow
of blood. The electrical deflection was still very distinct. In figure
4, taken from a different animal, a similar injection abolished secretion
entirely. The electrical deflection was still prominent and the super-
basal flow of blood during the period of stimulation was hardly affected,

208 ROBERT GESELL

0.03 M+

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EFFECTS OF ATROPIN

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210 « ROBERT GESELL

but the prolongation of the superbasal flow of blood occurred again.
Further injections abolished the upward deflection, but it will be noted
that stimulation of the chorda tympani then produced~a very small
downward deflection. Though atropin usually abolished the electrical
deflection this reversal was not an uncommon occurrence; but as a rule
it was of small magnitude and occurred even after. ange ne of
30 or more milligrams of atropin.

EA nie cy | ag

a;

boobs OF ARIUS 1

ee ea

Fig. 6

If the injection of atropin prevents the development of an electrical
deflection when the chorda tympani is stimulated, it might be logical
to expect the injection of atropin to abolish an electrical deflection which
is already in progress as a result of a continuous stimulation of the

chorda tympani. Figures 6 and 7 show the results obtained on this -

point. In record A, figure 6, the chorda was stimulated before atropini-

|

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IMNNTNCATENT TANTO TTOTTATOATNSATTTT TTT ga fT

Zz
io
Ay
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en
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HTH Et HEE HH HH FH

212 ROBERT GESELL

zation. At the cessation of stimulation a sharp downward deflection

occurred. In record B, atropin was injected during the period of stimu-
lation. When the atropin reached the gland the same sharp downward
deflection occurred, although the stimulation of the chorda tympani
continued. In figure 7, record B, where the atropin reached the gland
at relatively the same time interval as cessation of stimulation in record
A, the electrical deflections were nearly identical. From the electrical
deflections one might conclude, neglecting the volume-flow of blood,
that atropinization during stimulation of the chorda tympani and
cessation of stimulation call forth the same effects. |

It is obvious that we know too little about electrical phenomena in
living tissues to arrive at such a definite conclusion, yet I have tried in
an indirect way to put this conclusion to the test. It should be pos-
sible to grade the strength of stimulation, or perhaps more correctly
stated, the end effect of stimulation, in two ways—mechanically by
regulating the strength of shock delivered by the induction coil, and
physiologically by reducing the effectiveness of stimulation of constant

strength by the injection of graded doses of atropin. A comparison —

of the electrical, secretory and vasomotor response of the gland with
these two methods of gradation of stimulation seemed worth while
attempting. The results obtained on two animals are shown in figures
8 and 9. The chorda tympani was stimulated at regular intervals with

stimuli of equal duration. In the first observation in both experi-

ments the stimulation was too weak to elicit visible secretion. The
strength of stimulation was then increased with each observation up
to an arbitrary maximum. That maximum was then kept constant
for the remaining series of observations, but prior to each stimulation

small amounts of atropin were injected, as small as 0.05 mgm. Each ~—

increase in strength of stimulation elicited an increase in the amount

of secretion and a decided change in the electrical deflection. After —
the maximum strength of stimulation was reached each injection re- *
duced the amount of secretion and elicited just as decided changes in ~
the electrical deflection. The records show the results obtained and —

hardly need a lengthy discussion. To be sure, the corresponding
deflections obtained with increasing and decreasing activation as indi-
cated by the secretion are not superimposable, yet if we carefully
compare the tracings of figure 8 keeping three factors in mind—the
magnitude of the upward deflection, the reversal of the deflection to a
downward deflection, and roughly, the general contour—definite simi-
larities in the deflections of the two series appear. Considering the

EFFECTS OF ATROPIN 213

fact that identical deflections can be obtained only when many factors
remain perfectly constant and that two series of deflections—one ob-

3 eee ie ee Se fy omy eet Bint a Be a ee ee eee se a ee ¢ 5 . 3 Oe = ede
EL ale Seg oT eS ee Fra ieee ee ot ela oe 3 ic ie ea
i = = a moe a eo ee 7 = oe —--2

Be Be ek Ea Rae a

POPE TREE

Fig. 8

tained with increasing strength of electrical stimulation and the other
with decreasing strength of electrical stimulation—show dissimilarities,
as demonstrated before, it is significant that the two series of deflections
as obtained in this research have as many points in common as they do.

214 ROBERT GESELL

If the electrical method detects minute changes in metabolic activity
we should be forced to conclude that a small injection of atropin may
abolish visible secretion as normally elicited by stimulation of the
chorda tympani, and yet permit activation of the gland as is indicated
by the marked electrical deflection which may occur in the absence of
visible secretion. This conclusion agrees with the later work of Bar-

PH feet be oh Wee oe

Fig. 9

croft (4) on oxidations in the submaxillary gland after atropinization
and is in agreement with his theory of metabolite control of volume-
flow of blood. Another conclusion we should be forced to draw is that
larger injections of atropin may abolish all metabolic effects which the
chorda tympani has upon the submaxillary glands. This conclusion
agrees with the earlier results of Barcroft (3) on oxidations in the sub-
maxillary gland after atropinization. If correct, it reduces the impor-

EFFECTS OF ATROPIN 215

tance of metabolite control, this control then only complementing the
control by the vasomotor nerves.

In some experiments in which I have measured the oxidations of the
gland as affected by stimulation of the chorda tympani after the injec-
tion of 3 to 5 mgm. of atropin (5), I found that although occasionally
oxidations were increased as much as 15 per cent above the oxidations
during rest, as a rule, oxidations were not increased by stimulation of
the chorda tympani. (The Van Slyke method was used.) This amount
of atropin usually abolished the electrical deflections. Oxidations were
not studied after smaller injections. It would appear that the amount
of atropin administered might markedly influence the results.

We know that living tissues, such as the secreting submaxillary gland,
the thyroid gland, the kidney, and a nonglandular tissue such as muscle,
exhibit electrical changes when their supply of blood is markedly inter-

fered with. The inference might be that the electrical deflection is

due to disturbed oxidation. In keeping with this inference is the
observation that the metabolism of injured tissue is higher than that
of normal tissue; on this basis the current of injury has been attributed
to greater oxidations at the point of injury.

The delicacy of the electrical method of detecting metabolic activity
has been demonstrated in many ways by Waller (6) in his researches
on the Signs of Life. Yet it must be pointed out that a lack of change
in electrical condition of a tissue need not necessarily indicate an absence
of change of metabolic rate, for we know that a symmetrical structure
such as the web of the frog’s foot does not give an electrical deflection
when excited symmetrically, whereas the single layer of skin of the back
of the frog gives a large deflection. The magnitude of the deflection
does not always vary in direct proportion to the rate of salivary secre-
tion; in fact, occasionally the reverse happens. Apparently a balanced
action of two effects of stimulation comes into play. There is, however,
no evidence that symmetry of structure or perfectly balanced effects
come into play more after atropinization than before.

BIBLIOGRAPHY

(1) GeseLu: This Journal, 1919, xlvii, 411.

(2) Baytiss AND Braprorp: Proc. Royal Soc., 1886.

(3) Barcrort: The respiratory function of the blood, 1914, Cambridge Univer-
sity Press.

(4) Barcrorr: Journ. Physiol., 1901, xxvii, 31.

(5) Gmsexti: Not published.

(6) WauutzER: The signs of life, New York, 1903.

Pe

-

AlT.,

: THE AMERICAN ?
_ JOURNAL OF PHYSIOLOGY
Bp vOL. so - DECEMBER 1, 1920 No. 2

THE DISTRIBUTION AND QUANTITATIVE ACTION OF THE
VAGI AS DETERMINED BY THE ELECTRICAL CHANGES
ARISING IN THE HEART UPON VAGUS STIMULATION

E. W. H. CRUICKSHANK
From the Physiological Department of Washington University, St. Louis

Received for publication July 3, 1920

In a survey of the literature upon the vagus nerves, one can not but

_- be struck by the fact that so many workers in this field have paid so

little attention to a clear discrimination between the right and left
vagi. General conclusions have been drawn, with regard to vagus
activity, which are applicable only to the left vagus, and in the earlier
publications no attempt even was made to differentiate between vagus
and accelerator nerves, so that functions peculiar to different nerves
have been assigned to both vagi.

The idea that the vagus nerves may or may not exert contralateral
effects was not put to the test of experiment till 1911, when Garrey (1)
carefully considered the matter, using the whole and partially split
heart of the turtle and recording his results graphically. His con-
clusions, which are very definite, are as follows. He asserts first, that
the origin of the beat is to be found in the right caval veins, the inherent
rhythmicity of which he showed to be greater than that of the sinus

and auricles and then he shows that, in the whole heart, the right vagus

affects chronotropically, through its action on the right veins, every
part of the organ, while the left vagus may affect the whole, including
the right veins, but that usually it affects the left veins, the sinus and
the auricles, producing quiescence by decreasing the excitability, con-
ductivity and contractility of the auricles, leaving the rhythm of the

@ right veins unaffected. In the “ascidian preparation” of the turtle’s

heart, in which the beat travelled from right to left, very clear con-
clusions were arrived at, namely, that the right vagus stopped the whole
217

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 2

218 E. W. H. CRUICKSHANK

preparation, while the left inhibited the left auricle and right auricle,
the right basal veins still continuing to beat at their original rhythm.

This, according to Garrey, is due to the fact that the left vagus does -

not affect the pace-maker, but acts upon structure similar to that upon
which normal cardiac impulses act. The essential point determined
is that the vagi act chronotropically on their respective sides, upon those
parts which initiate the rhythm. This is the basis of the homolateral
view of the action of the vagi, which has been so strongly brought for-
ward by Garrey who, however, showed that such effects were not so
clearly marked with regard to the auricles, and he states “that the
experiments bring out clearly that in auricles as well as in veins and
sinus the vagus effects may cross to the contralateral side.”

Robinson and Draper (2), from an investigation of the action of the
vagus nerves upon the human heart, corroborate previous findings on
the mammalian heart, namely, that the predominant action of the
right vagus is a control of rate, through its inhibitory action upon the

normal pace-maker of the heart, and that of the left nerve is primarily |

a control of conduction from auricle to ventricle, through a direct
inhibitory effect upon the conducting system and that in gauging the
difference upon conductivity of the right and left vagi, the factor of
heart rate must be taken into account. Again, with regard to auriculo-
ventricular dissociations, it was shown that these are not caused by
diminution in the conductivity, but are essentially due to the inherent
high rate of rhythmicity of the ventricles, dissociation occurring as
soon as vagus stimulation reduced the auricular rate below that at
which the ventricles will contract by their own inherent rhythmicity.
This viewpoint is supported by experiments of Rothberger and Winter-
berg (3) who demonstrated true nodal rhythm by stimulation of the
right vagus and left accelerator nerves.

Cohn (4) in 1912 stated that negatively chronotropic effects may be
obtained upon stimulation of the left vagus and he made this significant
statement ‘that it is doubtful whether the distribution that has been
described is as refined as is necessary to explain the results” he obtained.

With regard to the importance of heart rate in gauging dromotropic
effects of the vagi, attention is drawn to a paper by Robinson (5) in
1916, in which he shows that there is no constant difference between
the right and left vagus nerves in their action upon conductivity be-
tween the auricles and ventricles during auricular fibrillation.

The first report upon the electrical changes in the heart due to vagus

stimulation was made by Gaskell (6). The type of experiment which

. ‘ = 7 ee
I — a Oe ee

VAGUS ACTION ON HEART 219

he describes depends upon the peculiar arrangement of the vagus nerve
in the tortoise heart, where a branch of the vagus runs with the coronary
vein from the sinus to the base of the ventricle. This “coronary”
vagus being free, the sinus and left auricle could therefore be cut off
without damage to the nerve supply of the right auricle, thus allowing
of a quiescent preparation. A demarcation current having been pro-
duced by ‘‘thermic section” and the right vagus stimulated, a deflection
was obtained in the same direction as that of the injury current, indi-
cating increased positivity of the uninjured part. These experiments
were carried out by means of a d’Arsonval galvanometer. _

Gotch (7) in 1887, using a capillary electrometer, could not obtain
this vagus effect, the position of the meniscus, upon vagus stimulation,
remaining at the position obtaining during diastole. It was suggested
by Burdon Sanderson (8), during a discussion of this subject, that the
capillary electrometer was probably not sufficiently sensitive to detect
such slight changes of potential as had been detected by Gaskell by
means of a much more sensitive instrument.

Einthoven (9) in 1908 criticised Gaskell’s results and doubted their
accuracy because, by means.of the string galvanometer he was unable
to detect any changes of potential whatsoever. That Gaskell was
correct was clearly demonstrated in 1911 by Meek and Eyster (10),
who with an Edelmann galvanometer, using the tortoise heart prepared
according to Gaskell’s method, obtained uniformly positive results.
Theirs was an exceptionally clear corroboration of the Gaskell phe-
nomenon, as evidenced by a rapid deflection of the string and its slow
return to the original position. If the heart were beating, this pro-
longed slow fall of the string to its original level would be cut short
by the first beat following upon inhibition and the return of the base
line to the zero position would be rapid. That this happens in the
beating heart was shown by Samojloff (11), who used the same type
of instrument. The hearts of decapitated frogs were utilized, an injury
current was produced by the application of a drop of 1 per cent KCl
to the apex of the heart, and leads taken, one from the apex the other
from an uninjured part of the heart. A monophasic variation was
obtained with each beat, and upon stimulation of the right vagus the
zero line was deflected in the direction of the injury current; the deflec-
tion was slow and emphasis was laid upon the fact that the return of
the string did not begin until a contraction supervened.

220 E. W. H. CRUICKSHANK

PART I
Electrical changes associated with the action of the-vagi

The fundamental principle involved in determining these changes
depends upon the following acceptation. |

With a demarcation current or injury current. In normal muscle
excitation is evidenced by an increased negativity of the part involved
and electrodes can be so arranged that such negativity gives rise to an
upstroke of the string of the galvanometer. When in the heart an
injury current is produced, the injured surface becomes electrically
negative to the uninjured part and the difference of potential thereby
occasioned gives rise to a deflection of the string in a direction opposite
to that which denotes excitation in normal muscle. A wave of excita-
tion passing over such a field would produce at the positive or unin-
jured part a decrease of its positivity with respect to the injured
spot and therefore cause a rise in the direction of, but smaller than,
that due to a normal contraction. If the vagus nerve be stimulated,
its effect is to reduce the condition of negativity and therefore relatively
increase the positivity, the result being a deflection of the string in
the same direction as that of the injury current.

In the normal or uninjured condition. To obtain monophasic vari-
ations from the action of the heart muscle, one electrode must be placed
on the heart, the other on a part of the body wall sufficiently removed
from the heart to allow of the activity of all parts of the heart muscle
being marked by an upward deflection. If now the electrical changes
of the heart, due to its activity, are not totally inhibited, these will

show themselves as upstrokes of reduced amplitude, arising from a

zero line at a lowered level.

It may be assumed that whatever occasions the beat produces a
sudden catabolic change, a change dependent upon a previous building
up of excitability or, to use Gaskell’s term, a process of assimilation.
There is no reason to suggest that such a process of assimilation should
be of such a sudden nature as that of the catabolic discharge or dis-
similation; the deflection caused by the one may be wholly different
from that caused by the other, the factor chiefly concerned being that
of velocity. It is supposed that these anabolic changes are inaugurated
by vagus action and therefore that electrical stimulation of the vagus
increasing these, it should be possible, using a galvanometer of sufficient
sensitivity, to detect the changes in potential arising therefrom.

Pe ee ee eT ee

LE ERO TE ENE pate RE Ses CTE oe

VAGUS ACTION ON HEART 221

During inhibition the heart suffers an alteration of its excitability
and its activity is depressed. It may therefore be feasible to accept
the interpretation of Samojloff’s curves, that contractions may be

resumed at an increased level of anabolism, provided the process has

reached completion and stopped, as otherwise it is difficult to conceive
of contractions supervening during a process which is essentially of
the nature of an inhibition. If the building up of excitability or the
inhibitory process has not reached its maximal development, then,
to explain the breaking through of heart beats, one must assume that
the vagi and the contractions act upon different mechanisms in the
cardiac muscle. Gaskell (12), McWilliam (13), (14), and Roy and
Adami (15) state that excitability is diminished during inhibition but
all that they can prove is, that the heart in inhibition was inexcitable,
and that, during a period when it was establishing a necessary condition
such that the subsequent stimulus should produce a contraction, that
is, a condition of excitability. Therefore their diminished excitability
is associated with what may be regarded as a refractory period of
inhibition. Just as there is a refractory period in catabolism so one
postulates a similar condition in anabolism. This would support the
idea suggested that the upstroke is indicative of the true inhibitory
period, i.e., the time till maximal deflection is reached, after which the
excitability of the tissue is such that stimuli may be effective, which
stimuli, however, may be delayed over a longer or shorter period,
according to the degree of diminution of conductivity, which is a mani-
fest effect of both vagi. :

‘The results of the experimental work to which reference has just
been made, demonstrate clearly the occurrence of electrical changes
in the heart during vagus stimulation. These changes then may be
employed to determine the distribution of the vagi in the heart, to
demonstrate the action of these nerves upon its various parts and to
decide if possible by what means vagal impulses are propagated. To
do this a preparation of the whole and also of the partially split heart
has been used.

Experiments carried out.with the d’Arsonval galvanometer

From previous work done, using the string galvanometer for deter-
mining the electrical changes occurring in the heart upon vagus stimu- .
lation, it was found that, in the normally beating heart, the string
galvanometer is too sensitive an instrument to withstand the action

222 E. W. H. CRUICKSHANK

current of the heart, when the string is slackened to that extent which
is necessary to give definite evidence of the positive variations. With
sensitivities such that 1 m.v. gives deflections from 5 to-10 em., the
results are in many cases not convincing. It was therefore determined,
seeing that it was impossible to carry out these experiments upon a
quiescent heart, to utilize a very sensitive d’Arsonval galvanometer
and, by means of a rheotome, to place it in circuit with the heart only,
during the very brief period of its quiescence between beats.

Methods

The rheotome. This consisted of a brass segmented wheel having
one continuous central contact and two, one on either side of the former,
in which there was placed a different length of fiber, so that by adjust-
ment of these, any length of non-conducting material could be readily
obtained. Thus by using three contacts, the two external being con-
nected, a definite period of time could be obtained during which the
-galvanometer could be thrown into the circuit. This period was chosen
so that the ventricular, auricular and sinus effects could be eliminated.
The circumference of the rheotome was 500 mm., the gap was, after
experiment, cut down to 45 mm.

The quiescent period of the heart. To throw the galvanometer into
circuit at this point of the cardiac cycle, a simple make and break
device was arranged whereby the relaxation of the ventricle completed
the circuit between six storage cells and a solenoid, which lifted the
catch checking the rheotome wheel. This allowed the wheel to rotate,
the retaining pawl being so placed that immediately it was lifted upon
ventricular relaxation, the galvanometer was put in circuit with the
heart. The rotation of the segmented wheel was so arranged by a
switch upon the power table, that one revolution was just completed
within the period of the cardiac cycle. The pawl, which was dropped”
upon contraction breaking the circuit, stopped the wheel for a very
brief moment, depending upon slight variations in the heart rate.

It was found that in the majority of cases it was necessary, in order
to obtain a quiescent period, to cool the heart, because, in a heart
beating at the rate of thirty per minute, the sinus was in action some-
times during a ventricular contraction, or following so closely upon
ventricular contraction that the rheotome method was rendered of
no avail. The sinus was cooled by means of a blind perfusion cannula,
through which was maintained a continuous flow of ice-cooled water

a ee a een

VAGUS ACTION ON HEART 223

of a temperature of about 10°C. The cooled point was maintained
upon a definite spot on the sinus, a spot previously determined as being
the seat of highest rbythmicity. :

The sensitivity and calibration of the galuanometer. The d’Arsonval

_ galvanometer used in these experiments was the type “R” of the Leeds

& Northrup Company, which had a sensitivity of 5 X 10-9 amperes
per mm. and a voltage sensitivity of 0.5 mm. per microvolt, with a
period of 5 seconds. Calibration of the instrument, with the rheotome
- running, gave readings for 0.1 m.v. of 7.8 cm. in 5 minutes and a return
to 0.5 em. from zero in 6.0 minutes. Therefore a deflection of 7.3.
em. = 0.1 m.v. and is equal, without the rheotome to a deflection of
22cm. From this it is seen that to obtain full values with the rheotome
method, it would be necessary for the vagus effect to last 5 minutes.
From the rate of rise of the curves and from their magnitude it will
be seen that the “‘vagus’ effect is a very marked one, is maintained
and is dissipated quickly by subsquent beats.

A difficulty which arises with the rheotome method is, after vagus
stimulation, to cut out the prolonged beats of the auricle which usually
encroach upon the quiescent period of the heart, even abolishing it,
and so cause a sudden return of the beam of light, which may reach
its original position or pass beyond it in two or three steps. One can,
however, by operating the rheotome with a key, cut out the auricular
beats occurring immediately after inhibition, the solenoid circuit being
broken upon the first sign of movement in the basal veins.

Compensation of the injury current. With a very slowly moving coil
galvanometer, with the time of stimulus very short, namely 0.18
second when the rheotome was making thirty revolutions to the
minute, the response to small currents may be so small as scarcely to
be noticed. In compensating, therefore, one must accurately gauge
the period of the heart cycle in which no electrical effects from auricular
beat are allowed to encroach upon the period of quiescence. The
sinus effect is so small that deflections caused thereby can be ignored,
but small inclusions of auricular effects would simulate over-compen-
sation. This, as well as the opposite effect’ of under-compensation,
must be guarded against, because the latter, if not accurately gauged, .
will give deflections in excess of those due to vagus stimulation, since
a deflection denoting an increased positivity is an extension of that of
the injury current.

Type of reading obtained. It is essential, if maximal deflections are
to be obtained, to stimulate the vagus, maintaining the quiescence of

224 E. W. H. CRUICKSHANK

the heart, till there is no further increase in the amplitude of the deflec-
tion. This may necessitate a vagus stimulation up to from 60 to 80
seconds, although maximal deflections are usually obtained well within
this period. The type of reading resulting from this method is shown
on the tracings, these records having been made by a device of Gesell
(16), in which the beam of light can be followed and the curve recorded
upon a moving drum. The record is in the form of a series of steps
which are of varying sizes and are in the form of curves as shown in
several tracings. It was not practical to follow accurately these fine -
swinging movements of the beam of light. The first few initial steps
are steep; they then rapidly become less and less in size till, at the
plateau of the curve, the small pendular movements synchronous with
each revolution of the rheotome only are in evidence. The plateau
may be maintained for a longer or shorter period and is, unless the
rheotome is controlled by hand, suddenly terminated by the first
auricular contraction.

Type of reading without the rheotome. In the partially split heart,
where the wave of contraction sweeps from right to left, it is of course
possible to prevent auricular or ventricular effects of the side under
observation, from affecting the galvanometer, but it has been found
that after 2 or 3 hours, when the activity of the heart has become
considerably lessened, the ventricle has little effect upon the galvano-
meter, the sinus none at all and the auricular contractions show as
small deflections, with a total amplitude of from 1 to 1.5 em. With
such a weakly contracting heart, beating at about sixteen per minute,
the mean of these deflections can be taken, because the beats are usually
so weak that any cumulative effect takes a considerable time to show
any alteration in the mean level of the beam of light. Thus the changes
occurring upon vagus stimulation are quite easily discernible. !

The whole heart

Table 1 shows that the right vagus is always markedly active upon
the right auricle and in many cases very slightly less so upon the left
auricle. The point of note with regard to the left auricle is that, in
about a fourth of the cases, the right vagus is more effective than the
left, and generally the left vagus is always less marked in its action upon
the left auricle than is the right vagus upon the right auricle, while
upon the right auricle the left vagus is, with three exceptions, decidedly
weaker in action than either the right or the left vagus acting upon

VAGUS ACTION ON HEART 225

its respective side. The tracings reproduced here as figures 1 to 5,
with attached explanatory notes, illustrate the basis upon which these
conclusions rest. All were obtained through the d’Arsonval galva-

nometer.
; | TABLE 1
Results with the d’Arsonval galvanometer, with an injury current
RIGHT VAGUS LEFT VAGUS
REMARKS
Right | Left Left | Right
auricle | auricle | auricle | auricle
cm. cm. cm. “cm.
1 | 4.5| 3.5] 5.5 | 3.0 |Heart cooled. Rheotome rate 24 revolutions
per minute .
2 | 7.5] 5.0] 4.0| 3.5 | Heart quiet with muscarine; no rheotome
3 | 4.5 | 4.0
4 10.5 | 5.5 | Heart rate 16 per minute
11.5 | 5.0 Temperature 10°C.
5 9.0| 8.0 Without rheotome; heart quiescent; right auri-
8.5 | 9.5 cle cut away
8.5 | 110
9.0} 9.5 :
6 | 2.5 1.0 | Gaskell’s preparation; coronary vagus intact
4 1.5 0.5
: aur 9 0.8} 1.0 ]- Sinus cut off from right auricle; coronary vagus
a Ol T.6 intact; left auricle quiet for short periods
ais 8 7.8 | 8.8 Heart rate 15 per minute, temperature 10°C.
a 9 8.5 | 6.0 Heart rate 20 per minute; heart cooled
_ 6.5 | 5.5 hy
10 | 6.5| 6.5 | 4.5 | 3.5 | Heart rate 20 per minute; heart cooled
a 11 6.5} 5.0} 4.0 | 4.5 | Heart rate 20 per minute; heart cooled -
. 12 | 6.2}| 5.5| 4.5 | 5.0] Heart rate 20 per minute; heart cooled
iY 13 | 3.5} 2.5] 3.3 | 2.8 | Heart rate 20 per minute; heart cooled
i 14 |} 5.5] 3.0] 2.5 | 2.0 | Heart rate 20 per minute; heart cooled
‘i 15 |} 2.0 2.0 Heart completely split :
. | 3.5 2.0 Heart completely split :
+ 2.0 1.0 |
: 17 | 4.8 3.3 Heart completely split
if 4.0 2.5
,: 18 | 5.5| 5.5] 4.2] 4.0 | Heart cooled
4 19 | 2.2} 1.8] 1.9] 1.6 | Heart cooled
q 20 | 2.5 2.0 | Heart cooled
fe 21 | 3.3] 2.0} 3.2,| 2.0 | Heart cooled
? 22 | 2.1} 1.0| 2.2 | 0.6 | Heart cooled
P! reer hee Pe Te 0.9 | Heart cooled
a 24 | 1.6 0.8 | Heart cooled
rt 25 | 2.9| 2.0} 1.9] 1.2 | Without the rheotome
e 26 2.6|/ 2.3] 2.1 | 2.0 | Without the rheotome

226 E. W. H. CRUICKSHANK

JEST LEO UDADURIDEOSSENGULEPOUAGUTIOIVIOIEUINEL/ER EEE IUINCHOORIOEEET

Fig. 1. Whole heart; right auricle, injury current; right vagus stimulation.
This is a typical result of the rise and fall during the quiescent period of the heart.
Here the only part of the curve not due to vagus activity is the first downstroke
occasioned by the commencing auricular beat. The vagus was stimulated for
36 seconds, the deflection obtained being 3.2 cm.

Fig. 2. Whole heart; right auricle, injury current; left vagus stimulation.
This record shows the steady rise step by step indicating increased positivity
of the left auricle. The inhibition of the left side was maintained for a period of
58 seconds, while stimulation of the vagus lasted for 60 seconds, producing a
maximal deflection of 2.6 cm.

VAGUS ACTION ON HEART 227

}
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————__———.

ead ISEERESUECERUSRUROURRESUCUDDCUUGHUCECUULEULODECUDRUUDNOSHOLESETUID!

Fig. 3. Whole heart; left auricle, injury current; left vagus stimulation. This
is a very good example of the steady step-like movement of the beam of light.
The vagus was here stimulated for 36 seconds, the duration- ch the rise was 24
seconds and the maximal deflection was 2.7 cm.

Fig. 4. Whole heart; left auricle, injury current; left vagus stimulation.
This tracing is of interest in showing the effect of one beat of the heart breaking
through in a short inhibition, which is then followed by a steeper and longer rise
giving a total deflection of 3.2 cm. Stimulation of the vagus was continued for
64 seconds and there was no apparent sign of shifting of the electrodes or. tempo-
rary stoppage of stimulation to account for the single beat.

228 E. W. H. CRUICKSHANK

agen Shin, H————

LAuR Whole He
3- ANaqus

Fig. 5. Whole heart; left auricle, injury current; right vagus stimulation,
The total deflection of 2.0 cm. was obtained in 30 seconds. The rise and fall are
both typical, the fall below the original base line being due, probably, to a dimi-
nution of the demarcation current.

Fig. 6 Fig. 7

Fig. 6. Partially split heart; rheotome; right auricle, injury current; right
vagus stimulation. In this case the injury current was not compensated. The
vagus acted for 12 seconds and gave a maximal deflection of 2.6 em.

Fig. 7. Partially split heart; rheotome; right auricle, injury current; right
vagus stimulation. Here the pendular movements were followed as accurately
as possible. This record shows exactly the type of movement which is performed
by the beam of light upon the scale, as the galvanometer responds to every brief
impulse which it receives with each revolution of the rheotome. The inhibition
was maintained for 26 seconds and the rise was completed in 15 seconds, the total
deflection being 2.3 cm.

VAGUS ACTION ON HEART 229

The partially split heart ©

With the rheotome. The type of results obtained from the partially
split heart when the d’Arsonval galvanometer is put into circuit by
means of the rheotome is seen in figures 6 and 7 and table 2, and de-
scribed in the legends to the figures.

Types of deflections obtained without the rheotome. Using the rheotome,
the results have, in the case of the action of the vagi on the contralateral
sides of the heart, been invariably negative. In fact there is to be seen
a slow movement of the beam of light in the direction opposite to that

TABLE 2

Results with the d’Arsonval galvanometer, with an injury current; split heart

RIGHT VAGUS LEFT VAGUS

REMARKS
Right | Left Left | Right
auricle | auricle | auricle | auricle

cm. cm. cm. cm.

Ot em These readings correspond with those obtained

0 previous to sagittal section. Therefore (?)
os iid faulty section

2
3
4
5
6
7
8
9
10
11
12

indicating a positive variation, due either to a diminution of the injury
current or to the effects of weak contractions from parts of the heart,
other than the auricle under obsevation. The best and most decisive
results in the partially split heart have been obtained, without the
rheotome, 2 to 4 hours after opening the pericardium and about }# hour
after making the sagittal section, because by this time the Heke run-
ning from right to left, have.lost. much of their vigor and rapidity.

The current of injury is not: compensated and from the tracings (figs.
8 to 13) it will be seen, that the movements, due ‘to contractions, are
very small indeed. oo

Oks,

Fig. 8. Partially split heart; without rheotome; right auricle, injury current;
left vagus stimulation. The movements of the beam of light were successfully
followed in this case and it will be noted that there is neither inhibition of the right
auricular beat nor alteration of the base line due to, a stimulation of the left
vagus lasting 52 seconds.

Fig. 9. a, b, c. Partially split heart; without rheotome; right auricle, injury
current; right vagus stimulation. These three are typical of results obtained
without the use of the rheotome. The height and rapidity of the deflections are
comparable to those obtained by Gaskell, and show a definite electro-positive
change. The deflections have a maximum of 3.8, 4.0 and 3.3 cm. with a time of
3.0, 4.0 and 3.5 seconds, respectively. The tendency, even in a weakly beating
heart, for the ‘‘vagus’’ effect to be repeated, is seen ‘in these tracings.

230

VAGUS: ACTION ON HEART 231

PUGUGs ORG eGe Ree eeeeeeeemeneuell

Fig. 10. Partially split heart; without rheotome; left auricle, injury current;
left vagus stimulation. The action of the left vagus upon its own side, as graph-
ically shown here, is similar to that obtaining in the whole heart. The response
is rapid and the time taken to attain a maximal deflection of 3.8 em. is 4.0 seconds:

VUUEUSEDUE A UTIDITUUU EE UC TUEUCCCCCUUUCe (DEELO SN CEEIEAUDEDUGTUERPULEOE NE |

Fig. 11. Partially split heart; without rheotome; left auricle, injury current;
left vagus stimulation. This shows activity of the left vagus upon the left
auricle three and one-half hours from the commencement of the experiment.
The pendular movements are due to the right auricular beats.

232 E. W. H. CRUICKSHANK

1, Aum
RVaqus
abit aD

Fig. 12. Partially split heart; without rheotome; left auricle, injury current;
right vagus stimulation. The survival of the left auricular contractions in the
partially split heart, upon right vagus stimulation, is clearly seen here. The
more rapid beats due to the right auricular rhythm are seen at the beginning of
the tracing and their almost immediate inhibition upon right vagus stimulation
is clearly shown, the slower left auricular rhythm remaining. That in this
‘‘ascidian’’ preparation the right vagus has no effect upon the uniform position
of the base line, which is the mean of the auricular deflections, is demonstrated
in this experiment, where the right vagus stimulation lasted for 58 seconds.

Annn

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eee annem cmeneticenineasininisetienamnnpeens nescence

UUOLULUUUUENCOUMOGROQUNUNURURTTNURRUNCDDRUOQUNU LOE

Fig. 13. Partially split heart; without rheotome; left auricle, injury current;
right vagus stimulation. The fact that the right vagus has no action on the left
auricle in the partially split heart is again shown here. After the deflection of
the injury current was obtained, the movements of the beam of light were fol-
lowed and the right vagus stimulated for 60 seconds. No alterations in the base
line took place. The right auricular contractions were inhibited and the latter
half of the tracing shows only the left auricular deflections, which with their
slower rhythm come into evidence toward the end of the tracing. This occurs
after a period of stand-still of very brief duration, during which time the inherent
rhythmicity of the left auricle becomes established.

‘
4
: 4
4
if
"4
4
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VAGUS ACTION ON HEART 233

PART II
The quantitative effects of the vagi *

The foregoing results with their demonstration of crossed vagal
effects suggested a quantitative study of these.

To determine what quantitative vagus changes may occur on both
sides of the heart the graphic method was used and the curves plotted
on codérdinate paper. As the rheotome revolved at a rate of either
twenty or thirty revolutions per minute, and as both vagi, in most of
the experiments, completely stopped the heart, the repeated contacts
were made at regular intervals, thereby giving one a means of comparing
the rate of deflection of the beam of light step by step, each step denot-
ing a definite period of time during wh‘ch the galvanometer was in
circuit with the preparation. To arrive at some idea as to the intensity
of the vagus action, it is necessary to compare the deflections, using
as a standard a definite period of time during which the nerves are
stimulated, and to do this the most convenient method is to take a
number of steps or a number of revolutions of the rheotome, but not
more than that required by the lowest curve to attain its maximal
deflection. Without the rheotome, to take a definite height as a stand-
ard and compare the time required to reach it, would be admissible,
but with the rheotome a standard height precludes any comparative
conclusions, because it manifestly gives the slower effect the advantage
of a greater number of contact periods.

The curves are plotted for the position of the beam of light either _
every 5 seconds or for every revolution of the rheotome; the abscissae
denote time in seconds and the ordinates deflections in centimeters.
The stimulation of the vagus, marked by a signal, was usually con-
tinued till the maximal effects were obtained, the time was recorded
by a Jacquet chronograph and the records’ were made by Gesell’s
method already referred to. The results obtained can best be presented

as descriptions of the curves.

Figure 14. Here the right vagus was stimulated for 36 seconds,
and the electrical change commenced 13 seconds after stimulation
began, and in 38 seconds the maximal deflection had been obtained.
The curve both in its gradient and height is typical of the right vagus
effect. In 15 seconds the beam of light had risen 2.9 em., after which
the change became more and more gradual, till 10 seconds later, the
maximal deflection of 3.3 em. was attained, where it was steady for
5 seconds and then fell at first quickly, later more and more slowly

EE Be Ta a 7

ee

VAGUS ACTION ON HEART 235

as the base line was approached. The right vagus effect upon the left
auricle shows a change commencing as soon almost as that of the right
vagus upon the right auricle. The gradient of the electrical change
is less marked though quite as definite as that of the homolateral effect.
The total stimulation lasted 52 seconds and the maximal height of 2

em. was reached in 30 seconds. The left vagus in this experiment had _,
a longer latent period but, like the right vagus, the period was prac- |

tically the same for both sides, 20 and 21 seconds respectively. This

is the case previously mentioned, in which a beat was interpolated 24 |

seconds after inhibition had been produced. The action current of
the heart caused an immediate return of the beam of light to just below

- zero, inhibition was again produced and in 5 seconds it is evident and

much more markedly so than in the previous rise. Comparing the
left vagus effect upon the right and left auricles, it is seen from this
figure that the second rise due to the left vagus is steeper than that
of the left vagus effect upon the right auricle. This is the usual result
obtained, though not without exception. The maximal rise and the

’ time taken for each vagus on both sides of the heart are as follows:

Right vagus acting upon right auricle for 18 seconds........ Pies tae 3.3
Right vagus acting upon left auricle for 30 seconds................ 2.0
Left vagus acting upon left auricle for 40 seconds.................. 3.2
Left vagus acting upon right auricle for 49 seconds............... 240

Figure 15. For comparative purposes the deflections occurring upon
each of five consecutive revolutions of the rheotome are plotted. The
marked ascendancy of the right vagus upon the right side of the heart
is well shown; also from the graph of the second rise of the left vagus
upon the left side, which has been traced in for comparison in the posi-
tion of the original rise, it can be seen that the very definite effect upon
its own side is greater than the right vagus effect upon the left both
in maximal deflection and in rapidity of its rise. The left vagus action
upon the right side is the least marked of all and this record, with the

_ second curve of the electro-positive change of the left vagus transcribed,

sums up very clearly the various effects occasioned by both vagi, each
acting upon both sides of the heart. |

Noting the deflections after five revolutions of the rheotome, the
heart being inhibited by both vagi and the time interval between the
stimuli being the same for all, namely, 2 seconds, the galvanometer
being in circuit with the heart for a period of 0.18 second, we have the
following results:

236 E. W. H. CRUICKSHANK
Zin
2
qm Naque. L Qu. (2 rise).

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SeCsiagdeae Gl a ak uae ak Se ce Okan eee ee

— © e o o——_
re) ° ° mania

Fig. 17

Vv

2 as eS

ET OTS NAS

SPSS HE A Sa i ee a ee a ae . . ,
SOAR, SAA SS ae aig Sey coca ae Pe Bi Bh oe nen Mea = Pen 2 i a Ba 5
- mae 1

VAGUS ACTION ON HEART |

Right vagus acting upon right‘auricle...... 2.6.0... 22 eee ee 1.9
Right vagus acting upon left. auricle................. 0 cece eee eee 0.9
Lett vague acting.upon left auricle... ... 22.2... cies eee eee eres Rl
Left vagus acting upon right auricle............... 0... cece eee ees 0.7

Figure 16. As in the preceding record, the greater latent period of
the left vagus as compared with that of the right is shown and here
the left vagus action upon its own side is very definite, especially with
regard to the velocity of the change, in which in this case it exceeds
that of the right vagus upon the right side. The slower contralateral -

effects, though of scarcely less magnitude are noteworthy, when one

recalls what has been suggested with reference to the anatomical dis-
tribution of the vagi. The maximal deflections and the time taken

- for their completion are as follows:

Right vagus acting upon right auricle for 25 seconds............... 2.2
Right vagus acting upon left auricle for 39 seconds................ 1.8
Left vagus acting upon left auricle for 14 seconds...... PET ETN Ss 1.9
Left vagus acting upon right auricle for 22 seconds................ 1.5

Figure 17. Comparing these effects in four steps of 24 seconds each,
with the exception of the left vagus upon the left auricle in which each
step equals 2 seconds, one sees from the record that they are very similar
to those in the preceding case. The right vagus was very active, having,
for both sides of the heart, a latent period of 2 and 3 seconds respectively.

‘The difference in the velocity of the change effected is not so marked

in the first 10 seconds as subsequently; yet the difference in the gradient
of the activities of the left vagus upon the left and the right auricle
is well marked. The latent period of the left vagus, while greater than
that of the right, is, comparable to the right vagus, practically the same
for both sides of the heart, namely 8 and 9 seconds for the left and
right sides respectively. In this experiment the stimulation of the
nerves lasted approximately 20 seconds in each case. The deflections
from five revolutions of the rheotome were as follows:

Right vagus acting upon right atiricle.. <.................00.5 0000. 0.9
ieht varus acting upon left auricle). Ji.5, 222. ee 0.7
ett vagus acting upon left auricle ...3)..)...% Ami ok ee ee 1.0
Left vagus acting upon right auricle.................... 00. eee e ees 0.6

Figure 18. This is arecord of the only instance in this series in which
stimulation of the left vagus nerve did not stop the heart beat but
caused a progressive slowing of the rate from 30 to.15 beats per minute

238 E. W. H. CRUICKSHANK

a dudes

Teael al
Bg
~~ -I4
a Le
line: BOD STE eae as 0,
6 Oe a ae => == Ze =< = a >
Fig. 18

R.Vaqus.:
-~ = — L.Qun

! \
| — L Vaqus, L.Qur.

i ate ti aaae Vagus,

77 Ea
j
ea
Ce ame | es a : + ia
ee eee

VAGUS ACTION ON HEART 239

in a period of five beats. After the sixth beat there was a pause of 7
seconds, the heart then became tumultuous, big, forcible contractions
of the auricles and ventricle, of long duration, following in no regular
sequence. After a series of three or four of these had passed, it was
again possible to place the galvanometer in circuit with the preparation
during the quiescent period, the duration of which became longer and.
longer till it was again encroached upon by a similar series of irregularly
timed, forcible, heaving contractions. While a similar alteration in
the rhythm was produced when leading from the right auricle and
stimulating the left vagus, the maximal effect was obtained in four
steps. The nerve was stimulated for 31 seconds and the maximum

‘reached in 12 seconds. The results for all four activities were as

follows:

j : cm.
Right vagus acting upon right auricle for 14 seconds...........:... 2.1
Right vagus acting upon left auricle for 10 seconds................ 1:2
Left vagus acting upon left auricle for 32 seconds.................. 1.6
Left vagus acting upon right auricle for 12 seconds................ 0.8

Figure 19. ‘These curves denote the positive variation obtained with

_ six revolutions of the rheotome, this number being that taken by the

smallest curve, left vagus acting upon right auricle, to reach its maximal
height. |

Right vagus acting upon.right auricle...... 2.0... io... ede eee cee ee 2.0
Right vagus acting upon left auricle................. 0000. cece eee 1.3
Left vagus otings-mmoe Olt BUricle |. ob. ig chip walle Ce bee cima e's 1.2
Left vagus acting upon Pree Suricle.. : os os cs See ees ees os 1.0

Figure 20. In this case the maximal deflections were recorded step
by step with the rheotome revolving once every 2 seconds. The vagi
produced complete stoppage of the heart in the times noted on the
chart and the deflections were remarkably regular in their rise and bear
out what previous records have shown, namely, that the right vagus
is the most predominant in action upon the right side of the heart,
the left vagus less so on the left side, while here the usually parallel
effects of the right vagus upon the left auricle and the left vagus upon
its own side, are clearly demonstrated. The deflections for fifteen
steps, that is for 30 seconds, were as follows:

Right vagus acting upon right auricle.................... ee eee eee 4.5
Pient veaeus acting upon left auricle... joes. wee. ee eee eens OO
7 ere vagun acim upon left auricles: oi seis w kes cee es be ce ee tes 3.6

Right vagus acting upon right auricle............ 0.0. cece eee ies 2.7

:
i
=
8
2
E
ei

240

2 vs

army ‘snbva'7 a3

a

any "7 ‘snbon i le:

om =m @

anny snbon y --4

amy y ‘subuyay

“Sw ~ “woyeeiiag

VAGUS ACTION ON HEART 241

Results without the rheotome. Figures 21 and 22. These records are
given as typical of the curves obtained with a practically quiescent
heart, in which deflections caused by electrical changes of weak con-
tractions could be disregarded. These results are from hearts which
have been bloodless for from 3 to 4 hours. The curves are plotted for
the position of the beam of light every second and the duration of the
stimulus is practically 10 seconds in all cases. One again sees the
dominant effect of the right vagus, with a latent period of 5 seconds and

WitKout Me
Rheotome-
‘2.
° R.Vaqus RGus, = ——
/ R. Vagus. L.Quun=—-——
/ L.Vagus. dun, = —-—-
| / \ L.Vaqus. R.Qur,=- —-o—
e
ey
1 |
af i /
5 |
‘ i* Ke
| }
& bi, \
a } / \
Time: Se : f A \ !
= emg acne) “pe Taame 9 ape

Fig. 21

_ 7 seconds for the respective sides of the heart in both cases. The maxi-

mal height is 2.6 cm. and 2.9 cm. respectively. The right vagus effects
upon the left auricles are remarkably alike in both cases, the time taken
to reach the maximum being 6 and 5 seconds, and the maximal height
attained being 2.3 and 2.0 cm., respectively. Such an effect, compared
with that obtained from the left vagus acting upon the left auricle,
shows the resemblance between the crossed effects of the right vagus
and the homolateral effects of the left vagus, with regard to the elec-

242 E. W. H. CRUICKSHANK

trical changes they produce in the left auricle.. The rapidity with which
the change is produced in the left auricle is greater for the left than
for the right vagus. From the latent periods and the heights of the
curves, one sees that the crossed effects are slightly less rapid in their
inception and in the velocity of their action. It is possible that the

ar

Wihout. Fe
Rheorone,

Fig. 22

latent period, in obtaining crossed effects, is directly proportional to
the degree of the contralateral distribution of the vagi. From the
usually short lapse of time, 1 to 3 seconds, occurring in the production
of the crossed effects, either by the right or the left vagus nerves, one
must conclude that, anatomically, the crossed distribution of each
vagus may, in many instances, be a liberal one.

ple

VAGUS ACTION ON HEART 243

With regard to the quantitative effects of the vagi upon the whole
heart, the results may be summed up in a table of the various effects
reduced to a percentage of the sum total:

Right vagus acting upon right auricle 40.9 26.6 35.0 36.3 31.2 30.0
Right vagus acting upon left auricle. 20.4 21.9 21.4 23.6 25.0 24.3

_ Left vagus acting upon left auricle... 23.6 30.1 29.5 21.8 25.0 25.7
Left vagus acting upon right auricle. 15.0 20.3 14.2 18.1 18.7 20.3

This gives an average result of:

Right vagus acting upon right auricle.................6..0005- 30 to 40

- Right vagus acting upon left auricle................0.....6--- 20 to 25
Left vagus acting upon left auricle..................020cce eee: 25 to 30
Left vagus acting upon right auricle.....................5.0.. 15 to 20

DISCUSSION

The experiments carried out with the d’Arsonval galvanometer
described in the first part of this paper, corroborate the work of previous
investigators with regard to the presence, in the heart muscle, of elec-
trical changes, which are brought about by, stimulation of the vagus
nerves. It has been shown that these changes can be produced by each
vagus nerve acting upon the opposite side of the heart. From the
figures quoted in table 1 and from records shown of experiments upon
the whole heart, it is evident that the action of the vagi is not mainly
homolateral. As far as the vagus effect is distinguished by an increased
positivity, by a change in potential, one must conclude that each vagus
nerve exerts its greatest effect upon its own side, and upon the opposite

_ side an effect which, generally, is definitely less than, but may, in the

case of the right vagus, be almost as great as that of its homolateral
action. In comparing the two vagi, it is seen from these results that
the right vagus produces a much greater contralateral effect than does
-the left; that the effect of the right vagus upon the left auricle is gener-
ally less than that of the left vagus upon its own side. In only two
cases did the left vagus acting upon the right auricle produce an effect
greater than that of the homolateral action of the right vagus. It is
seen that the contralateral effect of the left vagus is undoubtedly the
weakest or least marked of all four activities studied.

These results do not substantiate Garrey’s conclusions with regard
to the preponderantly homolateral effects of the vagi. There may be
a tendency to over-emphasize the homolateral view. In Garrey’s case
this may have resulted from his assumption that the beat originated

244 E. W: H. CRUICKSHANK

in the right caval vein and from the fact that he could find no chrono-
tropic effects thereon with left vagus stimulation. He states ‘that
the left vagus is less effective upon the normal rhythm of the heart
than the right vagus, due to the fact that it does not innervate the
right basal vein;’’ and again he states ‘that crossed effects can be
obtained, in some cases even to chronotropic effects, on the right veins
by action of the left vagus.”

If the left vagus can affect the center of rhythmicity in the left side
of the partially split heart, is it not, from these results and from Garrey’s
suggestion, justifiable to conclude that the left vagus can or may, as
a usual function, affect the pace-maker or center or centers of rhyth-
micity in the right auricle in the whole heart?

That such may be the case, more or less marked according to the
degree of crossed distribution, is strongly suggested by: the results
recorded above, in which, in almost every case, left vagus stimulation
quickly arrested the beat of the heart. As there were no evidences
of block one must conclude that this is due to an effect upon the center
of rhythmicity and that to stop the heart the vagus,must act upon
all heart tissue which is jnherently rhythmical. Thus only, in those
cases of complete stoppage, can left vagus inhibition be explained and
this point of view demands a contralateral distribution of both vagi.
With regard to the partially split heart, it is shown from the figures in
table 2 and from the tracings briefly referred to, that here only are the
effects of the vagi strictly confined to their respective sides.

This raises the question of the conduction of crossed effects which,
according to Garrey, occurs by means of nerve. In the turtle, the
crossed effects obtained in the whole heart must pass to the auricle
by way of nerve fibers located in the tissue of the sinus. If the vagus
effects were the result of a general conduction by means of both muscle
and nerve, ‘then, seeing that the muscle wave spreads around the
sagittally split heart from right to left, there should be no reason why
the vagus effect should not so pass.

In the partially split heart right vagus stimulation stops the whole
initially, then the left sinus assumes a controlling rhythmicity for the
left side, and if the right side be maintained in inhibition for some time,
the beats originating in the left sinus will travel from left to right, but
they will not pass from the ventricle to the right auricle. The passage
of such contraction impulses shows that no vagus control is exercised
upon the ventricle in the turtle. Also, it must be concluded that in
the partially split heart no vagus effects can be transmitted across from

—-

ESITE Ue shy ey

Pe ee a ae

SF EI ky TE Ee

VAGUS ACTION ON HEART 245

the right to the left side of the heart, since there is no alteration in
the electrical potential of the injury current of the left auricle when
the right vagus is stimulated. ;

In concluding that the ventricular muscle of the turtle plays no
part in transmitting vagus effects, it must be borne in mind that the
reason may not necessarily lie in the ventricular tissue but in the

inability of junctional tissue to transmit these effects. That vagus

effects do not spread by way of the ventricle would not preclude such
spread from right auricle to left auricle through cardiac tissue proper.
Erlanger (17) and Erlanger and Hirschfelder (18) have shown that,
after clamping the bundle of His in the dog and thereby producing
partial or complete heart block, it is possible to demonstrate some action,
though slight, of the vagus upon the ventricle. This goes to show that
vagal impulses to some extent travel by routes other than those which
subserve normal physiological conduction of the cardiac impulses.

That these impulses may not be associated with ordinary nerve fibers
is suggested by the work of Meek and Leaper (19), who show that
conduction in the heart, either by nerve fiber or by skeletal muscle,
manifests no marked difference in the degree of compression necessary
to destroy it. Garrey, in a study of the dissociation of inhibitory nerve
impulses, states, “that if normal physiological conduction from sinus
to auricle proceeds along nervous paths the blocking of these paths
should at the same time block other nervous paths, including all vagus
fibers which pass to the auricle through the clamped area, and through
this area only.”” He shows that this is not the case; that compression
sufficient to establish complete sino-auricular block, may not interfere
with the passage of vagus impulses. This would indicate that if vagus
effects are to be transmitted they must pass by way of tissue peculiar
to them. That such effects do not spread through muscle tissue is
proved by the larger effect upon the corresponding side in the whole
heart. This rules out the idea that junctional tissue may be the cause
of a non-transmittal of these effects in the partially split heart. _

From these results one is led to the conclusion that there is no case
for the spread of vagus effects by non-specific tissue, either muscular
or nervous, but only by tissue hypothetically peculiar to vagus con-
duction. It would seem, from the results both in the normal and par-
tially split heart, that for crossed effects in the whole heart, it is not
a case of strong or weak stimuli affecting muscle fibers or nerve network,
but rather a case of stimuli efficient enough to pass along paths of
vagus conduction, the degree to which contralateral function is served
depending upon the richness of the anatomical distribution.

246 E. W. H. CRUICKSHANK

With regard to the distribution of both vagi, evidence for a rich
contralateral supply to the heart is borne out by this work and in
support of this view reference has been made to the work of Cohn and
of Robinson. The latter investigator showed that in gauging the
action of the vagi upon cardiac conductivity, the rate of the heart had
to be taken into account. From this it is seen that, in judging both .
of chronotropic and dromotropic effects of the vagi, the factor of
greatest importance is the action of both right and left vagus nerves
upon the center or centers of rhythmicity.

If it is assumed that the vagus acts by reducing the general reactivity
of the tissue it happens to innervate directly, then the observation
frequently recorded in the literature that left vagus stimulation often
blocks the transmission of the contraction wave may be accounted for
upon the basis of differences in the distribution of the two vagi with
respect to the pacemaker. If in a given case the left vagus innervates
the whole of the base of the heart excepting the pace-maker while the
right vagus acts upon both parts equally, then stimulation of the left
vagus will reduce reactivity and consequently the conductivity of the
auricles, but not the rate of impulse initiation. The result would
simulate the diminution in conductivity. Stimulation of the right
vagus would produce the same reduction in reactivity and of the same
parts but at the same time would slow impulse initiation. As a result
the interval between successive impulse conductions might become
long enough to permit the subjacent tissues, despite their lowered
reactivity, to carry every impulse that came from the pace-maker. —
If the beats could be maintained at their original rate while the right
vagus was being stimulated, a diminution of conductivity similar to
that produced by left vagus stimulation would become manifest.

CONCLUSIONS

It is concluded that the positive variation of the demarcation current
that develops during vagus stimulation is a phenomenon, not due to, —
although usually associated with, stoppage of the heart. This is shown
by the facts:

1. That in the quiescent heart the electro-positive change is obtaitiedl

2. That no electro-positive change is obtained, when in the partiaily
split heart the left auricle temporarily ie beating upon right vagus
stimulation.

a

VAGUS ACTION ON HEART 247

Thus, looking at the question from two totally different standpoints,
we have clear evidence that the electrical change indicative of inhibition
is not occasioned merely by the cessation of muscle activity.

It is also concluded that in the turtle the distribution of the vagi
through the base of the heart is bilateral, but is not uniform in all
parts. In general the relative intensity of action is as follows: Right
vagus on right auricle > right vagus on left auricle = left vagus on
left auricle > left vagus on right auricle.

The greater tendency, noted in the literature, of stimulation of the
left vagus to produce block is not necessarily due to a selective action
of this nerve upon the conducting system; the result can be explained
quite as well through the relatively slight action of the left nerve upon
the pace-maker, which is located on the right side of the heart, while
the reactivity of the remainder of the heart is reduced.

I am greatly indebted to Doctor Erlanger for his continued interest
in this problem, and for much helpful and very suggestive criticism.

BIBLIOGRAPHY

(1) Garrey: This Journal, 1911, xxviii, 330.

(2) Ropinson AND Draper: Journ. Exper. Med., 1912, xv, 14.

(3) RoTHBERGER ABD WINTERBERG: Arch. f. d. gesammt. Physiol., 1911, exli,

343.

(4) Conn: Journ. Exper. Med., 1912, xv, 49.

(5) Roxpinson: Journ. Exper. Med., 1916, xxiv, 605.

(6) GaskeLu: Journ. Physiol., 1887, viii, 404; also Ludwig’s Festschrift, ‘‘Bei-.
_ trage zur Physiol.,’’ 1887, 114.

(7) Gorcu: Journ. Physiol., Proc., 1887, viii, 24.

(8) Burpon: Journ. Physiol., Peic.; 1887, viii, 26.

(9) ErntHoven: Arch. f. d. casedadit: Physiol., 1908, exxii, 517.

(10) Meex anv Eystser: This Journal, 1912, xxx, 271.

(11) Samosuorr: Zentralbl. f. Physiol., 1913, xxvii, 575.

(12) GasKxeuu: Phil. Trans. Roy. Soc., London, 1882, clxxiii, 993.

(13) McWituram: Journ. Physiol., 1885, vi, 192.

(14) McWititam: Journ. Physiol., 1888, ix, 167, 345.

(15) Roy anp Apamt: Phil. Trans. Roy. Soc., London, 1892, elxxxiii, 199.

(16) Geseuu: This Journal, 1919, xlvii, 1.

(17) Ertancer: This Journal, 1909, xxv, p. xvi.

(18) ERLANGER AND HirscHFeLpDER: This Journal, 1906, xv, 165.

(19) Merk anp Leaper: This Journal, 1911, xxvii, 308.

(20) Garrey: This Journal, 1911, xxviii, 249.

THE INFLUENCE OF GLANDS WITH INTERNAL SECRE-
TIONS ON THE RESPIRATORY EXCHANGE

I. Errect OF THE SUBCUTANEOUS INJECTION OF ADRENALIN ON
NORMAL AND THYROIDECTOMIZED RABBITS

DAVID MARINE anp C. H. LENHART

From the Department of Experimental Medicine and the ie a of Surgery,

Western Reserve University, Cleveland

Received for publication July 29, 1920

The demonstration by Asher and Flack (1), (2), (3) that stimulation
of the laryngeal nerves in rabbits with intact thyroids increases and
prolongs the rise in blood pressure following the intravenous injection
of a given amount of adrenalin has provided the first concrete evidence
_of a thyroid-adrenal relationship. The Goetsch (4) test in exophthal-
mic goiter and in clinical conditions resembling exophthalmic goiter,
as tuberculosis, cardiac neuroses, etc., is a practical diagnostic applica-
tion of Asher and Flack’s original observations. The effect of the
subcutaneous injection of adrenalin on the respiratory exchange in

man was studied in 1912 by Fuchs and Roth (5), who found a slight

increase in the oxygen intake and carbon dioxide output which they
looked upon as negligible and an increase in the respiratory quotient.
Later work by Bernstein (6), by Peabody and his co-workers (7), by
Tompkins, Sturgis and Wearn (8) and by Sandiford (9) have clearly
established that in normal men adrenalin injected subcutaneously in
0.5 ec. to 1.0 ec. (1-1000) doses causes an increase in the respiratory
exchange. The studies of Tompkins, Sturgis and Wearn and of Sandi-
ford are the more recent and extensive. The former, working with
soldiers, were able to demonstrate the increase in twenty-seven of
thirty-four cases. The latter studied forty-six cases including exoph-
thalmic goiter, simple goiter, myxedema, Addison’s disease and four
normals. She concludes that the subcutaneous injection of 0.5 ce.
1-1000 adrenalin invariably causes an increase in the oxygen intake
and the carbon dioxide output.

La Franca (10), working with dogs, found that phloridzin causes a
marked decrease in the respiratory exchange, while adrenalin causes a

248

\

— ee ee ee ae ee er
ee ee te eer hs ee re ge er tea

_

—

INTERNAL SECRETIONS AND RESPIRATORY EXCHANGE 249

marked increase both in the respiratory quotient and in the oxygen
consumption. Hari (11) using curarized dogs and injecting adrenalin
intraperitoneally observed a decrease in the oxygen consumption anda
rise in the respiratory quotient. Lusk and Riche (12), studying the
effect of adrenalin on the power of the animal to oxidize glucose, noted
an increase in the respiratory exchange in two normal dogs. Wilenko
(13) used urethanized rabbits and found no change in the respiratory
quotient or the oxygen consumption. :

In view of Asher and Flack’s observation it has seemed to us of
importance to compare the effects of the subcutaneous injection of
adrenalin in normal and thyroidectomized animals and also to compare
the effect on the same animal before and after thyroidectomy. The
results of these experiments are given in the following pages.

Method. Rabbits have been used because it is possible to remove
the thyroids without any physical interference with the function of
the ind parathyroids and also because accessory thyroid tissue is less
common than in dogs, cats or rats. We have used a Haldane (14)
apparatus, modified by substituting Williams’ absorbers and a motor-
driven pump. ‘This apparatus is readily adapted for the use of rabbits
and is simple and accurate.

All rabbits were deprived of food for 15 to 16 hours before beginning
the experiments. The adrenalin (P. D. Co., 1-1000) was not assayed.
The dose was arbitrarily fixed at 0.5 cc. per kilogram. Each experi-
ment consists of an hour period although in most instances the obsér-
vations were repeated two or more times without interruption. This
makes it possible to compare the results by hours or by longer units.of
time. 7
This study includes observations on six rabbits which had been kept
in the laboratory for several months under the same conditions before
being used in this work. Three rabbits (R 3-203, —-205, —206) were
“normal.” Three had had thyroidectomies—one (3-208) 51 days
before beginning the studies and two (3-201 and 3-204) during the
studies. The detail figures obtained in each of these animals have
been arranged in tables 2, 3, 4, 5, 6 and 7. For the brief discussion
which follows, only: the figures for the O, consumption per gram of
body weight per hour will be considered. These have been averaged
and arranged in table 1. :

a. Controls (before administration of adrenalin). The average. Os
consumption for the five rabbits with intact thyroids was 0.607,
0.572, 0.524, 0.477 and 0.436 gram per gram of body weight per:hour

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 2

250 ‘DAVID MARINE AND C. H. LENHART

before the administration of adrenalin. These figures might be termed
the basal rates and show the usual ‘‘normal’ variations for different
animals though the rate is relatively constant for a-given animal.
Rabbit 3-208 which had been thyroidectomized 51 days before begin-
ning the observation shows the ordinary effect of thyroidectomy on
metabolism first described in man by Magnus+Levy (16).

With rabbits 201 and 204 it is possible to compare the O2 consump-—

tion before and after thyroidectomy. The figures given in table 1
represent the first series of observations before thyroidectomy and the
last after thyroidectomy (80 and 31 days). The decrease in OQ, con-
sumption following thyroidectomy is striking. Reference to tables 6

TABLE 1

Average Oz consumption per gram per hour in cubic centimeters

ES BS ES Ex
<z2|<zZ2| 425 | 422
nie | SHS | eke | 88a

Picadas CONDITION OF THYROID dal 5 <8 e<s 58 o<39
"$H|o8H| "Sa | wSi
Boh | Sah | Bok | Bef
mom | Som | fom | Bow
& nD & &

203 | Intact | 0.607] 0.637] 0.697] 0.789] 0.680) 0.708

205 | Intact 0.436} 0.528) 0.549) 0.595 ;

206 | Intact 0.524!) 0.545} 0.605

208 | Thyroidectomy 51 days 0.355} 0.344|-0.417| 0.433) 0.457

204 || Intact 0.477) 0.651| 0.610} 0.530

204 || 30 days after thyroidectomy 0.348) 0.326) 0.498) 0.366

201 || Intact 0.572) 0.530) 0.681

201 || 31 days after thyroidectomy 0.402} 0.396} 0.502) 0.480

and 7 show that the decrease in the rate of metabolism following thy-
roidectomy is very slow—several days being required before the change
becomes manifest. This may be due to the fact. that the thyroid
hormone is very stable and is normally needed in exceedingly small
amounts. |
These observations are in harmony with the clinical observations that
the onset of symptoms of myxedema in thyroidectomized animals is

slow and also that there is usually a latent period of 24 to 48 hours.

following the feeding of desiccated thyroid before an increase in the
metabolic rate can be demonstrated. .They are at variance with the

effects on blood pressure as reported by Asher and Flack (loc. cit.) '

and by Levy (17). These authors showed that in acute experiments

“= s aein
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251

INTERNAL SECRETIONS AND RESPIRATORY EXCHANGE

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INTERNAL SECRETIONS AND RESPIRATORY EXCHANGE 257

lasting only a few hours the effect of adrenalin on blood pressure was
markedly decreased by thyroidectomy. The cause of these time
differences between the effect of adrenalin on blood pressure and on
metabolism in animals with and without thyroidectomy is not clear.

Ooper grm lst hour 2nd hour 3rd hour

per hour Control after 0.5cc after 0.5cc after 0.5cc

in cc, adrenalin adrenalin adrenalin
per kg.subc.per kg subc.per kg subc.

; 0.8

Ou?

oN ea

(2)
0.5

(3)

0.4

(4)

0.3

Fig. 1. Average O2 consumption per gram per hour in cubic centimeters.
1, Rabbit 203, thyroids intact; 2, rabbit 206, thyroids intact; 3, rabbit 205, thy-
roids intact; 4, rabbit 208, thyroids removed (51 days).

b. After adrenalin administration. In each case 0.5 ec. per kgm.
(P. D. adrenalin 1-1000) unassayed stock adrenalin was injected sub-
cutaneously in the flank. The observations were recorded in hourly
periods following the injection. In all instances a rise in the O2 con-
sumption was noted. The changes are shown graphically in text fig-

258 DAVID MARINE AND C. H. LENHART

ures 1, 2 and 3. It occurs as well in the thyroidectomized animals as
in “normals.” This is brought out best in figures 2 and 3 when the
effect of adrenalin before and after thyroidectomy may be compared in

O,per grm lst hour 2nd hour 3rd hour
per hour Control after 0.5cc after 0.5cc after 0.5cc¢
in CC. : adrenalin adrenalin adrenalin

per kg subc.per kg subc.per kg subc.

0.7

0.6
(a)

0.5

0.35

Fig. 2. Rabbit 201. Average O2 consumption per gram per hour in cubic
centimeters. a, Thyroids intact; b, thyroids removed (31 days).

the same animal. The percentile rise in O2 consumption appears to be
little changed by thyroidectomy. These results confirm Sandiford’s
observations in human myxedema. The adrenalin effect lasts for

hours. We have observed it for five hours, though usually the great-

INTERNAL SECRETIONS AND RESPIRATORY EXCHANGE 259

est increase is reached in the third hour. There is some evidence
that in the thyroidectomized animals the onset of the increased rate
of metabolism is delayed and also the reaction is of shorter duration as

Ooper grm : Ist hour end nour 3rd hour
per hour Control after 0.5ce after 0.5cc after 0.5cc

in CC. adrenalin adrenalin adrenalin
per kg.subc.per kg subc.per kg.subc.

0.7

0.6:

0.5
(a)

0.4

(db)
0.3 :

Fig. 3. Rabbit 204. Average O2. consumption per gram per hour in cubic
centimeters. a, Thyroids intact; b, thyroids removed (30 days).

shown in figures 2 and 3. Another observation on the effect of adre-
_ nalin in thyroidectomized animals is that frequently there is a decrease
in Oz consumption during the first hour after adrenalin. This was also
observed in ‘‘normals’”’ but very rarely.

260 DAVID MARINE AND C. H. LENHART

SUMMARY

Our results on rabbits confirm those of Sandiford on-man. Adre-
nalin causes a rise in the oxygen consumption both in normal and
thyroidectomized rabbits. The absolute rise may be greater in nor-
mals but the percentile rise may not be altered. Evidence is given
that in general the onset of the rise in O. consumption following adre-
nalin is delayed in thyroidectomized animals and also that it does not

last so long. Some evidence is presented showing that in rabbits as in

other animals the decrease in the metabolic rate following thyroid-
~ectomy is gradual and requires several days for its demonstration.
These results differ from those in which the it on blood pressure
was used as the indicator.

BIBLIOGRAPHY

(1) AsHEeR AND Fiack: Zentralbl. Physiol., 1910, xxiv, 211.

(2) ASHER AND Fuack: Zeitschr. f. Biol., 1910, lv, 83.

(3) AsHeR: Arch. gesammt. Physiol., 1911, exxxix, 562.

(4) Gortscu: Med. Rec., 1918, xciv, 567.

(5) Fucus anp Rot: Zeiteohs. Exper. Path. u. Therap., 1912, x, 187.

(6) BERNSTEIN: Zeitschr. f. Exper. Pathol. u. Therap., 1914, xv, 86. ;

(7) PraBopy, CLtoueH, Stureis, WEARN AND TomPKINS: Journ. Amer. Med.
Assoc., 1918, Ixxi, 1912.

(8) Tompxins, SturGIs AND WEARN: Arch. Int. Med., 1919, xxiv, 269.

(9) SanpiForD: This Journal, 1920, li, 407.

(10) La Franca: Zeitschr. f. Exper. Path. u. Therap., 1909, vi, 1.

(11) Hart: Zeitschr., 1912, xxxviii, 23.

(12) Lusk AND Ricue: Arch. Int. Med., 1914, xiii, 673.

(13) WiLENKo: Biochem. Zeitschr., 1912, xlii, 44.

(14) Haupane: Journ. Physiol., 1892, xiii, 419.

(15) Bootuspy AND SANDIFORD: Proc. ainer, Physiol. Soc., This Journal, 1920,
li, 200.

(16) Misuesane: Zeitschr. f. Klin. Med. 1897, xxxiii, 269.

(17) Levy: This Journal, 1916, xli, 492.

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STUDIES ON THE VISCERAL’ SENSORY NERVOUS SYSTEM

II. Lune Avuromatism aND Lune ReFLexes IN Reprinia (TURTLES:
CHRYSEMYS ELEGANS AND MALACOCLEMMYS LESUEURII.
SNAKE: EUTENIA ELEGANS) —

! A. J. CARLSON anp A. B. LUCKHARDT
From the Hull Physiological Laboratory of the University of Chicago

Received for publication August 3, 1920

LITERATURE

The first observations on the contractility of the reptilian lung on
direct stimulation of the pulmonary tissue was made by Paul Bert (1).
He showed furthermore that the musculature of the lung was under
the motor control of the vagus nerve. These findings have been abun-
dantly corroborated by the subsequent researches of various investi-
gators of whom Francois-Franck (2) deserves particular mention.!
Francois-Franck (3), (4) published a number of papers dealing with
the comparative physiology of the reptilian lung. The results of these
studies are incorporated in two monographs which as far as they touch
our work are the most comprehensive and important contributions on
the subject. In most forms the vagus is found to exercise a motor
control over the lung of the same side. In one lizard (lézard ocelle)
the lungs possess in part a crossed innervation the vagus exercising not

1 Some 32 years after the original observations of Bert, Maar (Skand. Arch. f.
Physiol., 1902, xiii, 269) published an article on the gaseous exchange in the lungs
of turtles following ligation and stimulation of the vagi and sympathetic nerves
under various experimental conditions. This author apparently was not familiar
with the fact that the lungs of the turtle are muscular organs innervated through
motor fibers carried by the vagus. The possibility that changes in gaseous ex-
change (increased oxygen consumption and increased CO: following ligation of
the vagus) might be accounted for by contraction of the lung (mechanical stimu-
lation of motor fibers) never occurred to this investigator. Practically all posi-
tive findings which he attributes to section and stimulation of fibers having a
secretory or inhibitory effect on gaseous exchange might be explained by the ac-
tivity or inactivity of the lung musculature and the passive influence of the
circulation through the lungs.

261

262 A. J. CARLSON AND A. B. LUCKHARDT

only a homolateral but also a contralateral control over the lungs.
Frangois-Franck (5) states that the vagi and not the sympathetic nerves
contain the motor fibers for the lungs. Jackson and Pelz (7), on the
other hand, state that stimulation of. the sympathetic in the neck
region with a weak current causes dilatation of the lung and that
stronger currents may give a contraction even more promptly and
vigorously than stimulation of the vagus itself. In some preparations
Frangois-Franck (4), (5) found that electrical stimulation of the vagus
in the neck gave rise to a contraction which was preceded by an in-
hibition. According to his experimental analysis this inhibition is
only apparent. He attributes it to a movement of neck muscles. If
the muscles of the neck were not involved during peripheral stimulation
of the vagus no inhibition preceded the contractions.. Personally, we
have never obtained an inhibition of the lung on direct stimulation of
the peripheral vagus. We have, however, in innumerable instances
observed an inhibition preceding lung contractions of central origin.
We shall return to this point later in a discussion of this phenomenon.

Kahn (8) and Francois-Franck (5) have described and pictured
rhythmical contractions of the lung due to central discharges from the

bulbar nuclei; for they stopped on section of the vagi. Kahn in his ©

analysis on turtles which had suffered high spinal transection showed
that these lung contractions followed the external respiratory act;
Francois-Franck noted these rhythmical undulations in the intrapul-
monic pressure even in curarized animals but only when the bulbar
centers and the vagi were intact. He was never able to obtain a tonus

rhythm in any lung separated from its center. He found on electrical —

stimulation of the peripheral end of the vagus that a secondary con-
traction may appear on the completion of first; or if the peripheral
vagus was tetanized for some time a series of contractions might appear
during the period of tetanization simulating tonus variation of the lung.
In both instanees, however, he was probably dealing with incomplete

tetany of the lung rather than with discharges from a peripheral nervous |

automatic mechanism as a result of the vagus stimulation.

A tonus rhythm appearing in a lung whose extrinsic nerves have been
cut would suggest a peripheral nervous mechanism possessing automatic
activity. On the anatomical side we have the observations of Leydig

(9) and Schulze (10) that the lungs of some turtles possess not only

ganglionic swellings on the course of the nerve fibers but actual accumu-

lations of ganglion cells. On the physiological side Fano and Fasola —

(2) have asserted that the oscillations of tonicity in the lungs of the

——— ss

VISCERAL SENSORY NERVOUS SYSTEM 263

turtle, Emys europaea, persist after complete isolation of the lungs
from their nervous center although they admit that under normal con-
ditions part of the tonicity is due to impulses reaching the lung through
their extrinsic nerves. Since such a peripheral automatism of the lung |
did not reveal itself in the turtle studied by Frangois-Franck (tortue
grecque) and only occasionally in our own preparations it would seem
that the question of:a peripheral automatic mechanism in the lung may
depend on the species as well as on the physiological condition of the
animal under observation. —

The published reports of lung contractions as a result of the stimu-
lation of various afferent nerves in the reptilia are very meager. Cer-
tainly Frangois-Franck, who made a most extensive study of the phys-
iology of the reptilian lung, refers only incidentally to rhythmical
lung contractions set up by stimulation of afferent fibers carried by the
vagi (3). In his second monograph (4) on the lizard (lézard ocelle)
he describes a similar contraction of one lung following ligation of the
vagus of the opposite side (mechanical stimulation). Since inthis
preparation there was intercommunication between the lungs andsince
he showed that in this species of reptiles the vagus contained motor
fibers not only for the lung of the same side but also for the lung of
the opposite side, it is possible to explain his supposed reflex contraction
on ligation of the vagus nerve to direct motor effects on the opposite
lung or to the passive distention of the lung of the opposite side of
ligation of the vagus. Both factors would give a record which might
be interpreted as an active reflex contraction of the opposite lung.

Prevost and Saloz (10) are the only investigators who, by a method
inferior to our own, have described reflex contractions of the lungs of
the turtle (tortue grecque). They found that trauma to the carapace,
mechanical stimulation of feet, tail, neck and anal region, caused marked
contractions of the lungs.

Coombs (12) has recently shown that stimulation of the optic lobes —
or the medulla in the turtle induced contractions in the lung, provided
the vagi are intact. This seems to indicate that the motor innervation
to the lungs passes exclusively by the vagi nerves, unless the sympa-
thetic nerves were sectioned by her method of lung isolation.

EXPERIMENTAL PROCEDURE

The solution of the problems under investigation required an accurate
recording of the lung tonus and lung contractions with the least possible
injury to or interference with the normal respiration and other processes

264 A. J. CARLSON AND A. B. LUCKHARDT

of the animal. In other words, the necessary aim and requirement so
far as possible was to secure physiological experiments.

1. Preparation of the animal. No anesthetics were used. Prins (1
to 24 hours) to any dissection or other procedures that would cause
pain, the animals were decerebrated by quickly drilling through the
skull a little to the side of the median line, introducing a suitable probe
through this opening and after cutting off the cerebrum from the mid-
brain by the transverse stroke virtually pithing the former. By using
a small drill and going 2 to 4 mm. lateral to the median line, the median
sinus is left intact and profuse hemorrhage avoided. By our method

of pithing the cerebrum very little hemorrhage was produced, so that -

from this factor alone there was little or no impairment to the general
circulation or interference with the blood supply of the medulla and
midbrain. A small iron hook was introduced into the trephine hole
in the skull, by means of which the head was secured to the post as

shown in figure 1. The hook being placed in an insensitive spot, the.

head and neck were thus fixed without the complications from continued
irritation of clamps or ligatures about head and neck.

By means of a small drill, holes were made near the edge of the
plastron anteriorly and posteriorly, and by strings through these holes
in the ventral plate the animal was secured on the turtle stand in the
manner shown in figure 1. In the species of turtles worked on, there is
a sufficient margin of the ventral plate at both ends for drilling the hole
and securing the animal on the stand in its normal posture without
touching or injuring the skin. }

A few animals were worked on without decerebration. In this case
the spinal cord was transected in the neck before any operations were
undertaken on the body of the animal. |

For convenience of dissection the turtle stand with the animal at-
tached was turned upside down and held in that position by a suitable
support while the trachea, the carotids, the jugular vein, the cervical
sympathetics, etc., were isolated, balloons introduced into the stomach
(via esophagus) and the cannulae and other devices placed in position
in the neck and anterior thoracic region.

Isolation of the lungs and other viscera was made vi removal of the
necessary regions of the dorsal carapace. Cutting through and remoy-
ing parts of the dorsal carapace is attended with considerable hemor-
rhage unless care is taken not to injure the intercostal arteries and veins
when the calcareous part of the body wall is sawed through or clipped
off with strong bone forceps. If suitable care is exercised in this regard,

|
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VISCERAL SENSORY NERVOUS SYSTEM 265°

the diploic veins controlled by surgical wax and the intercostal vessels
ligated before cutting away the peritoneum and other structures that
adhere closely to the calcareous part, the entire body cavity can be
laid open from the dorsal side, with very little immediate and with no
chronic hemorrhage.

25cm.

Ge 18cm Sem. | ; /

40cm.

Fig. 1. Diagram of preparation and fixation of the turtle for recording lung
contractions. A: tube to T-tracheal cannula for spontaneous or artificial respi-
ration in the intact lung. C: Cannula in the carotid artery for recording the
blood pressure and heart. activity. L: Lung, isolated, except for pulmonary
vagi and blood vessels, bronchus ligated off. Inflated in its normal position
in the body cavity. M: Tube to manometer for recording the spontaneous
respirations. R: Flexible steel rod for fixation of the head. S: Screws for thé
fixation of turtle to the top of stand by strings through holes in, the plastron}
T: Cannula and rubber tubing from tip of isolated lung to recording tambour,
VY: Cannula i in the jugular vein for intravenous injections.

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THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 2

266 A. J. CARLSON AND A. B. LUCKHARDT

- In a few experiments where it was desired to avoid the injury to
visceral sensory nerve connections involved in isolating one lung from
the dorsal side, the lungs were exposed and isolated by fixing the animal
dorsal side down, removing the entire plastron and dissecting away
the liver and greater part of the gut in the way described by Jackson
and Pelz (7). In animals fixed on the dorsal side and plastron removed
the circulation fails much sooner than if the animal is fixed in the normal
position and the viscera exposed by removing one side of the dorsal
carapace.

In all our preparations exposing the lungs and viscera from the
dorsal side, the dorsal carapace was left intact along the vertebral
column, a strip 1 to 13 cm. wide. This precaution, together with
reasonable care in isolating the lung from its attachments along the
median line, leaves the main central connections of the visceral sym-
pathetic nerves, except possible fibers to the lung, intact.

2. Isolation of the lung. As is well known, the lung of the turtle is
a bilobed organ, united by the two long (3 to 5 em.) bronchi with the
very elongated trachea. The lungs are exceptionally large in compari-
son with the size of the animal, filling the dorso-lateral region down to
the .pseudo-diaphragm separating the pelvic cavity and the urinary
bladder from the rest of the viscera. Except for the posterior end
(about one-sixth of the entire area) the lungs are so firmly attached by
fibrous septa and membranes, dorso-laterally to the carapace, and
ventro-medially to the visceral organs, that complete collapse of the
lungs is impossible without severing the lungs from these connections.
It is therefore obvious that accurate recording of lung tonus and lung
contractions requires either complete anatomical isolation of the lung
from structures whose contractions could influence the lung volume,
directly or indirectly, or else complete curarization of the animal. We
used both procedures.

Complete isolation of one lung, usually the left, was produced in the
following way. The animal being decerebrated, fixed on the turtle
‘stand, as described above, the dorsal carapace over the. lung was re-
moved, all the septa and membranes suspending the lung in the body
cavity were severed, care being taken not to injure the bronchus, or
the pulmonary vagi and blood vessels entering the lungs along the
‘bronchus. In this dissection the lung was not handled directly with
forceps or other instruments. It was handled by means of the many
membranes and tendons attached to it. It is not necessary to remove
these structures close to the lung surface since they can have no effect

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VISCERAL SENSORY NERVOUS SYSTEM 267

on the lung tonus and lung contractions after being severed from their
normal connections with the rest of the body. This applies even to the
large flattened striated muscle closely adherent and attached to the
anterior end of the lung. Directed median- and antero-laterally over
the dorsal lung wall and being innervated by fibers from the brachial
nerve plexus, the contraction of this muscle reduces the size of the lung
cavity and thus serves as a muscle of expiration (fig. 24), supplementing
the expiratory muscles of the flank and the walls of the body cavity.
This muscle is probably identical with the one described by Fano and
Fasola as occurring in Emys europaea (2).

This operation and isolation of the left lung does not cause collapse
of the right lung or vice versa. There is naturally some interference
with the adequate ventilation in the lung of the intact side by the
lowering of the intra-abdominal pressure and the incapacitation of the
respiratory flank muscles on the side of the operation, but the animal
-is usually capable of filling and emptying the lung on the intact side
to meet the respiratory needs of the body, especially if one takes care
not to puncture the delicate septa partially separating the left and the
right side body cavities along the line of the great retractor muscle of
the neck. | #

In figure 2: are shown by diagram the main vago-sympathetic nerve
connections. The pulmonary vagi branches, usually two or more in
number, are of sufficient size and length to be easily handled for experi-
mental purposes; and the bronchus may be ligated and cannulated with-
out injury to the pulmonary nerves or vessels, and the pulmonary
vessels may be ligated or cannulated without injury to the nerves.

There is a persistent tendency on the part of some anatomists and
zoologists, as well as physiologists, to speak of the ‘‘bronchioles”’ .or
bronchial musculature of the turtle’s lung. This is entirely mis-
leading. In species of turtles worked on by us the bronchus ter-
minates peripherally, not by the mode of branching into smaller and
smaller divisions (bronchioles), but abruptly in the general lung cavity.
We are told by the anatomists that the numerous septa subdividing
the lung cavity into irregular chambers have strands of smooth muscle
fibers like the external walls of the lungs. But the smallest passages
between these chambers are of larger diameter than the trachea itself.
These passages and septa are alveolated. If these internal septa and
irregular passages are to be termed “bronchioles” on the basis of their
musculature, the entire lung of the turtle is composed of ‘‘bronchioles.”’
These subdivisions in the turtle lungs are alveolar spaces and not part
of the dead space similar to the mammalian bronchioles.

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VISCERAL SENSORY NERVOUS SYSTEM 269

3. Recording of the lung contractions. The lung contractions were
recorded graphically by water manometers or delicate tambours through
air transmission in either case, suitable cannulae being tied either into
the bronchus or the free end (posterior tip) of the isolated lung, the
isolated lung being distended with air to a pressure of 14 to 3 cm. of

water. In such a preparation active voluntary respiration in the lung
on the intact side will still cause passive changes in the intrapulmonic
pressure in the isolated lung through the movements of the viscera
(on which the isolated and inflated lung rests) especially as a result
of very vigorous expiratory movements. We eliminated this source
of error in two ways. The isolated and inflated lung was suspended
outside the body cavity without traction on the pulmonary nerves or
interference with the lung circulation. Or a metal plate was adjusted
to the body cavity on the operated side, fixed rigidly to the cut edge
of the carapace, except anteriorly, and the isolated and inflated lung
placed on this rigid floor, where visceral movements could not influence
it. This method can be adjusted so as not to cause traction on the
pulmonary nerves, or interference with the pulmonary circulation.
The best, and possibly the most physiological method of eliminating
these passive lung factors, is partial curarization, that is, doses of curare
that will completely paralyze the skeletal muscles, but leave the muscles
of respiration largely intact: In our animals this dose varied from 4
to +ce. of 1 per cent solution of curare injected intravenously. Turtles
subjected to these doses of curare go on breathing regularly though not
vigorously, leg and neck movements are abolished, and the gentle
respiratory movements on the intact side either do not mechanically
influence the isolated and inflated lung on the operated side at all, or
else this passive influence is regular or so slight that it is no source
of error in the work. Oy }

When all these dissections were completed and connections for record-

_ Ing made, the dorsal opening in the carapace was covered with a thick
layer of cotton moistened in Ringer’s solution, except during such phases
of the work as required direct inspection of or working with the isolated
lung or its local nerve and blood supply.

4. Artificial respiration. In all cases of complete curarization, arti-
ficial respiration was carried out periodically, usually in the intact lung.
For that purpose a T-cannula was usually inserted in the trachea.
This tracheal cannula was also used in studying the reflex influences
of pressure changes in the intact on the isolated lung. The venosity
of the blood in the isolated lung was a satisfactory criterion of the need

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270 A. J. CARLSON AND A. B. LUCKHARDT

of artificial respiration. Needless to say, artificial respiration was also
resorted to in the animals not curarized, if it appeared that the spon-
taneous respiration through the lung on the intact side did not suffice
to avert asphyxia.

It is well known that the reptilia execute the same buccal respiratory
movements as the amphibia, although actual swallowing of the air
into the lungs does not occur. However, in labored respiration as in
mild or severe asphyxia, the reptilia carry on actual swallowing move-
ments as a part of the respiratory act. These swallowing movements
may thus be used as an index of external respiration, or rather attempt
at external respiration, in animals with the respiratory muscles im-
mobilized by transection or pithing of the spinal cord.

8. Maintenance of efficient circulation. If we start with an animal
vigorous and in good condition, and care is taken to avoid all but the
minimum hemorrhage in the experimental procedures, a good circu-
lation will be maintained as shown by direct blood pressure records or
by direct inspection of the isolated lung, for 12 to 24 hours or even
longer. In feeble animals the circulation fails much earlier. The
circulatory failure is due, not to the failure of the heart, but to low
blood pressure evidently due to transudation of the blood plasma into
the lymph and tissue spaces. This is not ordinary oozing of blood from
cut surfaces or hemorrhage due to injured vessels. In a turtle prepa-
ration in this condition intravenous injection of Ringer’s solution im-
proves the circulation only temporarily, the added fluid soon passes
out of the blood vessels into the lymph and tissue spaces, and repeated
-Ringer’s solution injections then render the preparation more edematous.

It. is not unlikely that this phenomenon is analogous to the passage
of plasma from the blood to the tissues in traumatic shock in mammals,
‘as reported by some investigators.

6. Additional preparations of the animal for studying some of the
lung reflexes. The turtle preparation described above requires no
additional dissection for the study of the lung reflexes induced by
inflation or deflation of the intact lung, stimulation of the nares, the
cloaca, the penis, the skin or the skeletal nerves. It is even possible
by careful manipulation to get at and stimulate the bladder, the rectum,
ureter, large and small intestine, etc., through the opening in the dorsal
carapace made for the isolation of the lung, without altering the tension
on the lung. wall mechanically. In the artificial stimulation of the
central end of the pulmonary, gastric and vagi branches on the side
opposite to the lung under observation it is usually necessary to make

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VISCERAL SENSORY NERVOUS SYSTEM 271

a small opening in the dorsal carapace on that side and partially collapse
the intact lung. In the same way inflation of the urinary bladder or
the rectum, direct stimulation of the gall bladder, stomach, etc., re-
quires an opening of the carapace posteriorly on the side iceeite to
the lung under observation, if one is to avoid mechanical errors.

7. On the basis of our own results, and with due regard to methods
used by previous investigators on various phases of reptilian respiratory
physiology, we feel that a turtle prepared as above with minimum
trauma, fixed in normal position without trauma, one lung isolated for
accurate observation, the other intact'and used in normal spontaneous
respiration, is as near its normal physiological state as the necessities
of the problem permit. Most phases of lung physiology in the turtle
cannot be satisfactorily studied with the animal fixed with the dorsal

side down, owing to the special anatomical conditions.

The serious and unavoidable difficulties, apart from that of a Lidic:

ratory located in a city obtaining turtles in prime condition, are a,

depressor effects or “central shock” due to sensory effects involved in
the operative trauma; b, ‘peripheral shock”’ or gradual failure of the
circulation due to passage of the blood plasma out of the blood vessels
into lymph and tissue spaces. It is generally held that reptiles show
little or no spinal or traumatic shock, and this was our main reason,
besides that of avoiding anesthesia, for working out the visceral reflexes
first in this form. Shock, spinal and general, may be minimal but. is
certainly not absent in the turtle.

8. Experimental procedure on the snake. No satisfactory wegk on
the contraction of the lung in these animals can be done without virtu-
ally removing the lung from the body, complete pithing of the spinal
cord, or complete curarization, thus immobilizing the body. Curari-
zation is, of course, not applicable in experiments on the relation of the
lung contractions to the external respiratory act. Hence we used the
method of complete pithing of the spinal cord after transection of the
cord two or three segments below the medulla. The animal was then
fixed, dorsal side down, the lung exposed and isolated by a ventral
median incision, taking care to avoid hemorrhage. The recording
cannula was inserted in the tip of the lung rather than in the trachea.
The trachea was ligated after isolation from the vagi and the neck blood
vessels. This preparation is suited only for studying the relation of
the respiratory movements to the lung contractions, the vagi action
on the lungs, and the lung reflexes evoked from the head region of the
animal. In the pithed snake the external respiratory movements ap-
pear in the form of attempts at swallowing air just as in the turtle.

272 A. J. CARLSON AND A. B. LUCKHARDT

In the species of snake used by us the lung is not a paired organ.
Alveoli and septa are present in the anterior third of the lung only,
the posterior two-thirds of the lung being a delicate apparently
muscular sac very much like the primitive lung of necturus. There
being only one lung, the necessary artificial respiration had to be car-

ried out periodically in the lung while suspending the recording. This
fact renders the snake much less suitable than the turtle dai the sass

of lung motor physiology.

The musculature of the snake lung is very much less dévelanan han
that of the turtle. Even delicate water manometers are not sufficiently
sensitive for recording the lung contractions. The snake preparations

also deteriorate very much faster than does the turtle. Because of —

these several handicaps the work on the snake was only carried to the
point of establishing the identity of the fundamental facts of si: mobnr
payaalagys in these two groups.

THE RHYTHMIC CONTRACTIONS OF THE LUNGS DURING NORMAL RESPI-
RATION AND AFTER PARTIAL AND COMPLETE CURARIZATION

1. Typical records of the active lung contractions that follow auth

external respiratory act or a group of respirations are reproduced in

Fig. 3. Water manometer tracings of the intrapulmonic pressure in the ce.
Animals decerebrated. A: Record from left lung; dorsal shell removed on left
side, lung isolated except nerve and blood vessel connections. Cannula in left
‘bronchus. The spontaneous respiratory movements at a passively influencing
‘the isolated lung. Each group of respiratory movements is followed by contrac-
_tion of the isolated lung. B: Tracing from the same animal as in A, 24 hours later
after recovery from curare. . Record from the intact (right) luna -Cannula i In
trachea. Each group of respirations is followed by contraction of the lung.
-C: Tracing from left lung, isolated as in A, after intravenous injection of a dose of
‘eurare (7 cc. of 1 per cent, to paralyze skeletal muscles). Signal equals gasping or
‘swallowing movements. These are followed by lung contractions. D: ¢, left
-lung isolated as in A; }, right lung in normal attachments. a, carotid blood pres-
sure (Hg.); signal scant respiratory movements; { cc. curare injected intrave-
nously leaving slight respiratory movements of mouth and flanks... Showing lung
‘contractions following respiratory movements. E and F: Spontaneous contrac-
‘tion ‘of lungs after a large dose of curare (} ec. 1 per cent) completely paralyzing
-all striated. muscles. E, lung isolated as in A. - F,; lung in normal attachment.
‘Showing rhythmic lung contractions in the absence of external evidence of respi-

‘ration. Gand H: Water manometer records from inflated balloon in the stomach —

of.the turtle; shell intact; stomach atonic and quiescent, but showing beginning
of feeble rhythm at ¢; a, spontaneous respiratory movements, quick up and down
‘ stroke, followed by bit contractions; b, shown as diminished tension in the stom-
-ach because of the lowered intra-abdominal pressure created by the active lung
contraction, the shell being intact. Time, 5 seconds.

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figure 3, A, B, C. The lung contraction follows the respiration after
a latent period of 1 to 3 seconds. In preparations in poor condition
the latent period is usually much longer. If the external respiratory
movements are of the Cheyne-Stokes type, the lung contraction does
not appear until the end of the last respiration in the group, especially —
if the respirations come fairly close together, except in marked asphyxia.
The lung contractions are usually absent when the external respiration
is rapid and continuous. This absence appears to be due to inhibition |
of the center controlling the lung contractions by some process in the
external respiratory act.

Under certain conditions acts of external respiration may be executed
without being followed by a contraction of the lung. This is especially
the case in animals in poor condition from any cause, in preparations
in partial traumatic shock from the operative procedure, or if for any
reason, such as apnea, the external respiration is feeble. By feeble we
mean feeble discharges from the respiratory center. The general rela-
tion obtains that the stronger external respiratory act, the stronger the —
lung contractions that develop during the respiratory pause. _

Graphic records of this strong lung rhythm may be obtained from
an inflated balloon in the stomach of the turtle, provided the stomach
is relatively quiescent and in low tonus (fig. 3, Gand H). The external
respiratory act induces passive changes in the intragastric pressure (fig.
3, G, a) just as in mammals, except reverse, that is, in the turtle expi-
ration causes increased intragastric pressure and vice versa. The
active lung contractions appear on the gastric balloon records as waves
of negative pressure or lowered tonus, owing to the lung contraction
increasing the general intra-abdominal negative pressure.

Tracings showing the lung rhythm as it appears after complete curari-
zation are reproduced in figure 3, E and F. If a paralyzing dose of
curare is injected intravenously in a preparation breathing spontane- .
ously and regularly and exhibiting lung contractions as in figure 3, A,
the brief central stimulation phase of the curare action, as shown by
violent body movements and cardiac arhythmia, is followed by complete
quiescence of the lungs lasting for one-half to two hours. This is not
due to peripheral paralysis of the pulmonary motor nerve fibers. It is:
evidently due to paralysis of the medullary centers by curare. Reflex
lung contractions can be secured before the spontaneous rhythm ap-
pears. But when the spontaneous lung rhythm reappears, it continues
for hours or indefinitely provided suitable artificial respiration is carried
out at intervals and a fairly efficient circulation maintained.

275

VISCERAL SENSORY NERVOUS SYSTEM

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276 A. J. CARLSON AND A. B. LUCKHARDT

The fact that the lung rhythm appears in completely curarized ani-
mals shows that it is not due to reflexes evoked by acts of external
respiration. It shows further that central and efferent nervous pro-
cesses of the external respiration go on during the curarized state,
because these lung contractions are side events in the normal respi-
ratory act. : : 7

These lung contractions following the external respiratory act have
been studied and described by Francois-Franck (3), (4) in several
species of reptilia. ‘They are evidently identical with the active lung
contractions following the respiratory act in frogs and salamanders,
recently reported by us (13) and (14). It may be of some interest to
note that in this regard the lung physiology of amphibia and rep-
tilia is similar or identical, despite the fact that the mechanisms for
effecting lung ventilation in these two groups of animals are entirely
different. !

2. As already noted, the lung contractions develop during the respi-
ratory pause, and if the respirations come close together they may not
appear at all, except in states of marked asphyxia. In fact, a lung
contraction once begun may be weakened or cut. short by an act of
external respiration coming on before completion of the contraction.
It would thus appear that while a single respiratory discharge from the
respiratory center will, as a side effect, induce a lung contraction after
a characteristic latent period, several respiratory discharges following
in succession closer than this latent period of the lung contraction
interferes with this development in such a way that the lung. contrac-
tion follows the last respiratory act only. We have made very great
efforts to determine the mechanism of this interference by satisfactory
experiments.

On most of our tracings from the isolated lung, the animal breathing
spontaneously by the lung on the intact side, there appears a tonus
relaxation of the isolated lung during the period of external respiration,
that is, prior to the active lung contraction. This may be seen in
figure 3, A, and figure 6, A. If all purely mechanical factors were
excluded, this would indicate a central inhibition of the lung tonus by
discharges from the respiratory center prior to the central processes
initiating the lung contractions. Francois-Franck (4), (5) has called
attention to the fact that on-stimulation of the peripheral end of the
incompletely isolated vagus the lung contraction produced by this
stimulation may be preceded by an apparent inhibition of the lung tonus,
in reality due to mechanical factors developed in the body cavity as the

VISCERAL SENSORY NERVOUS SYSTEM 277

result of the contractions of some muscles of the neck. Furthermore,
if the isolated and inflated lung presses against any structure in the
body cavity with a force equal to or greater than the internal pressure
maintained in the lung (14 to 2 cm. water) and if these structures are
so moved by the act of spontaneous inspiration that the pressure on
the outside of the lung is decreased, the net result would be an apparent
inhibition of the tonus of the isolated lung during the external respi-
ratory acts. We endeavored to eliminate this source of error by com-
plete curarization, by section of the spinal cord below the medulla,
and by suspending the isolated lung partly outside the body cavity.
In completely curarized preparations there is, of course, no extérnal
respiratory act, but the facts cited above go to show that the discharge
from the respiratory center, after an initial paralysis, is resumed and
continues rhythmically. Despite this fact inhibition of the lung tonus
prior to the lung contractions rarely if ever appears on the tracings
from our completely curarized animals. This may be due, however,
to a depression of the medullary centers by the drug (direct chemical
action, or indirect by altering or diminishing afferent skeletal nervous
impulses) to such an extent that the tonus motor action of the medul- .
lary centers on the lung is abolished. Without such tonic motor action
there could be no relaxation of lung tonus by central inhibition. » This
negative result on completely curarized animals is therefore no criterion
of what the discharges from the respiratory center may do in the direc-
tion of central inhibition of lung tonus, when such central motor tonus
is present. 7 |

We have secured tracings from animals partly curarized, from ani-
mals with section of the spinal cord in the neck, and from animals
without curare or spinal cord transection, but with the observed lung
suspended outside the body cavity, indicating a central inhibition of
the lung tonus by the discharge from the respiratory center, an inhibition
prior to the motor innervation of the lung. A tracing illustrating this

fact is shown in figure 6, B. Water manometers are usually not delicate’
enough to disclose the inhibition. This inhibition is probably pro-
portional to the amount of central motor tonus in the lungs.

From the point of view of utility or teleology this inhibition would
be expected, as greater relaxation of the lung tonus means less resistance
to lung inflation, and active lung contraction would counteract the
inspiratory effort. The possible utility of the active lung contraction
following the respiratory act is another matter. Useless, useful or
harmful, it is there, and we are at present concerned only with the
mechanisms of its initiation and control.

278 A. J. CARLSON AND A. B. LUCKHARDT

3. We desire to point out, however, that the above described rela-
tions of the external respiratory act to the active lung contractions are
similar to the relation of the swallowing act to the peristalsis of the
esophagus. If the swallowing acts are sufficiently far apart, each swal-
lowing is followed by a peristaltic wave of the esophagus initiated from
the medulla. If the swallowing acts are repeated at very brief intervals
esophageal peristalsis follows the last swallowing act only. The parallel
between swallowing-esophageal peristalsis and respiration-lung con-
traction appears complete, despite. the very diverse functions of the
two mechanisms. The lung has the same organogenesis as the esopha-
gus, and the primitive mechanism for external respiration is swallowing,
the active lung contractions following the respiratory act (airswallow-
ing) serving probably a real respiratory function. As the mechanism
of external respiration changes with the reptilia the organogenesis
and primary nervous relations of the lung remaining the same, the
lung inhibition and contractions associated with the respiratory act may

represent inherited mechanisms useless, if not at times actually harmful to —

animals provided with this later respiratory device. It may be a vestigial
- mechanism on the road to elimination in the process of evolution.

4. The influence of asphyxia on the bulbar center for the lung rhythm
appears to run parallel with the influence of asphyxia on the respiratory
center. In curarized preparations permitted to go into varying degrees

of asphyxia the lung rhythm increases in rate and intensity to the point .

of incomplete lung tetanus parallel with increasing asphyxia (fig. 4).
It is clear that asphyxia increases the lung tonus by central motor action.
The lung rhythm does not end in this state of incomplete tetanus. If
the state of asphyxia is permitted to continue, the incomplete tetanus
stage is followed by feebler lung contractions appearing at greater and
greater intervals (5 to 10 minute intervals in some cases) before the
final quiescence. This probably represents the well-known final feeble
activity of the respiratory center when the asphyxia is carried beyond
the point of stimulation to that of actual impairment of the center.
Further evidence of this complete parallel is furnished by the type
of experiments illustrated in figure 5. Here artificial respiration through
the intact lung, normal, A, and denervated, B, both preparations being
completely curarized, inhibits the tonus and rhythm in non-asphyxiated
preparations, A, evidently by inducing apnea, and diminishes the tonus
and incomplete tetanus in the asphyxiated. preparation, evidently by
decreasing the asphyxial condition of the blood. :

ee ee ee

279

VISCERAL SENSORY NERVOUS SYSTEM

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280 A. J. CARLSON AND A. B. LUCKHARDT

In non-curarized preparations, in which one can follow the action
of the respiratory center by means of the external respiratory acts, con-
tractions of the isolated lung, not related to any external respiration,
may appear during asphyxia. It appears that asphyxia may occasion-
ally disorganize the normal correlation between the respiratory center
and the vagi center controlling the lung contractions, and that asphyxial
conditions are capable of stimulating the latter centers directly. —

Fig. 6. Tracings of the intrapulmonic pressure in the turtle. Left lung, iso-
lated except pulmonary nerves and blood vessels. Left bronchus ligated. Can-
nula in tip of lung. Animals decerebrated. Right lung left in normal relations.
A: signal, spontaneous respiration. Showing inhibition of tonus of isolated
lung during respiratory movements followed by strong contraction of lungs.
Water manometer. B: animal partially curarized, having respiratory muscles
in feeble activity. Signal, spontaneous respiratory movements. Both tracings
from left isolated lung. a, record from delicate tambour; b, water manometer.
Showing lung contractions following spontaneous respiratory movements, pre-
ceded by tonus inhibition, as revealed by tambour, the water manometer not being
delicate enough to register the tonus inhibition.

THE ROLE OF THE EFFERENT PULMONARY VAGI FIBERS AND THE VARIOUS
PARTS OF THE BRAIN IN THE GENESIS OF THE RHYTHMIC
LUNG CONTRACTIONS

1. In the species of turtle studied by us the spontaneous lung con-
tractions are of central origin, and the motor innervation is through
the vagi, as shown by Kahn (8) and Frangois-Franck (5) for the com-
mon land turtle of Europe. This may be shown by experiments such
as reproduced in figure 7, A. In that case the animal was completely
curarized, graphic records being taken of the lung contractions both
from the isolated and the intact lungs. The intact lung has, of course,
its sympathetic nerve connection intact. Nevertheless section of the
vagus nerve on the side of the intact lung abolishes the strong con-

-tractions in that lung at once and forever. The section of one vagus

281

VISCERAL SENSORY NERVOUS SYSTEM

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SAMOA Pisa ee Ree ike

NYS

RS es eer

282 A. J. CARLSON AND A. B. LUCKHARDT

stops the contraction of the lung on the opposite side temporarily
through stimulation of afferent inhibitory fibers in the vagus trunk
depressing the medullary centers. It is thus clear that the motor
innervation of the lung rhythm is solely through vagi nerves. The
same fact can be illustrated by the use of atropine. Atropine paralyzes
the vagi motor fibers in the lungs. The tracing reproduced in figure8,
C, was secured from the left lung of a completely curarized preparation,
the lung being isolated from the body except for the pulmonary blood

Fig. 8. Water manometer tracings of the intrapulmonic pressure in the turtle.
Animals decerebrated. Left lung isolated, cannula in tip; right lung in normal
relations. A and B, showing lung contractions following the spontaneous respi-
ratory movements. A: a, section of right vagus in neck, followed by temporary
inhibition (central) of the tonus and contractions of the opposite lung, also tem-
porary inhibition of the respiratory movements. The latter reappear at X, but
are not followed by lung contractions. Tracing B is continuation of A after a 6
minute interval, the lung contractions gradually regaining normal vigor. C: ani-
mal completely curarized; lung in strong tonus and contractions, probably from
partial asphyxia. a, intravenous injection of } mgm. atropine sulphate followed
by permanent inhibition (peripheral action) of lung tonus and contractions.
Signal shows vagus stimulation is without effect on lung.

These tracings indicate a tonic condition of the medullary center controlling
the lung motor tissues. This center is more profoundly influenced by depres-
sant impulses (‘‘shock’’?) than is the respiratory center.

vessels and pulmonary vagus branches. Intravenous injection of 0.5
mgm. atropine sulphate abolished the lung rhythm promptly just as
it abolished the motor action of the vagus on the lung musculature.

The reader’s attention may again be directed to the fall in lung tonus
induced by peripheral vagus paralysis of atropine and by afferent de-
pressor impulses acting on the medulla (fig. 8, A and C): The tracings
in figure 8 show at least that under some conditions (partial asphyxia)
the medulla-vagi-lung motor mechanism is in a state of tonic activity,
in which the stronger rhythmic contractions. are superimposed much

Po ee ee tae eae Cage ES = - T S g A S.

VISCERAL SENSORY NERVOUS SYSTEM 283

like the constant tonus and the occasional peristaltic contractions of
the gut. We have so far been unable to demonstrate this tonic activity
under more nearly normal conditions; but our results point in the di-
rection of some normal motor tonus.

2. The inhibition of the contractions of the lung on the opposite
side induced by section of one vagus (afferent inhibition of a central
automatism) is usually more prolonged than shown in figure 7, A.
In preparations not curarized it can be shown that section of one vagus
temporarily inhibits both external respiration and the tonus and con-
tractions of the lung on the opposite side, the external respiratory
acts returning sooner than the lung contractions.

Section of the spinal cord in the neck also produces a very prolonged
inhibition of the medullary center initiating the lung rhythm (fig. 7, B).
This central inhibition or shock is shown not only by the temporary
abolition of the spontaneous rhythm, but also by the lowering of the
excitability of these centers to reflex stimulation leading to lung con-

tractions.

3. As reported by many previous investigators, stimulation of the
peripheral end of the vagus nerve in the turtle causes contraction of
the lung musculature (fig. 9, C). We have never seen any inhibitory
effects on the lung in the turtle from stimulation of the peripheral vagus.
This appears to us significant in view of the fact that in the frog the
predominant action of the vagi on the lungs is inhibitory, and in the
most primitive amphibian lung (necturus) all the efferent lung nerve
fibers appear to be inhibitory.

In the species of turtle studied by us the motor action of the vagi
on the lungs is strictly unilateral. Single induction shocks (make or
break) applied to the peripheral vagus will not cause lung contractions
unless exceedingly strong. Weak tetanizing currents applied to the
vagi induce an incomplete tetanus or rhythm that seems to indicate
a certain degree of refractory state in the peripheral mechanism. Strong
tetanization of the vagi will, however, induce curves of complete tetanus
closely resembling those of an OnTERBEY: muscle nerve preparation bia
9, C).

In view of the identity of origin and certain similarities in the nervous
control of the lung and the alimentary canal, we made some comparisons
between the vagi motor action on the lungs and on the stomach (fig.
10). The latent period of the vagi motor action on the stomach is
very much longer than its motor action on the lung. The turtle’s
stomach cannot be tetanized by vagus stimulation; the lungs can be

284 A. J. CARLSON AND A. B. LUCKHARDT

tetanized. The third marked difference is the quick failure of vagus
motor action on the stomach on repeated stimulation in comparison
with the repeated tetany of the lungs that may be induced by vagus
stimulation. These differences may be due, in part, to the fact that
in the turtle the vagi carry inhibitory efferents to the stomach in ad-
dition to the motor, while the efferent vagus lung action is solely motor.
The differences also suggest that the gut has retained more of its primi-
tive automatism (independent peripheral nervous system) while the

Fig. 9. Water manometer tracings of the contractions of the isolated lung of
the turtle. Animal decerebrated and completely curarized; left lung isolated
except for pulmonary vagi fibers and blood vessels. Cannula in left bronchus.
Stimulation with moderately strong tetanizing current, A, optic lobes, B, medulla,
‘C, peripheral end of left vagus, showing incomplete and complete tetanus of the
lung musculature.

differentiation in the lung has been toward the more simple relations
of a muscle nerve preparation.

4. In 1878 Martin reported that stimulation of the optic lobes in
the frog accelerates the external respiratory movements.’ Since that
time several investigators have concerned themselves with the problem
of accessory respiratory centers above the level of the medulla. - Re-
cently Coombs (12) reported that stimulation of the optic lobes or the
medulla in the turtle with weak tetanizing current causes lung contrac-
tions via the vagi nerves.

VISCERAL SENSORY NERVOUS SYSTEM 285

As we have shown, the normal rhythmic contractions of the turtle
lung are side events in ‘the external respiratory act: This being the
case, we would expect lung contractions from stimulation of any part

Fig. 10. Simultaneous water manometer tracings of the stomach and the lung
contractions on stimulation of the peripheral end of the vagi nerves in the turtle.
In A and B, upper record from stomach (balloon method); lower record from left
lung, isolated except for the pulmonary blood vessels and vagi fibers; cannula
in left bronchus. Signal, stimulation of peripheral end of left vagus with the
tetanizing current. Showing longer latent period, more rapid fatigue and ab-
sence of tetanus in the gastric response to vagus stimulation.

286 A. J. CARLSON AND A. B. LUCKHARDT

of the central nervous system that has motor connections with the
medullary respiratory center. This has turned out to be a fact, at
least as regards the optic lobes. In figure 9, A and B, are reproduced
typical tracings of the incomplete tetanus the lung induced by stimu-
lation of the optic lobes and the medulla with weak tetanizing currents.
The lung contraction curves induced from the optic lobes and from the
medulla in completely curarized turtles are practically indistinguishable.
Both centers fail quickly under this type of stimulation and there is
a single after-discharge (lung contraction) that appears more normal
than the contractions induced directly by the stimulation. Complete
tetanus of the lungs cannot be induced by central stimulation, evidently
because of central refractory states.

A tonus rhythm or spontaneous contraction of the lung aftersection
of the vagi nerves similar to that described by Fano and Fasola (2)
in the European turtle, has been seen occasionally in our turtle prepa-
ration, when we used an exceedingly delicate tambour as a recording
device. The contractions are feeble but slow and regular. They may
persist for hours (fig. 23, A). Itis probable that a peripheral automatic
motor mechanism of the lung is present in all species of turtle, although
it may differ in degrees in various species; but it requires not only deli-

cate recording devices but, above all, animals in prime condition to

reveal it. It is certain that parallel with the development of the new
type of external respiratory mechanism in the reptilia, the peripheral
lung motor automatism, so prominent in the amphibia, has retrograded
or has been changed to one of primarily medullary origin.

LUNG REFLEXES

1. Lung reflexes from sensory stimulation of the respiratory tract: a.
The pulmonary branches of the vagi contain two kinds of afferent fibers
acting on the medullary center, one type causing reflex lung contraction
in a quiescent preparation, or acceleration of the contractions in an
active preparation. The other type causing inhibition of the tonus in
the case of quiescent preparations or of the contractions in active prep-
arations. For the sake of brevity we call these motor and inhibitory
afferents, respectively. The motor afferents are stimulated by inflation
as well as by deflation of the lung (fig. 11) or by the weak tetanizing
current. Strong tetanizing currents, on the other hand, stimulate the
inhibitory afferents. In all: these experiments the lung inflation and
deflation, and the direct stimulation of the central and of the pulmonary

— srt CO, hr ee ee leer el _

287

VISCERAL SENSORY NERVOUS SYSTEM

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CARLSON AND A. B. LUCKHARDT

3

A.

288

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VISCERAL SENSORY NERVOUS SYSTEM 289

vagi, we used, of course the lung and vagus on the opposite side to the
lung serving to record the contractions. In no case did we note the
inhibitory afferents being stimulated by lung inflation or lung deflation.

Typical tracings showing the reflex lung contraction on lung in-
flation and deflation are given in figure 11. Tracings showing the
opposite reflex effects on weak and on strong tetanization of the
central end of the pulmonary vagi are reproduced in figure 12. The
pulmonary motor afferents can also be stimulated by other mechanical
means, for example, rubbing the collapsed lung of one side between
one’s fingers induces reflex contraction in the opposite side via the
medulla.

Reflex lung tetanus cannot be produced . lung deflation or inflation,
that is, the active change in the intrapulmonic pressure, and not the
state of continued inflation or collapse that acts as stimulus to the
motor afferents. ;

In preparations not curarized, but: breathing normally by means of
the intact lung, single inflation of the intact lung during a respiratory
pause usually gives a reflex lung contraction; but if the inflation is
made shortly after completion of a spontaneous contraction by the
isolated lung, the reflex may not be elicited. The same is true if a series
of lung inflations is produced with very short intervals between each
inflation. These facts evidently point to a condition of refractory
state of the medullary reflex center, unless the phenomenon can be
due to simultaneous stimulation of the inhibitory pulmonary afferents.

In turtle preparations in good condition deflation of one lung by
suction through the: trachea invariably initiates or accelerates the ex-
ternal respiratory movements. Lung inflation, on the other hand,
may start a respiratory movement or it may temporarily inhibit the
external respiration. It is thus evident that stimulation of the pul-
monary motor afferents by lung inflation may initiate reflex lung
contractions by acting on the medullary motor center directly, the dis-
charge of the respiratory center not being a necessary link in the chain.

We have found that a single inflation of the intact or isolated lung
aerates the blood, accelerates the heart and by so doing improves the
circulation through the medullary centers because of the increase in
the general arterial pressure. It is certain however that the contrac-
tion of the isolated lung following one or more inflations of the intact
lung does not occur because of the improved circulation of a more
highly oxygenated blood through the medullary lung center; for this
reflex contraction persists not only following the inflation of a lung the

290 A. J. CARLSON AND A. B. LUCKHARDT

pulmonary vessels of which have been occluded by a temporary ligature
but can be elicited for a considerable time after the complete excision
of the heart. On the other hand, it is not obtained from the isolated
lung following a single or several inflations of the denervated but other-
wise intact lung of the opposite side.

We designate the refiex inhibition of lung tonus and contraction by
strong tetanization as due to a separate type of sensory fibers, the
inhibitory afferents, at the same time keeping in mind the possibility
that this inhibition may in reality be due to abnormally strong or

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oh

Fig. 13. Water manometer tracings of the intrapulmonic pressure in the turtle.
Animals decerebrated and completely curarized. Records from left lung isolated
except from pulmonary blood vessels and vagi branches. Cannula in left bron-
chus. A and B: stimulation of central end of recurrent laryngeal nerve, showing
reflex inhibition of the lung rhythm. C: stimulation of central end of gastric
branches of right vagus, showing reflex augmentation of the lung rhythm.

abnormally rapid series of impulses over the motor afferents impinging
on the central automatic mechanism. This stimulation also inhibits
the external respiratory movements.

b. Stimulation of the central end of the inferior or recurrent laryngeal
nerves causes invariably reflex inhibition of the lung tonus and con-
tractions (fig. 13, A and B). At no time was reflex lung contraction
obtained from the central end of this nerve. Motor afferents for the
lung reflex are either absent or the inhibitory afferents are so pre-
dominant that the action of the former type is entirely suppressed on
simultaneous stimulation of the two types.

VISCERAL SENSORY NERVOUS SYSTEM 291

Observations on the effects of external respiration of stimulation of
the central end of the inferior laryngeal were not made but in con-
nection with this reflex depression of the lung contraction mechanism
we can state that mechanical tension or pulling on the trachea causes
profound depression of the respiratory center (fig. 14). Mechanical
stimulation of the larynx and glottis (rubbing with blunt probe) on
the other hand induces strong lung contractions.

c. Mechanical stimulation (gentle rubbing) of the posterior nares
causes very powerful lung contractions. If the stimulation is long-

Fig. 14. Tracing of respiratory movements (water manometer) in the turtle,
Animal decerebrated; cannula in trachea, rapid respiration due to partial as-
phyxia; z-x’, strong forward traction on trachea, inducing a temporary depression
of the respiratory center, probably through stimulation of the recurrent laryngeal
nerves,

continued the lung goes into incomplete tetanus. This stimulation
‘initiates lung contractions in quiescent preparations and accelerates
the rhythm in active preparations. When the nares are thus stimu-
lated in decerebrated but non-curarized preparations such stimulation
induces most violent respiratory efforts and general struggling.

In general the reflex lung contractions evoked from the nares are
more powerful than those produced by stimulation of any other region
of the respiratory tract. It is one of the last lung reflexes to disappear
as the preparation deteriorates from circulatory failure, and other

292 A. J. CARLSON AND A. B. LUCKHARDT

causes, in long continued experiments. Reflex lung contractions from
the nares can be obtained in preparations with the medullary center
in such poor condition (‘‘reflex shock’’) that the spontaneous external
respiratory act is not accompanied by lung contraction, all of which
point to the fact that the sensory nerve fibers of the posterior nares
make strong motor connection with the medullary center controlling
the lung contraction. Stimulation of ace posterior nares with chemical
irritants were not tried.

2. Lung reflexes from spinal sensory nerves: Weak tetanizing of the
central end of any spinal nerve causes reflex lung contraction. Most
of the tests were made with the sciatic and the large brachial nerves.
Weak stimulation accelerates the rhythm in an active preparation and

[~~ ye” 4 i. f \.

I

Fig. 15. Water manometer tracings of the intrapulmonic pressure in the turtle.
Records from isolated left lung, cannula in left bronchus, pulmonary vagi fibers
and blood vessels being intact. Animal decerebrated. A, animal completely
curarized; B, animal not curarized. Signal, mechanical stimulation of the nares,
showing incomplete tetanus (reflex), of greater amplitude than that of the rhyth-
mical lung contractions.

initiates a rhythm in quiescent preparations (fig. 16, A, fig. 17, 8

Strong stimulation induces incomplete lung tetanus (fig. 16, B at x-v’,
fig. 17, C). In quiescent preparations the incomplete lung tetanus may
be followed by brief periods of apparently spontaneous lung rhythm
(fig. 17, B).

If the preparations are in good condition gentle rubbing of the skin
causes lung contractions. Pinching, crushing or cutting the skin causes
powerful motor reflexes into the lung (fig. 16,°C), even when the reflex
excitability is not at its maximum. Strong lung contractions are
likewise induced by mechanical (rubbing with a blunt probe) or elec-
trical stimulation of the cornea.

Reflex inhibition of the lung tonus or the rhythmical contractions
were not obtained from the spinal sensory nerves by any mode of
stimulation.

a —

—————— ee

VISCERAL SENSORY NERVOUS SYSTEM 293

Fig. 16. Water manometer tracings of the intrapulmonic pressure in the turtle.
Animals decerebrated and completely curarized. Records from left lung, iso-
ated except for pulmonary blood vessels and vagi nerves. Cannula in tip of
lung, bronchus ligated; A: light stimulation; B: stronger stimulation of central
end of sciatic nerve, showing acceleration and incomplete tetanus of lung rhythm.
C: a, stroking of skin of hind leg with finger; b, pinching toes of hind leg, «x, cut-
ting skin of hind leg. : Showing reflex lung contraction on stimulation of cutaneous
nerves.

Fig. 17. Water manometer tracings of the intrapulmonie pressure in the turtle.
Animals decerebrated and completely curarized. Records from left lung, iso-
lated,-except for pulmonary blood vessels and nerves. Left bronchus ligated;
cannula in tip of lung. Preparations showing no spontaneous medulla-lung
rhythm. Signal, stimulation of central end of sciatic nerve with tetanizing
current. Showing reflex development of lung rhythm and lung tetanus.

294 A. J. CARLSON AND A. B. LUCKHARDT

3. Lung reflexes from the visceral sensory nerves: a. The alimentary
tract. Reflex lung contractions may be obtained from practically every

part of the alimentary canal, the most powerful being the lung contrac= ~

tions following stimulation of the lower end of the gut (cloaca, rectum,
large intestine). Mechanical distention with balloon, rubbing, pressure,
cutting or crushing this end of the alimentary canal causes contractions
in the lung. (fig. 18, B, C, Da, Dd; fig. 19, a, c). In fact, one may.
induce incomplete tetanus of the ee by strong stimulation of the
lower end of the gut.

Mechanical or electrical stimulation of the anal intestines also.

induces lung contractions (fig. 19, b, d). Similar lung reflexes are

induced by stimulation of the central end of the gastric vagi branches
(fig. 18, C), and by direct stimulation of the esophagus. But the
contractions of the empty stomach, even when most vigorous, do not
influence the lung motor mechanism. |
Inhibition of the lung tonus and sabatrowticate: were not seen as a

result of artificial stimulation of the gut. But it seems clear that the — |
afferent nerves of the alimentary canal, especially the oral and anal

ends, make motor connections with the lung medullary centers.

b. The genito-urinary tract. Powerful lung contractions are induced
by mechanical distention, pinching, crushing, tearing or electrical
stimulation of the urinary bladder (fig. 18, A; ; fig. 19, c, b). Electrical .
or mechanical stimulation of the ureters, penis, prepuce and testis
also produces motor reflexes into the lungs (fig. 18, Dc; fig. 19, f, j).
As in the case of the alimentary tract, no reflex inhibitions into the —
lung were secured from the genito-urinary tract. In several female
specimens the ovaries and the oviducts were stimulated, without any
reflex influence on the lungs.. Stimulation of the kidneys also yielded
nothing definite in the way of lung reflexes. The most powerful and
consistent lung reflexes in this group are those elicited from the urinary =
bladder.

c. The visceral sympathetic nerves. Outside the alimentary, the genito-
urinary and the respiratory tracts no systematic or long-continued work
was made on possible lung reflexes from the viscera, except for the
stimulation of the central ends of the main viseeral sympathetic nerves
and connections. _We may note, however, that the stimulation of the
central end of the cardiac vagi branches has no effects on the lung.
Reflex lung contractions were, on the other hand, obtained from the
gall bladder, liver (fig. 19, g), and from the spleen.

295

VISCERAL SENSORY NERVOUS SYSTEM

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LUCKHARDT

CARLSON AND A. B.

J.

A.

296

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VISCERAL SENSORY NERVOUS SYSTEM ‘297

The stimulation (electrical) of the central end of the visceral sympa-
thetic nerves was made in a great many preparations, some in ‘good,
some in poor reflex conditions. ‘The results are positive, that is, the
stimulation of the visceral sympathetic nerves gives reflex lung contra¢-
tions (fig. 20). The reflex lung contractions are particularly powerful
from, nerves 2, 3 and 4 (fig. 2), these probably representing the aerONp
of splanchnic mantles nerves of the higher animals.

Reflex inhibitions of lung tonus and of lung contractions were never
seen as results of stimulation of the central end of the visceral sympa-
thetics.

Fig. 20. Water manometer tracings of the intrapulmonic pressure in the turtle,
showing reflex lung contractions on stimulation of the central end of various
sympathetic nerves. Animals decerebrated and completely curarized. Record
from isolated left lung; cannula in left bronchus. Preparations showing no ‘ene
taneous lung rhythm. A: stimulation with tetanizing pparent a, nerve 2 (see
fig. 2); b, nerve 3; c, nerves 2 and 3; d, nerve 4; e, nerve 5; f, nerve 6; g, nerve 7.
G: @, simulation with tetanizing Kieeent of nerves 2 and 3, showing reflex initia-
tion of a temporary lung thythm. ;

Results at times positive at times negative were obtained from the
central end of the cervical sympathetic (fig. 2). In some preparations
the stimulation of this nerve induced what appeared to be a reflex
lung contraction, in other preparations, in equally good reflex condition,
the stimulation had no effect on the lung. It appears, therefore, that
afferent components in the cervical sympathetic nerve trunk are. fei
in number and variable in their central action, at least as regards their
influence on the medullary centers controlling lung tonus and lung
contractions.

As the preparations are deteriorating the lung reflexes evoked ty om.
the visceral sympathetic nerves usually fail sooner than those called
forth by stimulation of the spinal sensory or the afferent nerves of the
respiratory tract itself. The cloaca, rectum and urinary bladder are

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, No. 2

298 A. J. CARLSON AND A. B. LUCKHARDT

an exception to this rule, the lung reflexes evoked from these organs
usually persisting to the end of all reflex response of the animal.

_ 4. The influence of visceral sensory nerves on the respiratory cenier.
-We have shown that, excepting the inferior laryngeal (inhibition), the
cardiac vagi (no action), the cervical sympathetic (inconstant), and
strong stimulation of the pulmonary afferents (inhibition), sensory
stimulation, spinal and visceral, induces. reflex lung contractions.
Since lung contraction is a normal adjunct to the external respiratory
act it is pertinent to ask whether these lung reflexes are not secondary to
the initiation or acceleration of discharges from the respiratory center.
What effects have these sensory stimuli on the rate and intensity of the
external respiratory movements? The turtle appears to be no exception
to the rule that stimulation of spinal sensory nerves accelerates or
initiates external respiration. As regards the spinal sensory nerves,
therefore, we have a complete parallel between effects on external
respiration and on lung contractions. Both are positive.

In regard to the visceral sensory stimulation the results are not so
clear.. In the first place much of our reflex work was done on completely
curarized animals, preventing parallel observation on the external
respiration. We can state, however, that so far as our observations
go, stimulation of the abdominal sympathetic nerves induces reflex
contraction of the respiratory muscles as an invariable result. But
discrepancies appear when we come to the stimulation of some of the
visceral organs, especially the cloaca, rectum and urinary bladder.
As already pointed out the mechanical or electrical stimulation of these
organs causes reflex lung contractions and never any inhibition, at
least in curarized preparations. But the same type of stimulation of
these organs in non-curarized preparations may cause pure inhibition
of the external respiration, as witness the tracing reproduced in figure
21. We are dealing here with two possibilities, viz., a, stimulation of
some of the visceral sensory nerves induces opposite effects on the two
medullary centers depressing the respiratory center and stimulating
the lung motor center; or b, the visceral afferents may have both effects
on the two centers, the end result (depression or stimulation) depending
on the intensity and rate of the artificial stimulation, the central action
of curare tending to reveal only the stimulation action on the lung motor
mechanism. Further work is required to establish either or both of
these possibilities.

— ee ee,

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—-

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299

VISCERAL SENSORY NERVOUS SYSTEM

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300 A. J. CARLSON AND. A. B. LUCKHARDT

RESULTS ON THE SNAKE

1. Spontaneous lung contractions. HH the external respiratory acts
are not too close together, each act of’external respiration is followed
by a contraction of the lung. ‘These lung contractions are feeble and
of very short duration (fig. 22, A). If the attempts at external respi-
ration come close together lung contractions appear only occasionally
or rather during the respiratory pauses (fig. 27, Ca).

Fig. 22. Tracings (delicate air tambour) of the intrapulmonic pressure in the
snake. Spinal cord cut and pithed below the medulla. Lung isolated; cannula
in tip, trachea ligated. A: signal, spontaneous respiratory movements, showing
lung contractions following each attempt at respiration; a, artificial respiration
through cannula in tip of lung; b, pithing of brain followed by cessation of lung
contractions. B: a, mechanical stimulation of skin of the head; 6, mechanical
stimulation of the cornea; c, mechanical stimulation of nares; d, electrical stimu-
lation of central end of left vagus—showing reflex lung contractions. C: a, lung
contractions following spontaneous respiratory efforts; b, tetanizing of both
peripheral vagi; c, stimulation of the peripheral vagi with increasing number of
electrical shocks—showing that motor discharge to lungs following respiratory
movements is nearly maximal, as brief tetanizing of vagi produces only slightly
greater lung contractions and that the vagi-lung muscle reacts to electrical stimu-
lation like an ordinary nerve-muscle preparation. Time, 5 seconds.

These lung contractions in the snake appear to us too feeble to be
of any value in the respiratory exchange of the lung.

2. Lung reflexes. Reflex lung contractions can be induced by the
stimulation of the central end of one vagus, the other vagus being intact,
and by stimulation of the nares, the cornea and the skin of the head
region (fig. 22, B). The lung contraction following stimulation of the
central end of one vagus is invariably preceded by an act of respiration,
that is, a discharge from the respiratory center. The other reflex lung
contractions may appear without being preceded by attempts at ex-
ternal respiration. :

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ae ee Se

VISCERAL SENSORY NERVOUS SYSTEM 301

8. Réle of the pulmonary vagi. Section of both vagi abolishes the
lung contractions associated with the acts of external respiration.
Stimulation of the peripheral end of either vagus causes contractions of
the lung. These lung contractions appear to be confined to the upper
third of the lung, that is, to the alveolated part or lung proper. It
is a singular fact that, with one exception, all the paired lung reptilia
thus far investigated, the lung motor action of the vagi is strictly
unilateral, while in this species of snake both vagi act on the one lung.
And it appears that the one-lunged condition of most snakes is due not
to fusion of the original pair but to atrophy of one of the pair.

_ The lung motor fibers in the vagi of this snake can be stimulated with
single induction shocks more readily than in the turtle. There is also
less evidence of a tendency to an all-or-none motor response in the
snake lung. In fact so far as our experiments go the vagus lung mus-
eu ulature behaves much like an ordinary nerve preparation. :
‘Lung inhibitions from stimulation of the peripheral vagi were never
observed. Destruction of the brain or section of both vagi neither
decreased or increased the peripheral lung tonus.. The lung vagi motor
mechanism is either not in tonic activity or our ‘method of preparing
the animal abolishes this tonic activity through central ‘‘shock.”’
~ While our work on the snake was limited to eleven rather small
specimens, the results indicate the essential parallel of the lung motor
physiology in the snake and the turtle, namely, the vagi motor control
and lung reflexes from the head origin of the animals. We have never
observed a peripheral lung motor mechanism in the snake after section
of the vagi.

THE DIRECT INFLUENCE OF THE SYMPATHETICS ON THE LUNG MOTOR
ea TISSUES

Jackson and Pelz (7) have published tracings apparently showing
strong inhibition of the lung tonus on stimulation of the central end
of the cervical sympathetic nerve. These investigators also state that
strong stimulation of this nerve may induce lung contractions of greater
amplitude than that caused by stimulation of the peripheral end of
the vagus. We have made great efforts to verify the results of Jackson
and Pelz, particularly in view of the réle which such inhibitory efferents
may play in the lung reflexes.

We have been unable to follow nerve branches from the cervical
sympathetic into the lung as figured by Jackson and Pelz. The sym-

302 A. J. CARLSON AND A. B. LUCKHARDT

pathetic pulmonary branches described by them appear identical with
the motor fibers from the brachial plexus to the striated muscle
attached to the anterior and dorsolateral wall of the lung.

Stimulation of the central end of the cut cervical sympathetic may
induce respiratory movements followed by lung contractions, provided
the spinal cord and the vagi are intact. Orif the cervical sympathetic
trunk is stimulated near its course past the brachial plexus, contractions
of the striated lung muscle, previously referred to, may be produced
by an escape of current to its motor fibers. These are the only motor
effects on the lung that we have seen following the stimulation of the
central end of the cervical sympathetic nerve, and ‘neither are due to
stimulation of sympathetic efferents.

Fig. 23. Tambour tracings of the intrapulmonic pressure in the turtle. Ani-
mals decerebrated, fixed dorsal side down, plastron removed; stomach, liver and
heart excised; vagi sectioned. Cannula in bronchus. A: Tracing showing a
tonus rhythm in the lung independent of the vagi-medulla motor mechanism.
B: Signal indicates stimulation of the central end of the cervical sympathetic
nerve, showing slight inhibition (possibly reflex) of the peripheral lung tonus
rhythm, developed after vagus section. Time, 5 second interval.

If the course of the sympathetic fibers to the lung in the reptilia
is the same as in the amphibia and the mammals, these fibers should
join the vagi in the neck or thorax and reach the lung via the pulmonary
fibers of the vagi. We have stimulated the peripheral end of the cervical
sympathetic nerve before its union with the vagus in very many prepa-
rations without any effect on the lung motor mechanism.

In one preparation only did we note any inhibitory effect on the lung
from stimulation of the central end of the cervical sympathetic nerve.
This preparation was decerebrated, fixed on the dorsal side, plastron
removed, and eviscerated according to the method of Jackson and
Pelz. A peripheral tonus rhythm appeared in the lung after section
of the vagi. Tetanization (weak and strong) of the central end of the
cervical sympathetic nerve induced some inhibition of this tonus

303

VISCERAL SENSORY NERVOUS SYSTEM

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304 A. J. CARLSON AND A. B. LUCKHARDT

rhythm (fig. 23, B). Reflexes through the spinal cord were not .

excluded in this experiment.

The actual presence of sympathetic efferents to the reptilian fon is,
therefore, an open question.

SUMMARY |

1. Under normal conditions the external respiratory act inhibits the
lung tonus by central action, and is followed by a contraction of the

lung during the respiratory pause. These lung contractions are of .
central origin, the peripheral motor path being the vagi nerves (con-

firming Kahn and Francois-Franck).

2. The medullary center controlling the lung tonus and ‘the lung
contractions is not identical with the respiratory center, although
normally associated with it in function in such a way that discharge
from the respiratory center first depresses and then stimulates the lung
motor center. The two centers are influenced in the same direction
by asphyxial states, the lungs being put in a condition of incomplete
tetanus by asphyxia, but one center may act, automatically or reflexly,
without the other; and the lung motor centers are more profoundly
influenced by adfererit depressor nervous impulses (traumatic ‘‘shock”’).

3. The lung contraction developed during the respiratory pause is
not a reflex depending on the muscular and other movements in the
external respiratory act, as they persist after complete curarization,
transection of the spinal cord in the neck, pithing the cord, or complete
isolation of the lungs, save for the pulmonary vagi and blood vessels.

4, After section of the vagi a feeble tonus rhythm may appear in
the lungs, but the strong contractions associated with external respi-
ration are permanently abolished. Stimulation of the peripheral end
of the vagi causes contractions and tetanus of the lungs, confirming

the original observation of Bert. This vagus-lung action is unilateral. |

Stimulation of the peripheral vagus reveals no inhibitory action on the
lung motor mechanism. Stimulation of the optic lobes or the medulla,
the vagi being intact, causes lung contractions or incomplete lung
tetanus (confirming Coombs). The lung tetanus is less complete than
on direct stimulation of the peripheral end of the vagi, thus showing
a central refractory state.

5. The physiological relation of the respiratory and the lung motor
centers have a complete parallel in the relations of the activity of the
swallowing center to the medullary mechanism controlling esophageal
peristalsis. The action of the motor fibers of the vagi on the lung motor

ae

VISCERAL SENSORY NERVOUS SYSTEM 305

_ tissues appears to be more direct and less complicated than the motor
action of the vagi on the stomach, although there is some evidence of
a tendency to an all-or-none response and rhythm in case of weak
tetanization of the peripheral ends of the vagi even in the case of the
lungs.

6. Inhibition as a primary act on the lung motor center in the medulla
is produced by trauma to the vagi, stimulation of the central end of
the inferior laryngeal nerve, and by strong stimulation of the central
end of the pulmonary vagus. Trauma to the body causes a profound
depression (“‘shock’”’) of the lung motor center following an initial
stimulation.

7. Reflex lung contractions or incomplete lung tetanus can be in-
duced from stimulation of the sensory nerves of the respiratory tract:
(excepting the inferior laryngeal), that is by inflation, by deflation, or
mechanical pressure on the lung of the opposite side, by weak tetani-.
zation of the pulmonary afferents, mechanical stimulation of the larynx
and the nares. It is thus clear that the normal respiratory act by lung
inflation and deflation will by itself induce a lung contraction reflexly.
This afferent component from the lung is not necessary for the dis-
charge of the lung motor center associated with the respiratory rhythm,
but may act as a cumulative factor. The lung contractions and lung
tetanus induced reflexly from mechanical stimulation of the posterior
nares are particularly striking.

8. Mechanical and electrical stimulation of the alimentary tract
(esophagus, gastric vagi, large and small intestine, rectum, cloaca)
and the genito-urinary tract (urinary bladder, ureters, penis, prepuce,
testes) induces reflex lung contractions or lung tetanus, the lung tetanus
caused by stimulation of the cloaca, rectum and urinary bladder being
particularly marked. Reflex lung contractiom can also be evoked from
stimulation of the gall bladder and the spleen.

9. Stimulation of the central ends of the visceral sympathetic nerves
causes reflex lung contractions.

10. Stimulation (mechanical or electrical) of the cutaneous nerves,
the cornea and the central end of the sciatic or brachial nerves induces
reflex lung contractions or lung tetanus, depending on the strength of
the stimulation.

11. The conclusions in regard to lung motor rhythm, its central
and peripheral control, and the lung motor reflexes evoked from the
head and neck region of the animal, apply both to the turtle and the

snake. The lung reflexes from the viscera below the neck were not
studied in the snake.

306 A. J. CARLSON AND A. B. LUCKHARDT

12. It would thus appear that the predominant reflex control of the
lungs in these animals, at least under our experimental condition, is
motor, thus differing entirely from the amphibia, where the predominat-

ing control (automatic and reflex) of the lung motor mechanism is .

inhibitory. |
BIBLIOGRAPHY

(1) Bert: Legons sur la physiologie comparée de la réspiration, Paris, 1870.
(2) Fano AnD Fasoa: Arch. ital. d. Biol., 1894, xxi, 338.
(3) Francois-Franck: Arch. d. Zo6l. Exper., 1908, ix, 31.
(4) Francots-Francx: Arch. d. Zoél., Exper., 1909, x, 547.
(5) FRANgoris-FrRancK: Compt. Rend., 1906, lxi, 6.
_(6) Francois-FRANcCK: Compt. Rend. Soc. Biol., 1906, Ixi, 127.
(7) JACKSON AND Petz: Journ. Lab. Clin. Med., 1917, ili, 344.
(8) Kaun: Arch. f. Physiol., 1902, 29.
(9) Leypie: Lehrb. d. Histologie d. Mensch. und d. Tiere, Frankfurt, 1857 (as
quoted by OpPEt).
(10) Prevost BT Satoz: Arch. Internat. de Physiol., 1909, viii, 327.
(11) Scuuuze: Stricker’s Handbuch der Lehre von den Geweben d. Mensch. u.
d. Tiere, Leipzig, 1871 (as quoted by OPPEL).
(12) Coomss: This Journal, 1920, 1, 511.
(13) CARLSON AND RuckwAnor: This Journal, 1920, liv, 55.
(14) LuckHARDT AND CaRtson: This Journal, 1920, liv, 122.

ae

STUDIES OF THE RESPIRATORY MECHANISM IN CARDIAC
DYSPNEA

I. Taz Low ALvEoLtAR CARBON Dioxipe or CarpIAC DyspNEA

JOHN P. PETERS, Jr. anp DAVID P. BARR

From the Russell Sage Institute of Pathology, in affiliation with the Second Medical
Division, Bellevue Hospital and the Department of Medicine,
Cornell University Medical College

Received for publication August 9, 1920

It has been shown by many observers, working with various methods,
Beddard and Pembrey (1), Porges, Leimdoerfer and Markovici (2),
Peabody (3), Pearce (4)—that the alveolar carbon dioxide tension is
usually found to be low in cardiac dyspnea. The causes and meaning
of this phenomenon have never been clear. In cardiac dyspnea, in
contradistinction to most conditions that are associated with a low
alveolar CO2, no proportionate reduction of the alkaline reserve of the
blood has been found.

In 1917 one of us (5) studied the relation of the carbon dioxide tension
of alveolar air to the bicarbonate concentration of venous plasma. A
low alveolar CO,-tension did not prove to be a constant characteristic
of cardiac dyspnea. The alveolar CO. was sometimes normal. But
it was always lower than normal in relation to the alkaline reserve of
the blood as determined by the Van Slyke method. Van Slyke (6)
found that in normal subjects the alveolar CO.-tension maintained a
fairly constant relation to the plasma bicarbonate. If the milligrams of
CO, chemically bound in 1 cc. of plasma is multiplied by the constant
35, the result will be found to agree with the alveolar CO,.-tension
(Haldane) expressed in mm. Hg. with about a 10 per cent variation.
That is:

(Alveolar CO, in mm. Hg.) + (mgm. CO, chemically bound by 1 ce.
of plasma X 35) = 100 + 10.

This observation we corroborated. That such a ratio should obtain
in normal resting subjects seems reasonable. To a certain extent it
307

308 JOHN P. PETERS, JR., AND D. P. BARR

must represent the HeCO;/NaHCoO; ratio, if the alveolar CO. can be
assumed to be a measure of the arterial CO.-tension and the bicarbon-
ates of venous plasma an indication of the bicarbonates of whole blood.

In most normal resting subjects such assumptions will give rise to
no serious errors. The difference between the carbon dioxide content
of the arterial and the venous blood is not large and is comparatively
constant. In pathological conditions which interfere with the general |
circulation or the ventilation of the blood in the lungs, a disturbance
of these factors and consequently of the ratio may be expected.

In normal resting subjects the alveolar CO2/plasma bicarbonate ratio
varied between 0.90 and 1.10. In cardiac decompensation with dyspnea
and in some very advanced pulmonary conditions we obtained ratios
consistently below 0.85. To draw any definite conclusions as to the
cause of the phenomenon was impossible. We suggested that it might
be an expression of some defect of the normal mechanism for the elimi-
nation of carbon dioxide and pointed out the possibility of connecting
this with the pulmonary changes which had been demonstrated by
Siebeck (7), Peabody (8) and others.

Before any interpretation of the low alveolar CO,! of cardiac de-
compensation is attempted it is obviously necessary to ascertain whether
the method employed is applicable and whether the results obtained
can be said to represent the true alveolar tension. The prel minary
work was done with the Fridericia pipette (9). This has given rise
to some criticism. Repetition with a more orthodox Haldane method
has given substantially the same results. Although Pearce (4) has
criticised the use of the Haldane method, observations by his own
method are substantially in agreement with ours.

Siebeck (7), after a careful study of the respiratory mechanism in °
cardiac insufficiency, came to the conclusion that all determinations
of the alveolar CO, were useless in this condition. According to Sie-
beck (7) the alveolar aeration in cardiac dyspnea is very imperfect,
in consequence of which the expiratory air contains an excess of un-
changed inspiratory air. Apparently he means that attempts to deduce .
the arterial CO.-tension from the values obtained by the Haldane
alveolar method are unwarranted. But it is perfectly possible to

1 Although the reduction of the alveolar CO2-tension is not absolutely con-
stant, few patients fail to show it. In these few the carbon dioxide capacity of
the plasma is distinctly high, while in most instances it is at or slightly below the
normal level. It seems proper and simpler, for this reason, to speak of the low
alveolar CO: of cardiac dyspnea.

/

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 309

consider the alveolar CO, on its own merits as a functional entity
without any deductions about arterial CO,-tension. However well
grounded the anatomical conceptions of Haldane with regard to the
origin of the alveolar air in the normal lung may be, it is quite con-
ceivable that in pathological or physiological disturbances, conditions
may be so altered that any anatomical term loses its original significance.
_ Here a functional conception proves of greater value.

A normal expiration may be divided into two parts. The first part
is practically useless from the standpoint of respiration; it merely
serves to empty the dead space (the nose, mouth, pharynx, larynx,
trachea and bronchi) of the reom air with which it was filled by the
last inspiration. In normal resting subjects the volume of this dead
space is fairly constant, varying about an average of 130 cc. Behind
this lies a mass of air of nearly constant gaseous composition through- -
- out its whole extent and in close contact with the blood in the pul-
monary circulation. This is the air which serves for the effective
oxchange of gases between the blood and the outside air, and it is a
sample of this air that the Haldane method attempts to obtain.

The first object of these studies was to find out whether samples
obtained by the: Haldane method represented the effective respiratory
air. We have employed other methods in order to determine whether
they give results in agreement with the Haldane values.

Our studies have been confined to three methods: the Haldane (10),
the Plesch (11) and the Henderson (12) venous COs. Pearce’s (13)
method has been omitted because its use in severe dyspnea presents
considerable difficulties and also because it demands an amount of
apparatus that renders it less adaptable as an ordinary clinical procedure.

THE HALDANE METHOD

The Haldane technique for obtaining alveolar specimens, although
criticised for various reasons, is still considered the standard technique
for the determination of the arterial CO,.-tension in normal subjects.
It has not been generally adopted for clinical studies because of a popu-
lar opinion that it is difficult or impossible to obtain good samples in
any but highly trained subjects. This we have not found to be the -
ease. We are inclined to believe the observer needs more training than
the subject. !

In these studies we have adhered in all essential respects to the
orthodox Haldane technique, introducing only slight modifications

310 JOHN P. PETERS, JR., AND D. P. BARR

which tend to diminish subjective errors. One of these is by no means
original and consists of the introduction of a three-way brass stop-cock
designed by Mr. G. F. Soderstrom and shown in figure 1. This makes
it unnecessary for the patient to close the tube with his tongue. Fur-
thermore, no attempt has been made to collect inspiratory and expi-
ratory specimens, as Haldane advises. Instead, samples have been
obtained from the end of a forced expiration begun as soon as possible —
after the completion of a normal inspiration. When the respirations

A tie

2

Fig. 1. Three-way stop-cock. 1, tube for attachment of mouth-piece; 2, tube
leading to spirometer or left open to outside air; 3, tube for attachment of Haldane
tube, with side-arm C, to connect with gas sampling tube.

are rapid, as they are in cardiac dyspnea, it is almost impossible to
turn the valve at the proper moment to differentiate inspiratory and
expiratory alveolar air with any degree of certainty. The propriety
of using an expiratory specimen also seems questionable. The pro-
longation of expiration during dyspnea may be equivalent to holding —
the breath for the duration of an extra respiration. We have tried to
make the subjects force the air out rapidly enough so that the expiration
is not considerably longer than a normal one. The specimens as ob-
tained by this method are not all from expirations initiated at the same

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 311

point in the respiratory cycle, but at various points in the expiratory
phase between full inflation and deflation of the lungs.

In detail the procedure is as follows: The rubber mouth-piece with
the stop-cock and tube in place is first put in the patient’s mouth with
the stop-cock open to the air. He is encouraged to breathe through it _
to convince him that it will not “shut off his breath.” After he has
gained sufficient confidence the tube is removed from his mouth and
a simple spring nose-clip applied to his nose. When he is sure he can
breathe through his mouth alone without difficulty the mouth-piece
is replaced in his mouth. He is then instructed how to deliver a sample
on command and without altering his normal respirations. The
sampling tube is then attached to the side-arm of the stop-cock. The
operator stands at the right of the bed supporting the tube with his
left hand, with his right on the handle of the stop-cock. When he
feels that the breathing is normal and regular he tries to accustom him-
self to its rhythm, so that he may give the signal at the right moment.
The signal is given and the stop-cock turned at the same moment. At
the end of the forced expiration the cock is turned to the outside air
again before the subject has had time to gasp for breath. The operator
soon learns to know whether there has been a subjective error in the
technique on his own part or that of the patient.

From an inexperienced subject we are accustomed to take several
specimens. Although some of the first will occasionally be obviously
too low, it is generally possible to get good agreement on the third or
fourth attempt in even the most obtuse subject, as may be seen in
tables 1 and 2. In a total of 48 observations on 25 patients, involving
the collection and analysis of 127 samples, all but four patients gave
duplicate samples that varied by 0.4 per cent or less, an accuracy that
compares favorably with that found in trained subjects. Patients with
cardiac dyspnea proved no exception to this rule. Three of the four
patients (A. R., J. J. F: and D. W.) who gave unsatisfactory results
on the basis of this criterion, exhibited Cheyne-Stokes breathing. The
fourth (P. O. S.) was very ill, somewhat irrational and showed a slight
respiratory irregularity.

The results obtained by the Haldane method are in accordance with
those previously obtained with the Fridericia tube. The values are
comparatively low and much lower than should be expected from the
level of plasma bicarbonates. Two obvious criticisms of the method
might be made:

1. That the volume of the expiration is too small to clear the patient’s
dead space.

312 JOHN P. PETERS, JR., AND D. P. BARR
TABLE 1 bene!
Variations in duplicate Haldane specimens in subjects without respiratory or cardiac
disorders = . ;
NOR ee Lath Gunpinow ALVEOLAR | sonvED VARIA-|CEPTED VARIA-| ALVEOLAR
TION TION* - CO2.
per cent ‘mm.
( 6.16 0.22 0.22 43.1
5.94
5.25 0.06 — 0.06 37.4
5.31
5.72 0.02 0.02 40.6
5.74 :
5.39 0.13 0.13 tee
5.26
5.16 0.24 0.24 |. 87.6
5.40
5.39 0.18 0.18 39.0
DD. o> D., DOMME tee 5.57 ;
. 5.60 0.00 0.00 | 38.7
5.60
5.39 0.21 0.21 | 39.2
5.60
5.45 0.05 0.05 38.8
5.50
5.62 0.11 0.11 39.1
5.51
5.32 0.34 0.34 | 38.6
5.24
||. 5.58
A. tas ee 0.46 0.29 39.9
5.64
. 5.74
JesPg POMEL Bs uke ase 4 5.45
5.94 0.09 0.09 41.6
ties ik '

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 313

TABLE 1—Concluded

susmscranpconprmon | “2VECUAR |scevenvanta-|currepVamia-| ALVEOLAR
TION ; TION* COz2z
per cent mm
fincas fe. 0.26 0.26 — 40.5
5.62
SS ee are ‘
. 5.35 0.15 0.15 37.5
t 5.20 .
6.24 0.07 0.07 44.4
SS ra
6.31
4.70 0.12 0.12 94.8
W. S. pee porms! gs 4.89 a
5.88. 0.25 0.25 41.2
P. K., gastric neurosis...... 5.61
. 5.86
Be a 0.16 0.16 30.1
4.19
4.35
Jno. K., diabetes mellitus. .{
4.07 0.05 0.05 28.4
4.06
! 4,02
3.98 0.11 0.11 28.2
C. P., pernicious anemia. .. 3.91
7 4.02
4.94 0.32 0.32 34.7
H. R., polycythemia....... 5.11.
4.79

* Values in parenthesis in this and the following table have been discarded for
‘the most part on the basis of internal evidence, only. .

2. That the low values are produced by subjective errors; preliminary
- forced inspirations or gasping at the end of expiration.

To rule these out we attached the Haldane tube to a Tissot spirom-
eter which was equipped with a calibrated recording device which
will be described later. By this means we were enabled to obtain a
graphic record of the respiration during the entire time that the stop-
cock was turned, and could at the same time measure the volume of the

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 2

JOHN P. PETERS, JR., AND D. P. BARR

314

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320 JOHN P. PETERS, JR., AND D. P. BARR
TABLE 3
Relation of alveolar COz to volume of expiration
sunsecr roumen oy | Sea
ce, per cent
1016 5.09
J..C., chronic cardiac compensated.................. i as
790 5.38
797 4.08
864 4.00
W W., chronic cardiac decompensated ............... ‘ 615 3.97
665 (4.35
‘ 385 4.32
710 5.90
452 5.67
645 5.78
R. W., chronic cardiac compensated................- 4
758 6.10
651 6.22
‘ 516 6.52
1081 4.51
J. D. B., chronic cardiac decompensated............. _ pa
986 | 4.34
| 505 3.96
A. R., cardionephritic. Periodic breathing......... po" a
. 505 4.21
(758 3.08)
D. W.,* cardionephritic. Periodic breathing........ bell ap
952 3.59

* See figure 2.

expiration. Table 3 shows the values obtained from six experiments
done in this way. From the first five experiments it is evident that the
expiratory volume is sufficient to clear completely any but an enor-
mously increased dead space. If the dead space were increased to this —

a i i

eo ae

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 321

degree, however, the alveolar CO.-tension should vary with the expi-
ratory volume. ‘This relation does not obtain. In only one instance,
the second observation on R. W., is any such relation even suggested,
and in this case the total variation is only 0.23 per-cent.

In none of these cases is there any indication in the graphic records

of inspiratory activity during the time that the stop-cock was open.

That the method is capable of showing such subjective errors is demon-
strated in the record of D. W. (fig. 2). This patient presented symp-
toms of uremia with Cheyne-Stokes respiration. His tracings show
that the low values obtained from the first and third samples were due

DW.

PRreoeic.e.) DtyZ %
Ap TS2e.c. (3.08%)
(Nee cc. 4.077%

629 c.. (3. 22%).

Fig. 2

to inspirations while the valve was open and before the samples had
been withdrawn. The discrepancy in the two remaining values seems
to depend on the fact that one was obtained during an apneic, the other
during a dyspneic period.

The low alveolar CO, is not due to subjective errors on the part of
the patient nor to any considerable increase in the volume of the dead
space. |

The alveolar CO, after rebreathing. One of the most striking char-
acteristics of decompensated cardiac patients is the intolerance to an
increase of CO, in the inspired air (14).

322 JOHN P. PETERS, JR., AND D. P. BARR

It has been shown by Haldane (15) and others that when a person
rebreathes air the alveolar CO.-tension and the ventilation increase as
the CO, in the inspired air rises. When the alveolar CO,-tension has
risen to a certain point the patient becomes extremely dyspneic and
uncomfortable. This we may call the point of intolerance to COs.
If the alveolar CO, be low in cardiac dyspnea at rest it seems reasonable
to suppose that it should also be relatively low after rebreathing to the
point of intolerance.

For the rebreathing experiments a 100-liter Tissot spirometer, ac-
curately balanced and calibrated at all levels, was employed. To
separate the inspired from the expired air a two-way T-valve of the
- Douglas type was used. Between the mouth piece and this T-valve
was interposed the three-way brass stop-cock described above. The
opening with the side-arm was, as usual, fitted to a long, wide-bore rub-
ber tube for the collection of alveolar specimens. The other arm was
connected with the T-valve. (The total instrumental dead space was
about 50 cc.) In this way it was possible to collect specimens of alveolar
air during the course of a rebreathing experiment by merely turning
the stop-cock during expiration and directing the patient to breathe
out forcibly. The inspiratory air was withdrawn from the spirometer
through the opening in the top usually employed for the insertion of
the thermometer. Samples of the inspiratory air were drawn from
this tube near the T-valve through a small rubber side arm. Graphic
records of the respirations were obtained by means of a pen attached
to the counter-weight of the spirometer. As it moved in a vertical
plane only, direct calibration was possible. Simultaneous time records
were obtained. The apparatus is represented diagrammatically in
figure 3. ;

The spirometer was filled with 20 liters of room air for each experi-
ment. A larger amount was found to prolong the experiment unduly
and to introduce a considerable element of fatigue. Smaller amounts
rendered the succession of events too rapid to permit the introduction
of all the necessary procedures and accurate analysis of the results.
obtained.

The minute volume attained in the rebreathing experiments was
calculated from the average tidal air and the respiratory rate during

the last half-minute of the experiment, except in one or two cases where _

the limit of tolerance was reached so rapidly that it was necessary to
use a shorter terminal period for purposes of calculation. Of course,
assuming that the minute volume increases continuously throughout

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 323

B

Fig. 3. A, mouth-piece; B, 3-way stop-cock; C, Douglass valve; F, inspiratory
tube; G, expiratory tube; E, counterweight of spirometer; D, recording pen; H,
Haldane tube; a, sampling tube for alveolar air; 7, sampling tube for inspiratory
air.

_ i
=

324 JOHN P. PETERS, JR., AND D. P. BARR

the course of the experiment, the values obtained in this way must
be somewhat too low. On the other hand, the respirations never be-
come absolutely regular, and calculations based on too small a number
will be subject to considerable error. In rebreathing so large a volume
as 20 liters the respiratory increase is sufficiently gradual to permit
the use of the last half-minute, in the majority of instances, with the
introduction of no significant error.

After a satisfactory determination of the resting alveolar CO, and
minute volume had been made, a rebreathing experiment was begun.
The patient was urged to continue as long as he felt able. When he
had reached the limit of tolerance the valve of the three-way stop-cock
_ was opened to the Haldane tube during an expiration. The patient
was told to breathe out forcibly and the valve was again closed before
the following inspiration. ‘The patient was at once disconnected from
the apparatus and the alveolar sample removed for analysis. The
second operator at the same time removed a sample from the inspiratory
tube.

Of course, the coédrdination and coéperation of the subject were
necessary and there were opportunities for mistakes. Altogether eleven
successful experiments were made on two normal subjects; one out of

two was successful in a patient with a gastric neurosis; one in shell

shock; three in two compensated cardiacs; and five out of seven
attempted in four patients with cardiac decompensation. The results
appear in summarized form in table 4. ;

A very striking difference is apparent at once between the decompen-
sated cardiac cases and the others. The former were unable to tolerate
as much COQ, in the inspired air and their alveolar CO, did not rise to
as high a level. This was due to no lack of will power on the part of
the cardiacs because in one or two cases they continued rebreathing
to the point of exhaustion, while none of the compensated subjects
went beyond a state of moderate discomfort. The alveolar CO, at
the point of intolerance is therefore lower in decompensated cardiacs
than it is in normal persons. |

With the exception of A. J., the shell shock patient, the height of the
alveolar CO, at which intolerance was reached bore a general relation
to the preliminary alveolar CO.-tension. In this one case the low rest-
ing alveolar CO. was due to over-ventilation. This was clearly shown

by the fact that his initial reaction to rebreathing was a reduction of

the minute-volume, instead of an increase.

7

a
a
5 i

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 325
TABLE 4
The effect of rebreathing on the alveolar COz
AT END OF REBREATH-
ING EXPERIMENT
inj ledge Femi aind DIAGNOSIS AND REMARKS
CO2 _ |Inspiratory| Alveolar
air CO2 O2
per cent per cent per cent
8.36 8.52 | Normaladult. Continued rebreath-
6.05 8.01 8.16 ing to point of considerable dis-
comfort
baD..P. Bo 7.93 8.21 | To discomfort
; . 5.28 8.06 8.30 | To discomfort
5.73 } 7.84 7.95 -| To slight discomfort
( , 6.86 7.53 | To slight discomfort
7.90 8.32. | Normal adult. To considerable dis-
comfort
SMe Box 7.95 7.98 | To extreme discomfort
6.90 7.27 | To discomfort
6.86 7.05 | To considerable discomfort
ie: yf 6.61 Gastric neurosis
hay ok . See 5.50 6.07 6.85 | To moderate discomfort. No ex-
} haustion
5.50 5.90 7.30 | Chronic cardiac valvular disease,
4. T. deC.. ; compensated .
5.70 6.59 7.27 | Rebreathed to point of moderate dis-
comfort
5. Jo M.L 5.23 7.54 7.92 | Chronic cardiac. Compensated.
Continued rebreathing to point
of considerable discomfort
eon y 4.51 6.26 6.30 | Shell shock with effort syndrome.
te 4.72 6.30 6.89 | . To moderate discomfort only
7 JA 2 5.92 5.74 6.36 | Chronic cardiac, valvular disease,
anh 4.52 7.34 compensated while at rest. Re-
breathed to point of considerable
discomfort with some exhaustion
4.50 3.70 Chronic cardiac valvular disease.
OP ie Misa 4.50 3.92 5.55 Moderate dyspnea while at rest.
4.11 5:17 6.26 In the first two experiments con-
: tinued only to mild discomfort.
In the last experiment consider-
able discomfort. Somewhat ex-
hausted

326 JOHN P. PETERS, JR., AND D. P. BARR

TABLE 4—Concluded

AT END OF REBREA1H-
ING EXPERIMENT >
ee a viene i _ DIAGNOSIS AND REMARKS
Oz Inspiratory} Alveolar
air COz CO2
per cent per cent per cent
9. Jos, C,...:| 5:93 3.88 6.68 | Cardio-nephritic with cardiac de-
compensation. Considerable
dyspnea and hyperpnea. Con-
tinued to point of considerable
discomfort. Somewhat exhausted
10: CDi: 5.00 2.13 5.10 | Chronic cardiac, valvular disease
4.57 2.89 with marked decompensation.
Continued to point of marked
exhaustion
rh Pe i aye ee 4.17 2.94 Chronic cardiac, valvular disease
2:58 5.72 |. with decompensation. Continued
rebreathing to point of discomfort
with some exhaustion

In cardiac dyspnea, then, the alveolar CO: is low at the point of
intolerance. The relation of the alveolar CO, at this point to the resting
~ alveolar COz is similar to that found in normal persons. This is further
evidence that the air in the lungs in cardiac dyspnea really presents a
diminished CO>-tension.

THE PLESCH METHOD

Modifications of the Plesch method of obtaining samples of alveolar
air have been employed extensively in cardiac disease and other patho-

logical conditions by numerous workers. Both Porges (2) and Peabody ©
(3) used it for their studies on cardiac dyspnea. With this method, the —

subject rebreathes a limited volume of air, usually 600 to 1000 cc. a

number of times, usually from 5 to 10, in a period of time usually be- —

tween 20 and 30 seconds, which is supposed to be less than the duration
of a single complete circulation of the blood. With normal resting

subjects it gives values that lie a little higher than those obtained by |
the Haldane method and is assumed to represent the tension of tie

in the venous blood.
Usually the rate and depth of the respirations and the duration of

rebreathing can be varied within rather wide limits with only a slight _

:
r
{
3
’

ae as ee es. i
ae ae

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA oar

variation in the analyses obtained. This might reasonably be expected
even if the theoretical basis of the method were entirely wrong. It
has been shown that in normal subjects at rest, ung volume, carbon
dioxid output, arterial CO,-tension and venous CO,-tension vary within
narrow limits absolutely and in relation to ohe another.

The assumption that such an empirical method is-equally applicable
to the study of a condition in which all these factors are greatly dis-
turbed, is hardly justifiable.

We hair made a few observations on normal sibibitte a and on patients
with cardiac disease (see table 5) in which we tested the effect of varying

TABLE 5

Results of varying time and number of respirations on Plesch-alveolar CO>

NORMALS J.M.L. DECOMPENSATED CARDIACS
COMPEN-

SATED
D, .P.-B. J.P.| carprac |8.I.| D.O.C. |W.W. Jie

5 respirations in 25 sec-

onds.. eeduaee . .(6.17/6.04/5.24/6.29| 6.08 |4.48]4.85)5.41/4.70/5.03/4.82 -
10 PunieaGiinn in 25 sec-

OE FOIE ORL ENS 6.53)5.97/5-90/6.22| 6.76 |4.62/4.92/5.88/5.41/5.14/5.53
5 respirations in 35 sec- |

© RR pease ittiiae oa on 6.43/6.35/5.89/6.34) 6.90 15.99|5.67/5.63/5.81
10 respirations in 35 sec-

meee 2 ae .. .|6.34/6.41/5 .88/6.43) 6.92 |4.70 6.29/5.99/5.50
Average value........... 6.37/6.19/5.73/6.32| 6.67 ,|4.60/4.89/5.8915.44/5.33/5.17
Maximum variation... .. .|0.36)0.44/0.66/0.21) 0.84 |0.22/0.07/0.88|1.29/0.60/0.99
Haldane alveolar COs... .|5.28/5.33 5.61; 5.41 |8.86/3.72/3.75/4.34/4.11/4.11
Difference between , .

Plesch and Haldane.. .|1.09/0.86 0.71) 1.26 |0.74)1.17/1.99)1.10)1.22/1.06

the time and rate of rebreathing within certain limits. The time limits
chosen were 25 and 35 seconds; the number of breaths 5 and 10. The
apparatus used was essentially the same as that recommended by
Peabody (16): a three-way brass stop-cock described above, which
connected the subject either with the outside air or a rebreathing bag
containing 1000 cc. of room air. The bore of the valve and tubing
was 1.75 cm., the instrumental dead space, including the rubber mouth-
piece, 30 cc. Rebreathing was always begun after a forced expiration
and the patient was directed to make a maximum respiratory effort
with each breath. |

328 JOHN P. PETERS, JR., AND D. P. BARR

In the case of J. P. the total difference obtained within the limits
of variation of time and number of respirations employed was 0.21
per cent. In D. P. B. the differences were greater: from 0.36 to 0.66
per cent. Moreover, in one experiment on the latter (no. III) it appears
that the duration of the experiment has less influence than the number
of respirations. In the cardiac patients studied the differences were
slightly larger, in one case 1.29 per cent. Again variations in the
number of respirations appear almost as important as variations in
time. One would be rather at a loss to BOW which values to accept
under these conditions.

Of course 35 seconds and 10 breaths are both in excess of the limits
_ usually employed. The objection may be raised that we have not
limited the element of time and the number of breaths sufficiently.
To us it seems essential to determine whether limitation of experimental
factors produces an equivalent limitation of variation in all subjects.
One is not justified in producing an apparent constancy in results by
an arbitrary limitation of experimental variations.

THE HENDERSON-LAURENS METHOD

Y. Henderson (12) has recently published a method for the deter-

mination of the venous CO.-tension. It does not differ in principle

from previous methods, but demands less effort and intelligence on the
part of the subject and is therefore more applicable to clinical studies.
It depends on the principle of intermittent rebreathing. Laurens (17)
has pointed out that it is necessary to regard certain precautions in
rebreathing because diffusion of CO, is more perfect during respiratory
motions than it is while the chest is motionless. Our technic has been
modified, therefore, to meet his requirements.

The apparatus used was the same as that employed in the Plesch
studies. The rebreathing bag was filled with about 2000 ce. of expi-
ratory air. In most cases a second observation was made in which the
bag was filled with a mixture of air with 6.5 to 10 per cent COx. While
the patient was breathing room air quietly through the valve, he was
ordered to give a maximum expiration. When his lungs were com-
pletely deflated the stop-cock was turned. He then filled his lungs with
a mixture from the bag by a deep inspiration, retained the air in his

lungs about 10 seconds and expired into the bag forcibly. The stop-—

cock was then turned to the room again and he resumed normal respi-

rations. After a sufficient interval to permit the respirations to return

= ER I oo se eee hh

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 329

to normal. the same procedure was repeated. After each respiration
the mixture in the bag was tested for CO.. The rebreathing was con-
tinued until analyses after successive respirations showed that the CO,-
tension had reached a practically constant level. In normal subjects

_ this level is reached after about 7 rebreathings and the level attained
in rebreathing expired air and CO; rich mixtures is the same.

The results of these studies are shown in table 6. Not all of the

experiments are entirely satisfactory; but a more or less constant level _

is reached in cardiac subjects as well as in normals.

There is a surprisingly close agreement between the venous alveolar
values obtained by the Henderson-Laurens method and the average
of the values obtained by the Plesch method. The application of the
Plesch method, in the simple form usually employed, to the physiologic
study of cardiac disease, seems hardly warranted. The limitation of
the number of respirations and the duration of rebreathing is more or
less arbitrary and based on the study of normal subjects only. The
use of an average value obtained from a series of observations in which
both time and number of respirations has been varied seems preferable.
In normal and cardiac subjects values so obtained agree with those
obtained by at least one other venous method.

The difference between the Henderson and the Haldane alveolar
CO, is greater in subjects with cardiac dyspnea than in normal persons

or cardiac patients without dyspnea. In spite of this the actual values

found by the Henderson method in cardiac dyspnea are slightly lower
than normal. This is again evidence that the carbon dioxide tension
of the air in the lungs is reduced in cardiac dyspnea. .

It is not possible to argue that the difference between the Henderson .
and the Haldane alveolar CO, represents the difference of CO.-tension
between arterial and venous blood. The Haldane values can not be
interpreted as a measure of the arterial CO.-tension until the criticisms
of Siebeck have been answered. The only way to answer them is by
the direct determination of the arterial CO,-tension. ‘The objections
to the application of alveolar methods to the determination of the carbon
dioxid tension of venous blood are even greater. Christiansen, Douglas
and Haldane (18) have pointed out that direct determination of the
venous carbon dioxid tension by these methods is impossible. The
peculiar effect of oxygen on the carbon dioxid dissociation curve of the
blood necessitates the introduction of a correction for the oxygen
unsaturation of venous blood. An average correction can be applied
in the case of normal persons without any considerable error. In

wey

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JOHN P. PETERS, JR., AND D. P. BARR:

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RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 333

patients with cardiac decompensation, however, the oxygen unsatu-

ration of the venous blood is much greater (19) and more variable.
The use of a standard correction factor may therefore involve consider-
able error.

SUMMARY AND CONCLUSIONS

‘The Haldane method of obtaining alveolar air has been employed
in the study of a series of cardiac patients with dyspnea with no greater
variation in results than is found in studies of trained normal subjects.
The carbon dioxid tension of alveolar air thus obtained has been found»
consistently low in comparison with the carbon dioxid capacity of
venous plasma. The comparatively low values are not due to technical
or subjective errors nor to any considerable increase in the volume of
the dead space. The patient with cardiac decompensation maintains
his alveolar CO, at a lower level than does the normal and the level
to which he will permit it to rise under the influence of rebreathing is
proportionately reduced.

The Plesch method gives variable results according to the number
of respirations and the duration of rebreathing. If, however, an average
of a series of observations in which these factors have been varied is
employed the results agree with those given by the Henderson method
for venous alveolar CO.. The values found by these methods are some-
what lower in decompensated cardiac subjects than in normal persons;
but are high in relation to the values given by the Haldane method.

No attempt has been made to translate alveolar CO,-tension into
terms of arterial or venous CO,.-tension. The alveolar air has been

considered entirely from a functional standpoint as that portion of the

air in the lungs which is available for the exchange of gases between the
blood in the pulmonary circulation and the outside air. If the term
“alveolar air’ is used in this sense, it may be said that the alveolar

-CO,-tension of subjects with cardiac dyspnea is low in comparison with

the carbon dioxid capacity of the plasma. As the latter is variable,
but seldom abnormally high, the alveolar CO,-tension is usually not
only relatively but absolutely diminished.

BIBLIOGRAPHY

(1) Bepparp AND Pemsrey: Brit. Med. Journ., 1908, ii, 580.

(2) Porars, LEIMDOERFER AND Markovict: Zeitschr. f. klin. Med., 1913, lxxvi,
446.

(3) PzasBopy: Arch. Int. Med., 1916, xvi, 846.

334 JOHN P. PETERS, JR., AND D. P. BARR

(4) Pearce: Journ. Lab. Clin. Med., 1917, ii, no. 12.
(5) Peters: This Journal, 1917, xliii, 113.
(6) VAN SLYKE, STILLMAN AND CULLEN: Journ. Biol. Chem., 1917, xxx, 401.
(7) StmBeck: Deutsch. Arch. f. klin. Med., 1912, evii, 253.
(8) McCuiure AND PEaBopy: Journ. Amer. Med. Assoc., 1917, lxix, 1954.
(9) Fripericta: Berl. klin. Wochenschr., 1914, li, 1268. —
(10) HaLDANE AND PriEsTLEy: Journ. Physiol., 1905, xxxii, 225.
(11) Puiescu: Zeitschr. f. exper. Pathol. u. Therap., 1909, iii, 380.
(12) HenpEerson: Journ. Biol. Chem., 1917, xxxii, 325.
(13) Pearce: This Journal, 1917, xliii, 73.
(14) PeaBopy: Arch. Int. Med., 1917, xx, 433.
(15) CampBEeLL, Doueias, HALDANE AND Hosson: Journ. Physiol., 1914, xliii, 303.
(16) Peasopy: Arch. Int. Med., 1914, xiii, 497.
_ (17) Laurens: This Journal, 1918, xlvi, 147.
(18) CHRISTIANSEN, DouGcias AND HaupaNne: Journ. Physiol., 1914, xlviii, 244.
(19) Lunpsaaarp: Journ. Exper. Med., 1918, xxvii, 219.

STUDIES OF THE RESPIRATORY MECHANISM IN CARDIAC
DYSPNEA

II. A Note on tHE Errective Lune VoLuME IN Carprac DysPNEA

JOHN P. PETERS, Jr. anp DAVID P. BARR

From the Russell Sage Institute of Pathology, in affiliation with the Second Medical
Division, Bellevue Hospital, and the Department of Medicine,
Cornell University Medical College

Received for publication August 9, 1920

That the vital capacity of the lungs is reduced during cardiac decom-
pensation is a well-established fact (1), (2). A few experiments have
been conducted to find out whether this reduction is associated with a
change in the total air containing space of the lungs and to ascertain
the effect of such a change on the functional efficiency of the respiratory
mechanism. |

METHODS

The problem has been approached in two distinct ways: a, by the
application of the Lundsgaard (3) method for measuring the lung volume
by rebreathing oxygen; b, by a study of the effect of continuous re-
breathing of air on the volume of the tidal air and the comparison of
the latter with the vital capacity.

The lung volume of a few normal and pathological cases was measured
by the Lundsgaard (8) method of rebreathing oxygen. A graphic
record of the preliminary respirations and the vital capacity was ob-
tained with a Tissot spirometer by means of a combination of valves
similar to that used for rebreathing experiments and described in the
preceding paper (4). A rebreathing bag containing a measured amount
_ of oxygen was attached in place of the Haldane tube. In all cases the
subject commenced rebreathing from the position of complete expira-
tion. The volume of the residual air was calculated from the gas
mixture in the bag at the end of the observation. The volume of the
vital capacity was obtained from the graphic record. The values
thus obtained were compared with those obtained by the Lundsgaard
(3) method of chest measurement.

335

JOHN P. PETERS, JR., AND D. P. BARR

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RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 339

The apparatus and method employed in the rebreathing experi-
ments were described in the first paper of this series (4). By a study
of the tidal air of normal subjects and cardiac patients after the con-
tinuous rebreathing of air from a spirometer, we hoped to gain some
information on the effect of the reduction of vital capacity on the func-
tional efficiency of the respiratory mechanism.

RESULTS

The results of the lung volume determinations are collected in the

first three columns of,table 1. In each case the normal lung volume, as
calculated by the Lundsgaard (3) method of chest measurement is
reported first, in italics. Below it appear the values obtained from the
rebreathing experiments. In the fourth, fifth and sixth columns are
given the conditions of the experiments: the volume of oxygen taken
in the rebreathing bag; the number of respirations and the duration
of the rebreathing.

The first two subjects were normal. The third had chronic cardiac
valvular disease without decompensation. His vital capacity, residual
air and lung volume were almost normal. Number 4, at the first
observation, in the absence of dyspnea, showed only a slight reduc-
tion of the vital capacity. Three weeks later, with a recurrence of
dyspnea, his vital capacity was found to be only half as large. His
residual air was not far from normal. Unfortunately his residual air
was not determined at the time of the first observation.

Number 5, A. R., had hypertensive nephritis with some edema and
signs of uremia: drowsiness and Cheyne-Stokes breathing. He showed
no considerable hyperpnea; although his plasma bicarbonate was
reduced. His chest was entirely clear. His vital capacity was very
small but his residual air practically normal.

The last three were decompensated patients with dyspnea and showed
very low vital capacities. The volume of the residual air was also
considerably below normal in the last two. With the exception of the
last patient, who had a massive hydrothorax, the physical signs found
in the chest were insignificant in comparison with the diminution of .
lung volume.

These results indicate that, in patients with cardiac dyspnea, the
total volume of air in the lungs which can be detected by the usual
methods is considerably less than that found in normal persons. ‘The
largest part of the reduction in lung volume is due to a diminution of
the vital capacity. The residual air is unchanged or slightly reduced.

340 JOHN P. PETERS, JR., AND D. P. BARR

The results of the rebreathing experiments are given in table 2.
The subjects are arranged from above downward according to the mag-
nitude of their vital capacities, which appear in column 1. In column
2 is given the normal vital capacity as calculated from the Lundsgaard
(3) chest measurements. In column 3 is shown the average resting —
tidal air and in column 4 the tidal air at the end of a rebreathing experi- _

TABLE 2

Vital capacity and tidal air

: fF angseoan 3 TIDAL AIR ig
era ahads - Myla nisl CAPAGEFY araix ue me pg oF DIAGNOSIS AND REMARKS
GAARD BREATHING
1;2D..P. Bis) 463% | \3332* 580 2407 | Normal
oO weasieinish, » OO, lev eon 372 1929 | Normal
3. T. deC....|. 3839 1249 | Cardiac. Compensated
#.oPs Mevoes eee 371 1192 | Gastric neurosis
Dd ae 2729 389 980 | Shellshock. Effort syndrome
6.82 BH 2519 737 1236 | Cardiac. Slight dyspnea and
hyperpnea
fee: Te, GA Sree 2432 |. 2980 A421 Cardiac. Compensated
ag ay; Oe ra 2430 | 2930 439 1116 | Cardiac. Compensated
Ae © 5 8 RP Perey 4 et 315 581 | Cardiac. Severe decompen-
sation }
10. J. M. L...| 1789 390° 1309 | Cardiac. No dyspnea nor hy-
; perpnea
11. A. R......| 1789 |; 3640 604 Nephritic. Mild acidosis .
12, Feo! Wess 1714 | 3930 244 Cardiac. Some dyspnea
is aes De a) ier em bate Wy ft” | 304 Cardio-nephritic. Mild acid-
OSs1S ‘
14. W. W.....| 1452 | 4698 469 ‘| Cardiac. Some dyspnea
15: D. O. C:..).. 1808+ | ' 3540 423 638 | Cardiac. Marked dyspnea
TGS i Eeiec | eee 375 Cardiac. Severe dyspnea
17. J..M 1197 475 Cardiac. Extreme cyanosis.
Some dyspnea
18, soa C.. 2.) TIS? 385 492 | Cardiac. Marked dyspnea

* See note, table 1.

ment. The last we have called the “maximum tidal air’ because it
is the largest respiratory volume which the subject will exchange under
the influence of the strongest respiratory stimulus that can be applied,
carbon dioxid. ;
Peabody (5) found the average resting tidal air slightly reduced in
cardiac dyspnea. This our figures do not show. If such a reduction

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 341

exists it is small in proportion to the enormous reduction found in the
vital capacity.

The maximum tidal air, on the other hand, varies almost directly
with the vital capacity and the ratio between the two shows the same
range of variation in cardiac patients with dyspnea as in normals (0.35
to 0.55). Number 10 is the only exception to this rule. In his case
the ‘‘maximum tidal air’’ was very large in proportion to the vital
capacity. It was noted in the course of the experiment on this patient
that he could not be induced to exert himself to the full extent of his
strength when he blew into the spirometer. The low vital capacity,
in this instance, may be only an expression of lack of effort on the part
of the patient. This was completely overcome under the stimulus of
carbon dioxid and he showed a normal “maximum tidal air,”’ although
he did not continue rebreathing to the point of exhaustion.

In the case of the decompensated cardiac patients, however, there
was no lack of effort apparent during the determination of the vital
capacity. Moreover, two of them continued rebreathing to the point
of extreme exhaustion. The reduction of the ‘‘maximum tidal air’
and the vital capacity may be a functional matter, due to reflex inhibi-
tion. If so, this inhibition is so profound that it can not be overcome
by the strongest stimulus which can be applied. The increase in the
tidal air which normally occurs in response to the stimulus of carbon
dioxid is limited by the lowered vital capacity.

DISCUSSION

Siebeck (1), in 1912, measured the lung volume in a series of cardiac
patients by a rebreathing method. His studies showed that in cardiac
dyspnea the residual air was relatively increased, although the absolute
values he obtained seem to have been lower than normal. The reserve
air and the complementary air were both decreased. These results
are substantially the same as ours. However, Siebeck regarded them
as questionable because he was unable to obtain the same constancy
in his rebreathed mixtures in cardiac patients as in normals. In the
latter the concentration of hydrogen in the rebreathing bag became
constant after 5 respirations. In subjects with cardiac dyspnea the
concentration continued to change even after 10 respirations. This
Siebeck considered to be due to improper diffusion of air in the lungs.
He concluded that the actual volume of residual air was greater than
that indicated by the rebreathing methods.

342 JOHN P. PETERS, JR., AND D. P. BARR

As Sonne (6) has shown, the residual air can be measured with only
an approximate degree of accuracy even in the normal person. Whether —
Siebeck is right in believing that the amount of air present is greater
than that found, we are unwilling to argue. In two or three cases the
number of respirations, the time and the volume of air rebreathed were
varied without significant changes in the values obtained. This does
not agree with Siebeck’s findings. But any air that may be present
and undetected must be peculiarly useless for respiratory purposes.
A functional conception of the lungs is of more value than a purely
anatomical one. In a normal person residual air determinations may
be considered as measurements of the volume of air space in the fully
deflated lungs, which is available for the rapid diffusion of gases. In
this sense, certainly, the residual air is not increased in cardiac dyspnea.
Since the vital capacity is decreased the effective volume of the lungs
must be diminished. |

Whether this diminution is due to a true anatomical lesion or not,
we are unprepared to say. In some instances hydrothorax plays a part,
but it is by no means an essential part. This we may assume not
only because of the absence of physical signs of fluid, but also because
of the rapidity with which the vital capacity increases as compensation
is established (7).

Just what effect such a diminution of the lung volume may have on
the gas exchange must be largely a matter of speculation until the cause
of the diminution is determined. If it is not due to a true anatomical
lesion it is quite possible that some or all of the blood in the pulmonary
vessels is not in gaseous equilibrium with the alveolar air. This might,
in part at least, explain the discrepancy between the alveolar CO, and
the CO.-combining capacity of the plasma.

This discrepancy Pearce (8) has attributed entirely to the effect of
stasis in the general circulation. He has drawn a fine distinction be-
tween the causes of the low alveolar CO,-tension in congenital heart
disease, pneumonia and acquired heart disease. In congenital heart
disease part of the venous blood does not pass through the lungs, and
in consequence is given no opportunity to rid itself of the CO. which
it has received from the tissues. In consequence the arterial blood is
a mixture of aérated and unaérated blood. If there were no compen-
“ satory reaction a true carbon dioxid acidosis would occur. The respir-
atory mechanism, however, responds to the stimulus of the carbon
dioxid. The alveolar CO,-tension and the CO.-tension of the blood
which does pass through the pulmonary circulation is reduced suffi-

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 343°

ciently to restore the CO>-tension and hydrogen-ion concentration of
the arterial blood to normal.

In patients with pneumonia in whom Pearce found the same dis-
crepancy between the alveolar CO,-tension and the plasma bicar-
bonates, he advances a similar explanation. In this case, although all
the blood passes through the pulmonary circulation, he supposes that
part of the blood passes through non-aérated portions of the lungs.
On what seems to us insufficient evidence, he denies the possibility
of such a condition in acquired cardiac disease. But certainly the
reduction of the effective lung volume renders it not only possible, but
not improbable. Even if Siebeck is right and this diminution is not
due to a decrease in the actual air-containing space in the lungs, a
certain part of this air must be unavailable for the rapid diffusion of
gases and that portion of blood in the pulmonary circulation which
-comes in contact with this air must be less effectively ventilated than
normal. 3

That the blood flow is retarded in decompensated cardiac disease is
probable in the light of Harrop’s (9) observation of an increase in the
difference of the oxygen content of arterial and venous blood.

That this is the sole factor in the production of the low alveolar CO,
remains to be proved. It is doubtful whether the method of Christian-
sen, Douglas and Haldane (10), which was employed by Pearce for the
determination of the rate of blood ffow, can be interpreted quantitatively
in cardiac dyspnea. In this method it is assumed that the CO,-tension
of the alveolar air is the same as that of the arterial blood. ‘That such
a relation obtains in cardiac dyspnea demands definite proof. The use
of respiratory methods that are designed to determine the venous CO:-
tension is also open to question when applied to conditions in which
the venous oxygen unsaturation is excessive. Christiansen, Douglas
and Haldane showed that the effect of oxygen on the carbon dioxid
dissociation curve necessitated the introduction of a correction for the
oxygen unsaturation even in normal persons. The extent of this cor-
rection in cardiacs can only be guessed.

Although the effect of the small effective lung volume on the exchange
of gases between the blood and the alveolar air remains largely a matter
of speculation, its effect on the mechanics of respiration is fairly clear.
Here we have to deal only with the vital capacity, the portion of the
lung volume available for ventilation of the lungs. |

Apparently the reduction of vital capacity which occurs during
cardiac dyspnea has little effect in limiting the respiratory exchange

344 JOHN P. PETERS, JR., AND D. P. BARR

during rest. The volume of the resting tidal air is not diminished.
On the other hand, the amount by which the tidal air can increase
under the stimulus of carbon dioxid seems to be distinctly limited by
the reduction of the vital capacity. The reserve of the mechanical
apparatus of respiration is therefore greatly diminished.

CONCLUSIONS

1. In cardiac patients with dyspnea no evidence of an increased
residual air was obtained by the Lundsgaard method.

2. As the vital capacity is diminished, this means that the effective
lung volume, the volume of air in the lungs available for the exchange
of gases, is diminished.

3. The maximum volume of tidal air attained under the stimulus of
continuous rebreathing is lower than normal in cardiac dyspnea. ‘The
reduction in the maximal tidal air bears a close relation to the reduc-
tion in vital capacity.

BIBLIOGRAPHY

(1) Srepeck: Deutsch. Arch. f. klin. Med., 1912, evii, 253.
(2) McCuiurE AND PEaBopy: Journ. Amer. Med Assoc., 1917, lxix, 1954.
(3) LUNDSGAARD AND VAN StyxkeE: Journ. Exper. Med., 1918, xxvii, 65.
(4) Perers AND Barr: This Journal (no. I of this series).
(5) PEaBopy, WENTWORTH AND BARKER: Arch. Int. Med., 1917, xx, 468.
(6) Sonne: Journ. Physiol., 1918, lii, 75.
(7) West: Paper read before the Section on Medicine of the New York Academy
of Medicine, April, 1920.
(8) Pearce: Journ. Lab. Clin. Med., 1917, ii, no. 12.
(9) Harrop: Journ. Exper., Med., 1919, xxx, 241.
(10) CuristTIANSEN, DovauAs aND Haupans: Journ. Physiol., 1914, xlviii, 244.

STUDIES OF THE RESPIRATORY MECHANISM IN CARDIAC
DYSPNEA

Ill. Tue ErrectiveE VENTILATION IN CARDIAC DYSPNEA

D. P. BARR anv JOHN P. PETERS, Jr.

From The Russell Sage Institute of Pathology, in affiliation with the Second Medical
Division, Bellevue Hospital, and the Department of Medicine, Cornell
University Medical College

Received for publication August 9, 1920

In the previous papers of this series (1), (2) it has been shown that
the effective lung volume is decreased during the dyspnea of cardiac
decompensation. The alveolar CO, tension, as determined by the
Haldane method, is usually low and always lower than the concentra- ,
tion of the bicarbonate in the venous plasma would indicate. We have
shown that the air obtained by Haldane’s method is probably true
alveolar or exchange air. In this paper we shall present evidence to
substantiate this contention and shall discuss the influence of a low
alveolar CO, tension and of a diminished lung volume upon the effective
ventilation in relation to the dyspnea of heart disease.

Peabody, Wentworth and Barker (3) find that, although the level
of metabolism is normal or only moderately increased during cardiac
decompensation, the minute volume of respiration is much greater
than in normal:individuals. The increase is accomplished by a more
rapid respiratory rate with a moderate diminution in the volume of
each expiration. The percentage of CO, in the expired air is diminished.
_ Peabody recognizes that the greater observed minute volume may
represent no increase in the amount of effective air breathed. The
tidal air consists of two parts. The dead space, which fills the upper
respiratory tract is practically atmospheric air and is of no value in the
ventilation of the functioning portions of the lungs. It is only the
remainder of an expiration, the exchange air, which is effective for
purposes of respiration. In any given expiration, the relative propor-
tion of dead space to exchange air will determine the efficiency of
ventilation. If, as Peabody assumes, the volume of the dead space
is not changed during decompensation while the volume of tidal air

345

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 2

346 D. P. BARR AND JOHN P. PETERS, JR.

is diminished, the amount of effective air in each expraae will be
diminished. /

It is possible to arrive at a rough estimate of the effective minute
volume by subtracting an average dead space value from the volume
of the tidal air and multiplying the remainder by the respiratory rate.
Different observers, Haldane and Priestley (4), Krogh and Lindhard
(5) and Pearce (6), have found the volume of the dead space in normal
resting subjects to vary between 100 and 200 cc. with an average of
about 130 ce. Since there is no evidence that the dead space is changed
in cardiac dyspnea and experiments reported in a previous paper (1)
indicate that it cannot be greatly above the normal values, 130 cc.
has been used as the average volume of the dead space for purposes
of calculation.

To determine the effective ventilation, we made a considerable
number of observations on the minute volume of normal, individuals
and of compensated and decompensated cardiacs. The method did
not differ from that of Peabody except that a nose clip and rubber
mouthpiece were substituted for the Siebe Gorman mask. The instru-
mental dead space was 30 cc. The expiratory air was collected in an
accurately balanced Tissot spirometer for periods varying from five
to ten minutes, the volume of expiration and the number of respirations

~

being recorded for each minute. Volumes were reduced to standard ~

conditions of 760 mm. Hg. and 0°C. ‘To these observations we applied
the following formula:

(Tidal Air — 160') Respiratory Rate = Effective Minute Volume

The results are tabulated in table 1. |

For the purpose of comparing our results with those of Peabody
we have calculated the effective minute volume from his figures. The
dead space of the Siebe Gorman mask which he used has been estimated
at about 50 cc. Averages of the results are given in table 2

The results of the two experiments are in practical agreement. In

the decompensated cases, the minute volume of respiration is greater .

and the respiratory rate is increased. The volume of the tidal air is
sometimes slightly diminished but this is by no means invariable as
is shown by the large tidal air of J. B. and J. D. B. (table 1). The
effective minute volume is much greater during decompensation.

It might be argued that the increase in effective ventilation is due
to the higher level of metabolism and the consequent increase in CO:

To the average dead space of the individual has been added the instrumental
dead space of 30 cc.

se ee eae Ss ee ee ee

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 347

production which is observed in many dyspneic cardiac patients. That
this is not true can be demonstrated by calculations from Peabody’s
figures (table 3).

Figures for surface area show that the average size of the patients
in the two groups is practically identical. Differences either in the
production of CO, or in the effective ventilation cannot be due to dis-
crepancies in size. The CQO, production per minute is only 5.1 per
cent greater in the decompensated cases. The volume of effective air
breathed per minute is 30.3 per cent higher than in the cases without

TABLE 1

RESPIRA- |EFFECTIVE
SUBJECT baa ieee TIONS PER| MINUTE DIAGNOSIS AND REMARKS
MINUTE | VOLUME

cc. ce.

D. P. B. | 6,900 532 13.0 4,822 | Normal adult

6,789 580 11.7 4,913
6,040 592 10.2 |. 4,406
6,751 668 | 10.1 | 5,131
” fp gt 5,524 | .. 372 14.9 3,148 | Normal adult
4,898 389 12.6 2,885
7,127 520 13.7 4,943
G.C. D.| 6,386 484 13.2 4,277 | Normal adult
6,243 488 12.8 4,198

rik; 4,659 293 15.9 2,115 | Gastric neurosis. No sign of
7,764 371 20.9 4,420 | . cardiac or pulmonary disease
Cap. 7,866 333 23.6 4,083 | Normal adult. Respirations

rapid and variable. Obvi-
ously over-ventilating

dA. 6,474 439 14.8 4,106 | Chronic cardiac valvular dis-
7,611 | 408 18.6 4,626 ease. No dyspnea while at
rest in bed

J.M.L. | 4,553 390 | 11.6 2,675 | Chronic cardiac valvular dis-
6,133 438 14.0 3,893 | ease. No dyspnea while at
. rest in bed

RW: 5,826 244 23.9 2,003 | Chronic cardiac valvular dis-
ease. Breathing quietly
while at rest in bed. Rapid
and superficial during experi-

ment
J. W. 7,680 410 18.7 |. 4,675 | Chronic nephritis. Hyperten-
sion. No dyspnea while at
rest in bed

Average 6,401 442 15:2 3,973

348 D. P. BARR AND JOHN P. PETERS, JR.
TABLE 1—Concluded
SUBJECT Titan bars IONS PER “MINUTE rane AND REMARKS
ce. cc.
J.B, 13,170 737 17.9 | 10,310 | Chronic cardiac valvular dis-
10,210 612 16.7 7,540 ease. Dyspnea and cyanosis
while at rest
Jos. C 13,590 385 35.3 7,936 | Chronic nephritis with hyper-
14,140 350 40.3 7,638 tension. Severe cardiac de-
compensation with marked
dyspnea
C.D, 9,982 315 31.8 4,916 | Chronic cardiac valvular dis-
ease. Severe dyspnea and
orthopnea
Ee | 6,930 375 18.5 3,970 | Chronic cardiac valvular dis-
ease. Massive hydrothorax.
Dyspnea and orthopnea
D. 0. C 8,069 423 19.1 5,020 | Chronic cardiac valvular dis-
ease. Marked cyanosis and
moderate dyspnea while at
: rest. Right hydrothorax
W. W. 9,528 469 20.3 6,279 | Chronic cardiac valvular dis-
ease. Moderate dyspnea —
while at rest
TER. 11,393 311 36.7 5,542 | Chronic cardiac valvular dis-
10,110 357 28.3 5,575 ease. Marked dyspnea and
orthopnea while at rest
P.O.S 9,087 279 32.6 3,879 | Chronic cardiac valvular dis-
ease. Right hydrothorax.
Marked dyspnea and orthop-
nea while at rest
J.D. B. | 14,810 821 17.4 | 11,501 | Chronic cardiac valvular dis-
12,800 538 23.8 8,996 ease. Marked dyspnea and
’ orthopnea while at rest
J. M. 8,652 475 18.2 5,733 | Chronic cardiac valvular dis-
9,843 496 19.8 6,653 ease. Extreme cyanosis and
some dyspnea while at rest
Average 10,788 463 25.1 6,766
dyspnea. Only a small part of the increase in effective ventilation

can be accounted for by a greater CO, production.

Furthermore, since the dyspneic cardiac breathes a much greater
amount of COs, each volume of his effective or alveolar air must
contain a smaller percentage of COs.

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA

349

TABLE 2
COMPENSATED DECOMPENSATED
CARDIACS - CARDIACS
MME WORUIIBS LOG." Pte eras oe wp ves cies saa 5,901 8,521
EN OO. So tt a iss bg 8 tilde was vee 466 398
NES THUBL cs Vee a CO 13.2 21.8
Effective minute volume (cc.).................. 3,530 4,600

* Minute volumes have been recalculated from the CO, produced per minute

and the percentage of CO: in the expired air.

TABLE 3

COMPENSATED
CARDIACS

DECOMPENSATED
CARDIACS

Surface area (square meters)................... 1.72 1.77
CO: production per minute (cc.)..............: 196 206
Effective minute volume (cc.)...............+. 3,530 4,600

It is possible to show more directly the probability of a reduced

alveolar CO, tension. In the paper describing their method of obtain-
ing alveolar air, Haldane and Priestley (4) indicated a means of deter-
- mining the volume of the dead space. The data necessary for the
calculation were the volume of an expiration, the percentage of CO,

in the expired air and the percentage of CO, in the alveolar air. These
were combined in the following formula:
Formula I
Tidal air — Tidal air X per cent CO, in expired air send ehace

Per cent CO: in alveolar air

By a simple inversion of their formula, the percentage of CO: in
the alveolar air can be deduced.
Formula IT

Tidal air X per cent CO, in expired air
Tidal air — Dead space

= Per cent CO; in alveolar air

Both of these formulae were applied to observations on a number
of normal individuals and on cardiacs in varying degrees of decompen-
sation. Minute volume determinations were made as in the previous
experiments. At the close of an observation, the air was thoroughly
mixed in the spirometer and a sample was taken for analysis. Haldane

D. P. BARR AND JOHN P. PETERS, JR.

350

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RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 351

samples of alveolar air were taken before and after the: minute volume
_ determinations. A specimen of blood was drawn from an arm: ‘veiti
for determination of the bicarbonates in the plasma.» The. results
obtained from the plasma were converted into terms of sist sibehiel of
alveolar CO, according to the method of Van Slyke (7). epee en
In formula II; average dead space values were candosiilh for. the
calculation of the probable percentage of CO. in alveolar air: :-The
results are recorded in table 4 (column 6). They agree closely with
the results of direet observation by the Haldane method (column ‘7)
and consequently in the decompensated cardiac lie. far pete the- sicuiaed
indicated by the plasma (column 8). O04 :
- In formula I, the observed alveolar CO, per ent and ide atacindiiee.
indicated by the plasma have been applied to.calculate the. probable
dead space.. Substitution of the observed values give volumes of dead
space which are within the range of normal (columin 9) while substitu-
tion of percentages indicated by the a give sheen: maprpbable
results (column 10). |
Formula II has also been applied to ae 8 ‘hinmene _ Averages
are eres in table 5. FO wekitryst

TABLE 5
. COMPENSATED DECOMPENSATED |
CARDIACS' @ 4 CARDIACS . ..;
Remetar ait (oblyorey 4 MEG. CORE ah SEL IED Cd gal) AST) | oagaaaite
CO: in expired air (per cent).-....... A ORS TERS, : 3: 35 2 2.44 °°
Calculated alveolar CO, per cent (using dead | .- : ot #
) 8pAee,Ot; 180,061)". ded so lees... sa rere 5 4) Babes . 40 ’

. ‘Avorige dead space of 130 cc. to which is added the extra dead space of 50
ec., the capacity of the Siebe Gorman mask. 4

- Both in Peabody’ s figures and in ours, the percentage of on in os
alveolar air is lower during decompensation.

DISCUSSION

In this series of papers the question of the concentration of. CO,
in alveolar air during cardiac decompensation has’ been. approached
from several angles. Of the direct methods, that-of Haldane which
has been found applicable to dyspneic patients gives values for alveolar
CO, percentage much lower than it does in normal individuals. . Dupli-
cate samples, however, show quite as close agreement in dyspneic

352 D. P. BARR AND JOHN P. PETERS, JR.

cardiacs as in trained normal subjects. Experiments have shown that
the low content is not due to subjective errors nor to an increased dead _
space. After breathing increasing percentages of CO:, the alveolar
CO, percentage of dyspneic cardiacs is still relatively lower than it is
in normal individuals under the same circumstances. The percentage,
moreover, is not increased by increasing the depth of expiration. An
expiration of maximum depth contains no greater concentration of CO,
than does one of a volume just sufficient to clear the dead space.
Whether the specimen of air obtained by the Haldane method is from
the alveoli or from other portions of the lungs is not of great importance

_ in the present discussion. Whatever the anatomical source of the

sample may be, it is the only air which by the greatest effort the de-
compensated cardiac can expire. It is the only air which can be func-
tionally effective in the elimination of CO, from the body. If this is
true the CO, contained in this air should completely account for the
total CO. elimination. In this paper it has been shown by the use of
Haldane’s dead space formula that the percentage of CO, found in the
Haldane specimen is the percentage theoretically required to account
for the CO, eliminated.

The accumulated evidence, both direct and circumstantial, indicates
that the percentage of CO, is low in the effective air of dyspneic cardiac
subjects. The cause of this important phenomenon is not clearly ©
understood. Its chief importance in the present discussion lies in its .
effect upon the ventilation in cardiac disease.

A low percentage of CO, in the effective air necessitates.a larger
effective ventilation to accomplish CO, elimination. Even when the
metabolism is normal, the decompensated cardiac exhibits hyperpnea.
As long as he lies quietly in bed, this usually involves an increase in
the rate of respiration with little or no change in the volume of each
respiration. Under these circumstances the low effective lung volume,
which is the constant accompaniment of decompensation, is not a
factor of great importance in the causation of dyspnea. It probably
exerts a greater influence during conditions involving a greater pro-
duction of COs.

Concerning the mechanism of ventilation during exercise, we have
accumulated no direct evidence. A good indication of the response
is furnished, however, by experiments upon the rebreathing of COs.
Under these circumstances the cardiac is subjected to a most powerful
stimulus to respiration. The mechanism should, by analogy, be similar
to that occurring during exercise or any other condition involving a

RESPIRATORY MECHANISM IN CARDIAC DYSPNEA 353.

rapid production of CO, within the body. It has been shown that
the cardiac maintains a relatively low alveolar CO, percentage during
rebreathing. To accomplish this he must breathe a larger volume of
effective air. Under the stimulus of COs, the volume of the tidal air
increases both in cardiacs and in normal individuals. The increase,
however, is in proportion to the vital capacity. In both groups the
tidal air reaches a maximum at one-third to one-half of the volume of
the vital capacity. Thus, a normal individual with an original vital
capacity of 4000 cc. may show under the stimulus of CO, a tidal air of
1500 to 2000 cc. In a decompensated cardiac whose vital capacity may
be only 1500 cc. the tidal air will not rise above 500 to 750 cc. with
maximum CQO, stimulation. The volume of respiration is strictly
limited. Any attempt to increase it is accompanied by marked sub-
jective dyspnea.

Two factors in cardiac disease help to explain the dyspnea which is
its most constant subjective symptom. The low percentage of CO.
in the effective air makes an increase in ventilation essential. The
diminished effective lung volume makes any large increase difficult
or impossible. The first is active under all conditions while the cardiac
is decompensated. The second exerts its chief influence when the
production of CO; in the body is increased.

CONCLUSIONS

1. Air obtained from decompensated cardiacs by the Haldane alveo-
lar method is true exchange air. It corresponds to the alveolar air
obtained by the same method in normal resting subjects. It is the
only air effective for the elimination of COs.

2. During cardiac dyspnea, the bicarbonate content of the plasma.
gives no indication of the percentage of COs. in the exchange air.

3. The minute volume of effective or exchange air is increased during
cardiac decompensation.

4, This is not explained by the higher level of metabolism.

5. The greater effective ventilation is necessitated by the low con-
centration of CO, in the exchange air.

6. Great increases in ventilation are impossible because of the di-
minished effective lung volume of decompensated cardiacs.

354 | -D.-R, BARR AND JOHN P, PETERS,

Picat ee BIBLIOGRAPHY

(i) Perers anv Barr: This Journal, this series, hpi é
(2) Peters aND Barr: This Journal, this series, no. II.
(3) PeaBopy, WENTWORTH AND Barker: Arch. Int. ‘Aish 191
(4) HaLpANE AND PrizstLey: Journ. Physiol., 1905, xxxii, 240. dite
(5) Kroau anp Linpuarp: Journ. Physiol., 1917, xliii, 73. ;
(6) Pearce: This Journal, 1917, xliv, 391. PR
7) VAN Surns, SrruEMAN Ap CunuEn: Journ. Biol }

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STUDIES ON THE BRAIN STEM

LV. On THE RELATION OF THE CEREBRAL HEMISPHERES AND THALAMUS
To ARTERIAL BLoop PRESSURE

F, T. ROGERS
From the Hull Physiological Laboratory, University of Chicago

Received for publication August 9, 1920

In a previous: report (1) attention has been directed to the poikilo-
thermous condition that follows the removal of the cerebral hemispheres
and thalamus in birds and mammals. In an attempt to discover the
causes of this condition it was suggested that possibly a general fall
of arterial blood pressure with resulting cutaneous dilatation of the
blood vessels might be an essential factor. To test this point a method
was devised for measuring the arterial pressure in the pigeon and studies
of the blood pressure made before and after reduction to the cold-
blooded condition. These tests did not give the anticipated results
but led to the discovery that the removal of the cerebral hemispheres,
which alone does not reduce the bird to the cold-blooded condition,
leads to a permanent slight fall in arterial tension. The plan of the
work was therefore extended to a study of the arterial pressure in the
normal pigeon, the normal variations, effects of various procedures on
the normal pressure, and then, the effect of various types of brain
lesions on the arterial pressure.

Asher (2) in his review of the vasomotor mechanism, in 1902 wrote:
“There are no known facts which indicate the presence of vasomotor
centers in the parts of the brain above the medulla oblongata. The
higher cerebral centers influence the vasomotor tone exclusively in a
reflex manner through the medullary vasomotor center.’’ This inter-
pretation is based on the original experiments of Ludwig’s students,
Dittmar and Owsjannikow. These classic experiments in 1872 and
1873 demonstrated the localization of the vasoconstrictor centers in
the floor of the fourth ventricle.

Since then innumerable studies have disticdntaated that reflex influ-
ences from the cerebral hemispheres above and the spinal nerves below

355

356 | F. T. ROGERS

may play on this center. (Literature cited by Sachs.) It has become
common knowledge'that loss of the cerebral hemispheres does not neces-
sarily disturb the arterial pressure in acute’experiments. It therefore
came somewhat as a surprise to find that in a series of decerebrate
birds, kept for several weeks or months after removal of the cerebrum,
the arterial pressure was uniformly lower than in normal birds.

Porter showed in 1907 that in curarized rabbits and cats removal of
the cerebral hemispheres in acute experiments leads to a profound
fall in the blood pressure without depression of the reflex excitability
of the vasomotor center.

If a blood pressure tracing be made on a dog before and after decere-
bration by Sherrington’s method, it-is frequently found that the pressure
is somewhat less after decerebration than before. The complications
however of the muscular rigidity that follows, render difficult the
interpretation of the effects of the operation on the vasomotor center.

All these experiments are acute traumatic experiments subject to
all the uncertainties of shock effects. In the birds many of these
difficulties can be obviated, for they can be kept alive easily, for an
indefinite time after the operation, and spastic paralytic phenomena
are wholly lacking provided the hemispheres only are removed. In
the pigeon therefore we have a warm-blooded animal which easily
withstands the shock of cerebral ablation, lives indefinitely thereafter,
and in which the permanent effects on the blood pressure can be'sindiad!

There was the further inducement that the pigeon in comparison
with the mammals combines a relatively low type of cerebral develop-
ment with the warm-blooded condition and it was hoped that some
light would be thrown on the nature of the nervous mechanism of heat
regulation in its earlier development.

Methods. The measurements of arterial pressure were at first made
in the ordinary way of cannula and mercury manometer. It was soon
found that a more convenient and just as reliable method was furnished
by substituting a hypodermic needle for the cannula and this method
was used in all the work here reported. All measurements have been
made on the brachial artery using a hypodermic needle, size 19 or 20
with a bore of about 1 mm. diameter. This furnishes a tube which
when inserted into the artery slightly stretches it and fits tightly enough
to allow no escape of blood. (In very large birds it may be necessary
to use a larger needle.) This was connected to an ordinary small size
mercury manometer made of glass tubing 4 mm. in diameter, with
stiff-walled rubber tubing. In order to avoid any errors due to possible

STUDIES ON THE BRAIN STEM Sou

mechanical effects of friction or differences in level between the bird
and the level of the mercury, the manometer was set in a fixed position
with the level of the mercury in the plane of the tip of the breast muscles
when the bird was fastened on its back. The same manometer, two
needles of the same size, and the same size and length of rubber con-
nections, were used throughout the series. Hence any mechanical
errors will be nearly uniform throughout the comparative series of
pressure determinations. Clotting of the blood was prevented by
using 7 per cent sodium citrate throughout the apparatus. A lesser
concentration was not always satisfactory, but with citrate solution
of this concentration the tracing can be continued indefinitely through
all variations of blood pressure of zero to 200 mm. When the needle -
was removed from the artery the blood vessel was doubly ligated. This
causes no apparent trouble to the bird, so the collateral circulation must
be extensive. In only one case did gangrene follow the ligation and in
this case the ligature involved both brachial artery and vein. All
readings were made with the birds under ether anesthesia; no other
drugs were used in this study. This introduced, of course, the effects
of the anesthetic added to that of the cerebral lesion. No other method
however seemed available for it is important that there be no struggles
by the animal as these will promptly change the level of the arterial
pressure. In order to check these variations due to the action of the
anesthetic the routine procedure was adopted of etherizing the bird,
putting it on its back with wings spread and the needle inserted in
the artery. The pressure was then raised in the manometer to a value
a little less than the anticipated pressure. Some care was necessary
here not to exceed the arterial pressure and thereby kill the bird by
forcing citrate into the circulation. This happened twice in the series
of experiments. The arterial pressure was then recorded for all depths
of anesthesia, varying from light to deep, using rigidity as an index
of light, and abolition of the corneal reflex as index of deep anesthesia.
The readings of pressure given in the tables are, therefore, those of the
extreme variations under anesthesia, but not including variations due
to struggles of the animal. In many cases the pressure maintained
a nearly constant level and only one figure is given in such cases.

_ In order to determine whether or not there were errors due to pos-
sible differences in size of the arteries of the two wings, readings were
made on a series of birds comparing specifically the pressures in each
of the two brachial arteries, in the same bird, allowing intervals of
several days between determinations, during which time the birds

358 3 - KF, T. ROGERS

were kept confined in the cages used throughout the series of experi-
ments. ‘These readings (table 5) agree closely. Of course in the case
of specific anatomical anomalies such a comparison would be of no
value. One such case has been seen, namely, a double instead of a
single brachial artery. In a series of determinations however this

TABLE 1
| Arterial pressure in normal pigeons
; : Anesthesia variations
NUMBER OF PIGEON ARTERIAL PRESSURE NUMBER OF PIGEON ARTERIAL PRESSURE
Ee mm. mm.
4 122-142. 190 104-114
114-118 188 94-120 .
116-118 158 110-130 |
150-160 156 154-176
122-132 178 92-140 ©
153 108-142 161 78-130
170 136 163 108-116 —
172 103-118 162 106-108
171 135 179 122-160 .
3 116 169 100-110 ©
177 96-108 193 -— 96-98
181 92-108 193 ~ 96-102
126-152 194 122-140
185 116 194 106-124 .
164 134-176 195 110-148
182 92-118 195 104-128
184 88-128 159 150-170
183 86-112 104-120
186 102-118 168 122-144
187 90-110

Average pressure, 118 mm.

Average limits of variations, 109-130 mm.

The figure 109 is the average of all the lower of the two shadingn given for the
majority of the animals. Similarly 130 is the average of the higher reading given.
The figure 118 is the average of these extremes combined with the single figure
given for several birds.

factor is neutralized in the averages given, and by the consideration
that the arteries of both wings were used indiscriminately throughout
the series of determinations.

Normal blood pressure. The average arterial blood pressure in the
brachial artery under ether anesthesia of thirty-nine normal adult
pigeons was found to be 118 mm. (table 1). The extreme limits for

STUDIES ON THE BRAIN STEM 359

all stages of anesthesia were 78 to 176 mm. The average limits for
the series are 109 to 130 mm. (fig. 1). :

EN DE vi A er

Fig. 1. Arterial pressure in normal pigeons, ether anesthesia. A: Light anes-
thesia: 22 more ether given so as to put bird in a state of deep anesthesia. Time
in one minute intervals. B: Traube-Hering waves in pigeon under light anes-
thesia. Time in 30 second intervals.

Note: All figures of blood pressure are reduced one-half, except figure 3, which
is reduced one-third. A true scale for measuring pressure in these tracings is
given in figure 4.

Mechanical stimulation of the brachial nerves, ammonia to - the
nostrils, and asphyxia, produced the usual types of vasomotor responses.
The effects of these procedures are given in table 2 and figures 2 and 3.
Electric stimulation of these nerves was not employed. Mechanical
stimulation of the nerves by pinching with forceps or traction caused
brisk changes in arterial pressure, both pressor and depressor. effects.

360 F. T. ROGERS

A lowering of blood pressure by cardiac inhibition seemed particularly
easily elicited by traction of the brachial nerves. 'Traube-Hering waves
of pressure have been frequently observed as also have been the shorter
respiratory waves (fig. 1, B).

TABLE 2
Variations of arterial pressure in normal pigeons
RANGE OF VARIATIONS OF PRESSURE INDUCED BY
NUMBER OF PIGEON ) piaaons rr se ema peg Ammo ‘Agi (in-
a soit ia Pee . oo in cars
mm. mm. mm. : mm.
110 14 24
~ 130 34 |
110 40
153 125 30 | .
158 ~ 120 50 16 ae
169 105 22 22
178 116 12 |
179 140 8 26 22
156 164 16 16
170 110 32

* This includes both pressor and depressor effects.

TABLE 3
Effects of slight hemorrhage on arterial pressure
ARTERIAL PRESSURE
NUMBER OF PIGEON cas Pp
; Before bleeding 10 St ar i: bleed-
cc.
159 5 150-170 126-128
173 1 110 * 102-118
155 2 130-150 134-148

_. The removal of small amounts of blood leads to a fall followed by
‘quick recovery (table 3). Thus the loss of 1 to 2 cc. of blood in the
normal pigeon leads to a fall quickly followed by compensatory changes
| which bring the pressure back to normal. The loss of 5 ec. of blood
“causes much more profound effects but in ten minutes-the blood pressure
has again reached the average level although below the previous level
in the same bird (pigeon 159, table 3).

STUDIES ON THE BRAIN STEM 361

Fig. 2. Blood pressure, normal pigeons. A: 1, mechanical stimulation of
brachial nerves; 2, ammonia vapors to nostrils. B: Deeper anesthesia than in
A. 1, mechanical stimulation of brachial nerves; 2, ammonia to nostrils; V,
vomiting. 7

Fig. 3. Blood pressure, normal pigeon. Depressor effects following mechani-
cal stimulation of brachial nerves.

362 F. T. ROGERS

The effect on blood pressure of deprivation of food—but not water—
was tested on four birds. These birds were starved for four to eight
days. At the end of that time the arterial pressure was normal in
three of the birds and slightly lowered in one of them (table 4). The -
effects of confinement were tested by comparing the pressures of three
birds which had been in the laboratory cages for over a year. The
average pressure of these birds. (six determinations, table 5) was i
mm. compared with the average pressure of 118 mm-

TABLE 4

Influence of starvation on arterial pressure. Cerebrum intact

ARTERIAL PRESSURE
NUMBER OF PIGEON STARVATION PERIOD ; Vers
Before starvation After starvation
days mm. mm,
183 7 88-112 80-86
184 7 104-114 120-122
182 8 90-108 95-98
164 + 116-135 120-126

Comparative pressure determinations in the two brachial arteries of the same birds

TABLE 5

(birds have been in laboratory cages for one year)

NUMBER OF PIGEON ingens ppb Sami RIGHT WING . LEFT WING
days mm. mm.

193 3 96-98 96-103

194 3 122-140 106-124

195 3 104-128 110-148

Average pressure in these birds, 115 mm.

By this series of determinations of arterial pressure on thirty-nine
normal adult pigeons the average pressure has been determined, the
normal variations noted, and the effects of such factors as anesthesia,
starvation and the routine reflexes observed. Preliminary to the
production of cerebral lesions it was important to note whether or not
the loss of the small amount of blood necessary to the operation would
alter the level of the arterial pressure.. As noted in table 3, the loss
of 2 cc. of blood is so quickly compensated for as to justify the con-
clusion that if in operating not more than 2 cc. is lost the normal level
of arterial pressure is maintained.

STUDIES ON THE BRAIN STEM 363

Effects of decerebration. A description of the varying conditions
that follow the removal of the hemispheres or hemispheres and thalamus,
or partial lesions of both, has been given elsewhere (7). It will suffice
here to state that the classic picture of decerebrate behavior in the
pigeon is obtained only if the thalamus be not traumatised in the process
of decerebrating (8), (9). Thalamic injury is associated with temper-
ature disturbances, with the behavior of the animal varying as its body
temperature changes. The technique of decerebration is therefore
important in a comparative series of studies on the rdéle of these two
parts of the brain. The general anatomy of this region of the brain
is described in a previous paper (1). In decerebrating the upper part
of the skull above the cerebral hemispheres was removed taking care
not to puncture the dura mater. After a little practice this is easily
done. A bridge of bone over the very small longitudinal sinus was
left intact so as to diminish bleeding and to serve as a support for the
skin after removal of the brain substance. The dura was then cut
longitudinally and reflected, the anterior cerebral arteries were cauter-
ized and the hemispheres removed in toto. In this way the operation
can be completed with the loss of less than 1 ce. of blood. If more
than 2 cc. of blood was lost in operating it is indicated in the history
_ of the animals. If the thalamus was to be destroyed, this was done
with an electro-cautery, after removal of the hemispheres. The
autopsy findings in the brain with histologic description of the parts
of the brain remaining after recovery from this type of injury have
been described in the preceding report to which reference has been
made (7).

It is common knowledge that the removal of the hemispheres some-
times leads to a slight immediate fall in blood pressure which has been
attributed to shock, hemorrhage, mechanical stimulation, ete. The
same effect is true of the pigeon. In order to avoid these acute effects,
the birds have been kept for time intervals of one week to four months
after decerebration before the arterial pressure was measured (table
6 and fig. 4). These studies have been carried out on fifteen pigeons,
allowing time intervals for recovery from any acute shock effects.
In ten of these birds the arterial pressure was measured before decere-
bration; in five this was not done. For the latter animals some indi-
cation of the change in pressure may be gathered by comparing with
the average normal arterial pressure value given above.

The average pressure after decerebration, thalamus being left intact,
allowing three to seventy-five days for recovery, in fifteen pigeons, was

364 F. T. ROGERS

found to be 99 mm., in comparison with the average pressure in thirty-
nine normal pigeons, of 118 mm. This reduction seems to be uniform
and constant, and continues for months after loss of the hemispheres.
It is not due to loss of blood at the time of decerebration for the amount
of blood lost was so slight that compensation quickly occurs in the
normal animal. It is not due to failure of compensation in the decere-

TABLE 6
Effects on arterial pressure of removing the cerebral hemispheres,. thalamus not
traumatised .
ARTERIAL PRESSURE
NUMBER’ | qme FOR |
PIGEON yapinerenie Sucien. deneetiics, Baie vis
tion tion
days mm. mm.
179 5 120 102 Much bleeding at time of operation
178 5 120-144 120 Good condition
156 5 154-176 | 96-112 | Good condition
158 8 108-128 | 96-110 | Good condition
168 16 118-124 | 92-120 | Good condition
106 75 96-108 | Good condition
155 3 126-136 | 96- 98 | Good condition —
166 16 120-122 | 104-114 | Good condition
_ 167 5 116-124 | 96-102 | Good condition
152 16 82-98 | 90— 95 | Good condition
162 “3 104-108 | 88- 94 | Good condition
157 5 80— 84 | Good condition
116 11 86- 90 | Good condition
21 96 Good condition
6 87 Good condition

* The words. ‘‘ time for recovery”’ indicate the time interval elapsing between
the removal of the cerebral hemispheres and the final blood pressure determina-
tions. The words ‘‘good condition’’ indicate that the bird exhibited no skeletal
muscle incoérdination, body temperature was normal, feathers fluffed in the
characteristic decerebrate manner and the bird exhibited decerebrate restless-
ness. Any variation from these characteristic effects usually indicate thalamic
or medullary disturbances or incomplete decerebration.

brated bird, for when pressure tracings were made allowing the blood
to force the mercury from the zero level, it rises to the level of arterial
pressure as quickly in the operated birds as in the normal ones. (Com-
pare figs. 5 and 8 A.) With decerebration plus thalamic lesions, slower
compensation may be a factor (see below). This fall in pressure is
not due to mere disturbances of food supply for, as tested in normal

STUDIES ON THE BRAIN STEM 365

birds (table 4), complete starvation for periods of three to eight days
» did not cause greater variations in pressure than the normal anesthesia
variations. It might be due to one or more of the following factors.

Fig. 4. Blood pressure, pigeon 162: A: before, and B: eight days after removal
of the cerebral hemispheres. Thalamus intact and body temperature normal.
Time in 30 second intervals. B: x, ammonia to nostrils; b, mechanical stimula-
tion of brachial nerves.

Fig. 5. Blood pressure, pigeon 158, eight days after decerebration. A: ammo-
nia to nostrils; X: mechanical stimulation of brachial nerves, repeated three
times. There was no preliminary increase in pressure in manometer before mak-
ing this tracing, so the mercury was forced up from the zero line wholly by the
arterial pressure.

First, metabolic depression following loss of the hemispheres associated
with decreased activity of the animal; or second, loss of a tonic activity
of the hemispheres on the vasoconstrictor centers; or possibly due to
depressed skeletal muscle tone with resulting capillary dilatation.

366 F. T. ROGERS

In order to test the first of these possibilities the blood pressure deter-
minations were made on a normal pigeon before and after blinding and
starvation. Blinding the bird by excision of the eyes brings about a
condition of quiet and lessened muscular activity that simulates that
of decerebration. The combination of starvation for four days asso-
ciated with this inactivity of blindness did not lower the average arterial
pressure (see table 4, pigeon 164). Of course prolonged starvation
for a long period of time will alter the pressure, witness pigeon 183,
table 4, and pigeon 188, table 9. Inasmuch as all decerebrate birds
were fed by hand and kept in as good condition as possible, it seemed
that one week of absolute starvation would represent as great a meta-
_ bolic disturbance as might be induced by loss of the hemispheres. The
result of this test therefore suggested that the fall in arterial pressure
is due to the loss of reflex or tonic influences from the corchrae? on some
part of the blood pressure regulating mechanism.

In the decerebrate pigeon, thalamus not traumatised, the usual types
of vasomotor reflexes may be elicited by stimulation of spinal nerves,
ammonia, etc. (fig. 5). Asphyxia produces the usual rise in pressure.

Decerebration with destruction of the thalamus. Combined decere-
bration and thalamic cauterization abolishes the ability to maintain
and regulate the normal body temperature of the pigeon (39° to 41°C.).
As stated in the introduction, it was thought that possibly this condition
might be due to a generalized fall in blood pressure. As is evident
from table 8, this is not the case. If the body temperature of such a
bird is kept near the normal level by keeping the bird in an incubator
at 30°C. the arterial pressure of such a bird is very near the pressure
of the warm-blooded decerebrate pigeon. Thus the average values of
blood pressure, decerebrate pigeons (body temperature normal) and
decerebrate-thalamic destruction (cold-blooded animals) are as follows:

TABLE 7
pinos poupis or | nent 7e | ae
°C, mm.
NODA. chs Ob ss bc cok is ene Oe ok a eee 39 39-41 118
DecoreBrate s. oi oi ok Bens ani cick oes cope 14 3)41 99
Decerebration and thalamus destroyed......... 7 3441 99
Decerebration and thalamus destroyed......... 7 26-33 87

STUDIES ON THE BRAIN STEM 367

It is conclusively evident from this table that the loss of temperature
regulation is not due primarily to a lowered arterial pressure, but that
the changes of arterial pressure are secondary to the body temperature
changes. As the body temperature rises or falls the blood pressure
does likewise (table 8 and fig. 6). To this rule one exception was found,
pigeon 174. In this animal the pressure did not fall as the body temper-
ature fell, but. acted in an inverse manner. Attempts were made to
duplicate this but were unsuccessful. This was a bird in which the
operation of decerebration was associated with much bleeding. The
writer is inclined to attribute the high arterial pressure in this bird
to a possible intracranial pressure complication or incomplete destruc-
tion of the thalamus.

Fig. 6. Blood pressure in pigeon 191. Poikilothermous bird; decerebrate and
thalamus destroyed. A: Body temperature of pigeon, 35°C. B: Body temper-
ature of pigeon, 28°C. 2x, mechanical stimulation of brachial nerves.

In the poikilothermous pigeon the vasomotor reflexes vary in intensity
as the body temperature varies (figs. 6 and 7). Both pressor and de-
pressor effects were obtained by stimulation of the brachial nerves.
As the body temperature falls the pressor reflexes particularly decrease
in intensity. The depressor effects seemed to be due to cardiac inhibi-
tion and were present at low body temperatures at which the pressor
effects were very much reduced (compare figs. 6 and 7). Ammonia to
the nostrils caused a rise in pressure which is associated with respiratory
distress. This effect was present at all body temperatures tested (28°
to 41°C.), but was more sluggish at lower temperatures than at normal
body temperature (compare figs. 5, 7 and 8).

TABLE 8

Arterial pressure in pigeons rendered poikilothermous by destruction of cerebrum

and thalamus

NUMBER : ‘ . BODY E
Br OF os DATE PROCEEDING fic Sd Bit teed sa pone ae’
days “Cs mm.
(| June 22 Operation 39
191 {| June 28 Blood pressure 6 35 93.
| June 28 Blood pressure 6 28 58 |
March 9 Operation 39
175 March 14 Blood pressure %
1 p.m. 5 36 | 102-106.
6 p.m. Om ae 83-86
March 5 Operation, much bleeding 40
March 10 __—| Blood pressure
174; 2 p.m. 5 34. | «121
4 p.m. 5 31. | 124
6 p.m. 5 29° =|: 124-134
February 7 | Operation — 39.
14 February 22: | Blood pressure 15 29 92
March 8 Blood pressure 29 41 85
March 13 ‘Bird dead : :
113 February 7 | Operation eee sae
February 20 | Blood pressure 13 23. 92
Operation 3 By Se - 78
Operation 29 ee 85. ‘
| March 9 Operation 39
176 March. 12 Blood pressure 3 295 405 BO
\| March 13 Dead |
180 {| March 18 Operation, much bleeding 39 phy:
\| March 29 Blood pressure 11 34 | 102-106
March 1 Blood pressure tracing, fol- 39 100-110
169 lowed by. operation on
: brain
March 4 Blood pressure 3 31 72-76 |

Body temperatures of the operated birds fixed by varying the temperatures of
bird cages.

Average arterial pressure—body temper
Average arterial pressure—body temperature 28-31°C.—was 87 mm.

368 -

ature 34-41°C.—was 99 mm.

369

STUDIES ON THE BRAIN STEM

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370 F. T. ROGERS’

Compensation after loss of a small amount of blood occurs more and
more slowly as the body temperature is more and more depressed
(fig. 8). In this figure the pressure in the manometer was at zero
when the artery was connected with it. The slow rise to the level of

arterial pressure is in marked contrast to the quick ascent and main- _

tenance of level in the normal bird.
Effects of localized lesions. The major part of the cerebral ooithex
had. been destroyed by electro-cauterization in three birds a year pre-

TABLE 9

Effects on arterial pressure of removing one cerebral hemisphere

“oF DATE PROCEEDING oauuedie REMARKS —
June 10 | Blood pressure then 98-118 |
190 operation . Sian
June 23 | Blood pressure 96-122 | Bird in good condition—
; eats and drinks
June 8 | Blood pressure then 92-102
188 operation
June 23 | Blood pressure 80-96 | Emaciated, does not eat
May 25 | Blood pressure then 100-118
186 operation
| June 8 | Blood pressure 108-120 | Good condition, eats and
[ drinks
(| May 25 | Blood pressure then 94-110
operation
187 {| June7 | Blood pressure 82-92 | Poor condition, does not —
eat .
|| June 9 | Bird dead of starvation

vious to the determination of arterial pressure. All of these birds |

gave normal blood pressure values (table 10).

In one pigeon both occipital poles of the cerebral hemispheres were
removed and the dorso-median part of the thalamus cauterized without
severing the forebrain bundles. Four months later the arterial pressure
in this bird was normal (pigeon 118, table 10).

In one bird extensive cerebellar traumatism was done a year before
the blood pressure determination. The bird regained its ability to
codrdinate its muscle activities in the usual way. Blood pressure

—s

- table 10).

STUDIES ON THE BRAIN STEM

av

- determination in this bird showed a lower alshavtal tension (pigeon | 193,
At autopsy the bird was found to be tubercular.

In a series of four birds the right cerebral hemisphere only was

removed (table 9).

After this operation some birds promptly recover

and in a few days feed themselves in a normal way. The principal

TABLE 10

Arterial pressure after minor brain lesions —

NUMBER

. ARTERIAL
ton: DATE OPERATION PRESSURE REMARKS
mm.
; || April 28: Occipital cortex re-
; moved; thalamus
118 - cauterized :
MS a August 28 Blood pressure trac- 120 | Bird in good condi-
| ing tion
August 17,1918 | Cortez cerebri cau-
79 terized
July 3, 1920 Blood pressure trac- | 122-140) Bird in good condi-
renee it) ing tion
August 17, 1918 | Cortex cerebri cau-
73 | terized
July 3, 1920 Blood pressure trac- | 104-148} Bird-in good condi-
| ing : tion
" February 19, 1919} Cortez cerebri cau-
62 AS terized —
|| July 38,1920 | Blood pressure trac- | 102-124
ing
(| May 15, 1919 Gross traumatism
. of cerebellum’ :
193 <| July 6, 1920- Bird_ recovered. | - 96-102} Bird is tubercular
Blood pressure
L tracing

obvious disturbance in such an animal is complete blindness (at least

temporarily) of the opposite eye. In some cases these birds go into a
condition resembling complete decerebration (probably due to vascular
disturbances in the remaining hemisphere). These animals will not
feed themselves, assume a decerebrate attitude and die of starvation
unless fed by hand. In this series of four birds two recovered and two

372 F. T. ROGERS

died of starvation. The blood pressure readings in the two birds that
recovered were normal. The combination of hemi-decerebration and |
starvation led to lower blood pressure. — |

It was found, therefore, that no localized cerebral or thalamic injury
led to permanent depression of the arterial pressure. Complete loss —
of either hemispheres, or hemispheres and thalamus, leads to distinct
arterial depression.

Artificial stimulation of cerebral.cortex. Electric stimulation of the
cortex of the cerebral hemispheres gave a slight rise in blood pressure
with no movements of the skeletal muscle. Stimulation was done
under light ether anesthesia. Stimulation of the thalamus causes a
sharp rise in arterial pressure, but also leads to muscular activity.
Whether this rise is a true vasomotor reaction or a mechanical one due
to striated muscle contraction was not determined, but Sachs and others
have shown that vasomotor reflexes are readily induced by artificial
stimulation of the thalamic nuclei.

DISCUSSION

The results given above lead to the suggestion that the cerebral
hemispheres exert a continuous tonic activity on the mechanism
whereby arterial pressure is maintained. Whether this be through
the vasomotor, skeletal or some other systems individually or combined
is not determined. Porter’s findings on curarized decerebrate rabbits
indicate a tonic action on the vasoconstrictor centers. The relatively
slight influence of the cerebral hemispheres on the skeletal muscle of
the pigeon! suggests that in this case lowered blood pressure is also
due to a loss of vasomotor tone rather than to changes in the skeletal
muscle. eae |

The possibility of a depression of arterial pressure due to unknown
changes in metabolism, after loss of the cerebrum, can not at present
be excluded. Some evidence has been presented that this alone is not
sufficient to bring about the vascular changes described, witness the
negative effects of starvation, inactivity, ete. These experiments —
however do not conclusively exclude a contributing metabolic factor

1 There are no cortical motor points in the cerebral hemispheres of the pigeon
except possibly for the eye (Ferrier). Electric stimulation of the exposed cortex
in the unanesthetized pigeon causes no muscular movements. Removal of the
hemispheres likewise leads to no paralysis. These facts suggest that the fall in
arterial pressure is not due to secondary changes in skeletal muscle.

STUDIES ON THE BRAIN STEM 373

after brain injury. Further studies in metabolism after cerebral lesions
must be made in spite of the negative results reported by various
. observers. |

However this may be, the inference can not be avoided that any
functional depression of the cerebral hemispheres should be followed
by lowered arterial tension. This might be due to sleep, anesthesia
or destruction (provided there be no increase in intracranial pressure).
Certainly there are differences in detail of the mechanisms in each case
but it is noteworthy that in each case of cerebral depression there is
lowered arterial pressure. No localized cerebral vasomotor centers
are postulated. Indeed localized injuries of either hemispheres or
thalamus caused no change of arterial pressure. The lowering of
arterial pressure described follows the loss of large amounts of cerebral
substance rather than the loss of particular areas of the hemispheres
or thalamus. To this extent, this report reaffirms for the vasomotor
mechanism, the old teaching of Flourens that, in the bird, the effects
of cerebral injury are due not to the loss of local-centers but are pro-
portional to the quantity of brain tissue rendered non-functional.

SUMMARY

A method is described for studying the blood pressure in the pigeon.
The average pressure in the brachial artery of thirty-nine normal
- adult pigeons, under ether anesthesia, was 118 mm. mercury. The
average limits of variations, due to variations in anesthesia, were 109
to 130 mm. Pressor and depressor effects on the blood pressure may
be readily induced by stimulation of the spinal nerves. Ammonia to
the nostrils causes a sharp-rise in blood pressure. Respiratory waves
and Traube-Hering waves of blood pressure occur as in mammals.
The loss of small quantities of blood is quickly followed by compen-
satory changes bringing the pressure back to normal.

Starvation for three to seven days does not appreciably alter the
pressure in normal birds.

Removal of the cerebral hemispheres in Gfiedii birds led to a fall of
the average arterial pressure to 99 mm. (loss of 17 per cent). This
lowered pressure persisted for time intervals up to four months after
decerebration and never regained the level before operation. Removal
of the cerebral hemispheres and thalamus leads to a similar or greater
fall in arterial pressure varying as the body temperature varies. The
ereater the fall in body temperature, the greater the depression of the
arterial pressure.

a

374 F. T. ROGERS

The poikilothermous condition. i in the bird, following excision of the
thalamus, is not primarily due to lowered arterial pressure.

In the pigeon rendered poikilothermous by combined decerebration -

and destruction of the thalamus, the vasomotor responses to mechanical
stimulation of spinal nerves, ammonia to the nostrils, and compensatory
recovery of pressure after slight hemorrhages, are all depressed or take
place more slowly, varying with the depression of body temperature.

The arterial pressure is not appreciably disturbed by removal of a
single cerebral hemisphere, localized lesions of both hemispheres, or
localized thalamic lesion (without cerebral destruction) provided these
injuries are not associated with starvation.

These experiments suggest that the cerebral hemispheres and thala-
mus exert a continuous tonic stimulating action on the subcortical
blood pressure regulating mechanism. ‘This action is not one of local-
ized cerebral centers but varies according to the amount of brain
substance destroyed, rather than the particular area destroyed.

BIBLIOGRAPHY

(1) Rogers: This Journal, 1919, xlix, 271.

(2) AsHER: Ergebn. d. Physiol., 1902, ii, 346.

(3) OwsJANNIKOW: Ludwig’s Physiologische Arbeiten, Leipzig, 1872-1874.
(4) Dirrmar: Ludwig’s Physiologische Arbeiten, Leipzig, 1873.

(5) Sacus: Journ. Exper. Med., 1911, xiv, 409.

(6) Porter AND Storey: This Journal, 1907, xviii, 181.

(7) Rogers: Journ. Comp. Neurol., 1919, xxxi, 17.

(8) ScurapeER: Arch. f. gesammt. Physiol., 1889, xliv, 175.

(9) Munx: Uber die Funktionen der Grosshirnrinde, 2nd ed., Berlin, 1890.

OR eS ee

EXPERIMENTAL STUDIES IN DIABETES

Series I]. Toe INTERNAL PANCREATIC FUNCTION IN RELATION TO
Bopy Mass anp Meraspoutism!

5. The Influence of Fever and Intoxication

| FREDERICK M. ALLEN
From the Hospital of the Rockefeller Institute for Medical Research, New York

Received for publication August 10, 1920

The accuracy with which the metabolism of cold-blooded animals
can be regulated through the temperature was one of the reasons for
the attempt to produce diabetes in them at the outset of this investi-
gation. When the production of a satisfactory type of diabetes proved
impossible, the research was thrown back entirely upon mammalian
experiments, where the disturbing factors are greater. Some obser-
vations were made concerning the effects of fever and of external cold.

No discussion of the literature will be undertaken, beyond reference
to a review (1) of the earlier literature, and a more recent paper by
Freund and Marchand (2), which show that elevation of body temper-
ature is generally accompanied by elevation of blood sugar, but terminal
_ collapse may be accompanied by hypoglycemia; the rise of body temper-
ature in itself tends to increase sugar tolerance, and lowered tolerance
or glycosuria are attributable to intoxication or sometimes to pancreatic
damage. Infections are known to be one of-.the worst agencies in
aggravating human diabetes. The effect of aseptic elevation of temper-
ature seems not to have been tested. It was desired in the present re-
search to compare several forms of infectious and non-infectious fever
in their influence upon partially depancreatized dogs. The only results
actually permitted by circumstances consisted in records of a number
of animals which acquired chance infections, and one research concern-
ing the gas bacillus. The observations will be classified according to
_ the site of the infections.

1 The first four papers of this series are published in the American Journal of
the Medical Sciences.

375

376 ) FREDERICK M. ALLEN

Distemper. For this purpose canine distemper is closely comparable
to human tuberculosis. Diabetes plainly increases the susceptibility
of dogs and still more of puppies to this infection, in the sense that they.
both acquire it and succumb to it more easily. Tuberculosis seriously
lowers the food tolerance and increases the tendency to glycosuria
and hyperglycemia in human patients even in the earlier non-febrile
stages, and this effect becomes still greater in the febrile stage. In
numerous observations in dogs covering all stages of distemper and all
degrees of diabetic tendency, precisely the opposite effect has been
found. It is true that distemper is characterized by early failure of
appetite and digestion. The resulting emaciation constitutes a very
radical undernutrition treatment, and a similarity thus exists to Joslin’s
frequently quoted Case R (8), in which the emaciation of tuberculosis
on a regulated diabetic diet evidently improved the assimilation. But
the infection has been observed in dogs with definitely known tolerance,
which continued for some time to take a diet close to the limits of
tolerance. There have been other examples such as dog C3-77, 1 year
old, weighing 10.5 kilos, and subjected on April 13, 1916, to the removal
of all but 3's to =5 of the pancreas (estimated remnant 1 gram).? Glyco-
suria began immediately and by April 17 had reached 2.9 per cent, fast-
ing. It then ceased as the first signs of distemper appeared in the form
of conjunctivitis. The dog refused food and wasted away in the usual
manner till killed on May 7, at a weight of 6.3 kilos. The pancreas
remnant weighed 1.35 grams. The islands showed very slight vacuo-
lation in a few cells, such as might persist from the initial glycosuria,
but far less than would result from 3 weeks of active diabetes. The.
remnant was so small and the diabetic tendency so strong that sugar-
freedom on fasting would have been impossible if the infection had
introduced any aggravation, but the result seemed to be identical with
that in a non-infected animal.

Pneumonia. Dog B2-18, after removal of 4% of the pancreas on
December 2, developed moderate glycosuria on diets containing carbo-
hydrate, in a cold environment. December 6 the dog was found to be
unwell and febrile, but continued to eat the diet through the illness to
December 9, and the glycosuria continued unchanged. Death occurred
from double pneumonia on December 13.. The autopsy urine contained
1.65 per cent sugar. The vacuolation in the pancreatic islands was
similar to that of non-infected dogs at the same stage.

2 All operations were performed under ether anesthesia.

oa at

_ EXPERIMENTAL STUDIES IN DIABETES 377

Dog C3-73 similarly underwent operation leaving 75 to 7; of the
pancreas on April 3, and died of pneumonia on April 18. ‘The final.
urine on April 17 contained heavy sugar, but death was apparently

preceded by anuria.

Dog D4-72 was left with a remnant of $ to 7y of the pancreas on
January 12, and died of pneumonia on January 19. The appetite
continued and the glycosuria was not appreciably changed with the
onset of fever. Nothing was eaten after January 17. The autopsy
urine still contained a trace of sugar. There was thus a diminution
resembling the effect of ordinary fasting, not the great increase which
usually accompanies infection in any severe human case.

Cat A1-93 was left.with a remnant of $ to § of the pancreas on January
21, and died of pneumonia on January 26. _ Because of refusal of food,
there was only a transitory trace of glycosuria, as would be the case.
in a non-infected cat fasting after such an operation.

_ Other examples might be given in which infection failed to cause
glycosuria when the removal of pancreatic tissue was not quite sufficient
to produce it in a non-infected animal, and still others in which extreme
prostration prevented glycosuria which must otherwise have occurred.
In no instance was any evidence of aggravation of diabetes seen. ©

Pleurisy. Dog B2-22 had been used in another department for
collection of leukocytes by intrapleural injections, and on December
8, x5 of the pancreas was removed without knowledge of the existence
of a large purulent pleurisy. Fever, malaise and other symptoms were
found after the operation. The dog ate small quantities of bread and
milk on December 9 and 10, and glycosuria of 2 to 3 per cent continued
to December 12. Death occurred with sugar-free urine on December
13. .

Subcutaneous abscesses. In connection with subcutaneous injections
and other procedures a considerable number of abscesses have been
observed in dogs with various degrees of diabetic tendency. The
infections themselves have been of varying magnitude, from small
collections causing no systemic symptoms to large ones accompanied
by depression, anorexia and fever above 105°F. The organisms present
were sometimes identified as staphylococci, streptococci or mixed
bacilli. The same rule held as above, namely, that glycosuria might
cease with fasting and prostration, or in less extreme cases might con-
tinue unchanged, but.a marked aggravation such as is familiar in human
cases was never seen. :

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 2

378 FREDERICK M. ALLEN

Infected glands. Dog D4-92 on February 5, 1917, was subjected to
removal of all but + to 4 of the pancreas. Bread and soup were eaten on
February 8, and 100 grams glucose added on February 9, still without
glycosuria. Thereafter nothing was eaten and remarkable symptoms
of confusion and ataxia appeared, increasing on February 12 to general
convulsions and suggesting rabies. The dog was chloroformed on
February 13, and the brain examination was negative for meningitis
or Negri bodies. The autopsy otherwise was negative except for a
little creamy pus found oozing from between the pectoral muscles,
leading to caseous-appearing glands in and about both axillae. The
type or origin of the infection was not determined. Glycosuria
remained absent.

Rabies. Several partially depancreatized dogs died of rabies. One
of these was dog B2-02, which, as previously mentioned (4), had been
carefully studied and was known to have latent diabetes. No glycosuria
resulted in any instance. The negative results were of interest in a
condition attended with such pronounced nervous excitation, and in
which convulsions may give rise to very marked hyperglycemia (5).

General peritonitis. This naturally involves cessation of glycosuria
in most cases because of fasting and prostration. With sufficiently c
large experience, examples are encountered which indicate that the
infection in itself does not alter the glycosuria. Some such were de-
scribed previously (6), and the following have been observed since.

Dog B2-13. November 24, 1913, removal of +? of:pancreas. There was glyco-
suria of 0.25 per cent in 60 cc. of urine following operation, and 0.2 per cent in 330
cc. after eating 150 grams meat on November 27. Otherwise there was fasting and
freedom from glycosuria up to death from peritonitis on November 29.

Dog B2-21. December 4, 1913, partial pancreatectomy leaving a remnant of
73 to zy. After bread feeding on December 5, heavy glycosuria began, and con-
tinued to death from peritonitis on December 11. Bread was eaten daily to
December 9. The autopsy urine was 100 cc., with 2.85 per cent glucose. The
pancreatic islands showed the slight vacuolation proper to this early stage of
diabetes.

Cat A1-82. December 19, 1913, removal of § of pancreas. The cat billed
food but acted well and cleaned her fur up to December 22, and died of perito-
nitis December 23. Glycosuria began with a faint reaction on December 21, rose
to 2.5 per cent on December 22, and was 4 per cent in the last 55 ec. of urine on
December 23. The pancreatic ialande showed incipient vacuolation in a arg
of cells.

Cat Al-87. January 8, 1914, removal of % of pancreas. There was slight
continuous glycosuria with very little eating from January 9 to death from peri-
tonitis on January 12. The islands were free from visible vacuolation, as would
be expected in a non-infected animal with such brief and mild diabetes.

EXPERIMENTAL STUDIES IN DIABETES 379

Peritoneal and pancreatic abscesses. -In addition to previous examples
(6), the following may be mentioned.

Dog D4-65. December 21, 1916, an Eck fistula was unsuccessfully attempted,
and some sutures were left on the veins. January 2, 1917, 3% of the pancreas
wasremoved. The dog was lively and immediately developed glycosuria on bread
feeding. This ceased on January 8, and was restored by addition of 100 grams
‘glucose daily. On January 10 emaciation, fever and weakness first became
noticeable, but the diet was still eaten without change in the heavy glycosuria.
With a change of diet to 1 kilo of beef lung on January 12 glycosuria immediately
ceased. Beginning January 14 food was refused, and the dog was killed January
15, at a weight of 9.8 kilos as opposed to an original 13.5 kilos. A grape-sized
abscess of creamy pus at the site of the Eck operation was the only discoverable
cause of death. It had not altered the course of the diabetes from what is the
rule with non-infected dogs under the same conditions.

Dog D4-57. December 7, 1916, removal of { of the pancreas. The Saal com-
plete absence of diabetes was demonstrated thereafter. March 1, 1917, addi-
tional tissue was removed, possibly sufficient for mild diabetes. Malaise, fever
and complete refusal of food followed. Glycosuria was absent on March 2, 3 and
4, but present just before death on March 5 to the extent of 0.8 per cent in 182 ec.
of urine. The plasma sugar at this time was 0.625 per cent, CO2 capacity 69.2
vol. per cent. Autopsy showed the pancreas remnant to be riddled with small
abscesses, and though there was no necrosis the inflammatory injury had evi-
dently brought on a severe degree of diabetes which would otherwise have been
lacking. Infection has never been found to produce acetonuria or other evidences
of acidosis in any animal.

Dog F6-14 was subjected to removal of about 3 of the pancreas in three succes-
sive operations. January 31, 1918, an attempt was made to produce diabetes
by circulatory stasis of the remnant, as described in a later paper. Only slight
and transitory glycosuria resulted on a diet of bread and soup with 100 grams
glucose. February 8, operation showed an abscess containing about 5 cc. of
creamy pus between the pancreas and the duodenum. The cavity was cleaned
and stasis repeated. Glycosuria was still impossible to maintain, and on March
9 stasis was applied for a still longer time, no infection beingfound. Glycosuria
was then continuous up to March 19 on bread diet with 100 grams of glucose, but
ceased then on plain bread and soup feeding. March 20 the abdomen was again
opened, and the pancreas was found buried in a large mass of adhesions, which
- when delivered outside and opened was found to contain a very large abscess.
The pancreas remnant, which in its whole length formed one wall of the abscess,
was much inflamed but not digested. Nothing was done except the cleaning up
of the infection, and the tolerance continued exactly as before; i.e., glycosuria
was absent on bread feeding and present with addition of 100 grams of glucose.
On April 10 the abdomen was again opened, and a tiny abscess in the omentum
appeared as the only remains of the previous infection. Stasis was again applied
to the pancreas remnant, and the dog died within 24 hours, whether from infec-
tion or from pancreatic intoxication was undetermined.

380 FREDERICK M. ALLEN

The long history of this animal, with alternate presence and absence
of a low grade infection, seems to prove that in this instance the infec-
tiom had no important influence upon the tolerance.

Other examples of this sort might be given. There was particular
interest in the cases in which diabetes was produced by inflammation
instead of by simple resection, because of the supposed closer imitation
of the clinical etiology. It was conceivable that inflammation might
damage the islands in function as well as in structure, so as to render
- them more susceptible to toxic influences. The negative results raised
a question concerning some fundamental difference between clinical
and experimental diabetes, or a mere difference of constitutional reaction
to infection on the part of man and animals. The above general obser-
vations sufficed positively to exclude any such marked aggravation of
diabetes in animals as occurs regularly in human cases with the fever
and intoxication accompanying infection. There remained the need
of making a more exact test of the tolerance in experimental diabetes
as influenced by infection, and this opportunity was afforded by the
experiments with the gas bacillus reported in the next paper. These
seemed to indicate that the difference between clinical and the experi-
mental diabetes may be one of degree rather than of kind.

CONCLUSIONS

1. The serious aggravation of diabetes, which occurs almost invari-
ably in human cases in the form of a strongly increased tendency to
glycosuria and acidosis, is never seen in dogs. Even when the infection
is an abscess bordering or invading the pancreatic tissue, no influence
is evident beyond that explainable by direct injury of parenchyma.
This contrast between clinical and experimental diabetes is very marked,
but according to the more exact tolerance tests in the succeeding paper
it may represent a difference of degree rather than of kind.

2. Infection and fever have also no specific influence in diminishing
the diabetic tendency of dogs. Care is necessary in interpreting such
observations, in order not to confuse the direct influence of fever or
infection with the consequences of fasting or prostration, which tend
so strongly to suppress glycosuria in dogs. One suggestion of a con-
stitutional difference between species may be found in the tendency
of human patients to acidosis and of dogs to cachexia.

3. The aggravation of human diabetes is a reaction to intoxication
rather than to fever, as shown by its occurrence in the afebrile stage

EXPERIMENTAL STUDIES IN DIABETES 381

of tuberculosis and by other evidence. The present observations con-
cerning infectious fever, with the previous ones concerning the pyrexia
of exercise in dogs, prove that no specific aggravation of diabetes or
lowering of tolerance results from the metabolic alteration attendant
upon elevation of body temperature in experimental animals.

BIBLIOGRAPHY

(1) ALLEN: Studies concerning glycosuria and diabetes, 1913, 38, 563, 564.

(2) FrreuND AND Marcuanp: Deutsch. Arch. klin. Med., 1913, ex, 120.

(3) BENEDICT AND JosLin: Carnegie Inst. Washington, Pub. no. 176, 1912, 55.
Also Jostin: Treatment of diabetes mellitus, 2nd ed., 1917, 409.

(4) AtLEN: Journ. Exper. Med., 1920, xxxi, 384.

(5) ALLEN AND WisHart: Journ. Biol. Chem., 1920, xliii, 140.

(6) ALLEN: Studies concerning glycosuria and diabetes, 1913, 482 (dog 66); 492-
495; 760 (dog 179).

EXPERIMENTAL STUDIES IN DIABETES

Series I]. Toe INTERNAL PANCREATIC FUNCTION IN RELATION TO .
Bopy Mass anp METABOLISM

6. Gas Bacillus Infections in Diabetic Dogs

MARY B: WISHART anv IDA W. PRITCHETT :
From the Hospital of the Rockefeller Institute for Medical Research, New York

Received for publication August 10, 1920

In the course of this diabetic research four dogs died of gas bacillus
infection. Two of these instances were merely post-operative. peri-
tonitis, with the gas bacillus predominating.

Another was dog E5-74, a bulldog mongrel, aged 5 years, in splendid .
condition, weighing 20.7 kilos. July 3, 1917, two-thirds of the pancreas
were removed, and most of the remnant was cut off from duct communi-
cation.! On July 20, the remnant was subjected to circulatory stasis
for 14 hours. Glycosuria was present on bread diet with addition of
150 grams of glucose daily up to July 30, when it ceased, and the dog .
was turned loose in the yard with other dogs on bread diet. The
behavior meantime was normal. About August 5, swelling in the neck
became noticeable and the dog was slightly depressed. August 7, the
swelling was much larger, and a deep abscess was opened surgically,
releasing a considerable quantity of thick bloody pus containing gas
bubbles. Cultures from some of the necrotic debris gave a pure growth
of B.: aerogenes capsulatus. Death occurred August 9, after still
greater invasion of the neck. There was no glycosuria or vacuolation
of pancreatic islands, though both these conditions might have been
prevented by the terminal emaciation and cachexia.

Dog B2-49, a female mongrel aged 3 years, in medium nutrition
at a weight of 25.4 kilos, underwent partial pancreatectomy on March
27, 1914, leaving a remnant of } to $ about the main duct. As pre- —
viously mentioned (1), prolonged carbohydrate over-feeding was used.
in the attempt to break down tolerance, 300 or 400 grams glucose

1 All operations were performed under ether anesthesia.
382

EXPERIMENTAL STUDIES IN DIABETES 383)

being added to the diet of bread and soup daily. There was neither
glycosuria, diarrhea nor any evident ill-health, until the animal was
unexpectedly found dead on May 9. There were adhesions in the
right pleura, from supposedly sterile intrapleural injections in another
department long before the animal was taken for diabetic work, and
these probably furnished the start of the infection. Gas bacilli were
found abundantly in smears and cultures from the principal viscera,
seemingly alone. The greatest change was in the spleen, which was
blown up to resemble a lung. The pancreas remnant was normal and
free from vacuolation. In other words, neither the prolonged sugar
feeding nor the infection produced any change in either islands or
acini.in this non-diabetic animal.

A study of gas bacillus infections was in progress. at this time under
the direction of Dr. Carroll G. Bull. As gas bacillus infections are
rare in dogs, it was decided to follow up the above accidental obser-
vations by experiments upon diabetic dogs with a view to two questions;
first, whether such animals are abnormally susceptible to such infections
by reason either of the excess of circulating sugar or a specific diabetic
lowering of resistance; second, whether an aggravation of the diabetes
is demonstrable by such infections. The conditions were favorable
for both problems; for the first problem because the growth of the gas
bacillus is notably favored by the presence of sugar, and some test was
thus afforded of the theory of excess of sugar as the cause of diabetic
susceptibility to infection; for the second problem because of the proof
(2) of the production of a soluble toxin by the gas bacillus, so that'a

“systemic effect capable of influencing the diabetes might be expected
from a local infection. Accordingly experiments with intramuscular
injections of pure cultures of the Welch bacillus were performed upon
three diabetic dogs. The dosage used was intended to produce the
maximum possible local effects and general intoxication without exces-
sive prostration. Still larger doses might have overwhelmed the ani-
mals suddenly and completely, but would thus have demonstrated
nothing of value for either bacteriology or diabetes.

In the first experiment (table 1) glycosuria practically ceased with
the anorexia accompanying infection on September 12, as usual with
dogs and in contrast to the usual aggravation of symptoms in human
patients with infection. Nevertheless a lowering of tolerance was shown
by the heavier glycosuria when the diet was taken on September 13: -
Illness and fasting again resulted in sugar freedom after the injection of
September 14, but a more marked lowering of tolerance was evident in

384

MARY B. WISHART AND IDA W. PRITCHETT

TABLE 1

Dog E5-88. Male; Welsh terrier mongrel; old but strong, in excellent nutri-
tion; weight 11.25 kilos. August 24, 1917, removal of pancreatic tissue weighing
18 grams. Remnant about main duct estimated at 1.6 grams (, to j;). The
diabetes was checked by undernutrition and fasting, so that at the time of the
experiments the dog weighed 9.5 kilos and took a bread and soup diet with very —

slight glycosuria.
RECTAL URINE
DATE TEMPERA- REMARKS
bp pe Volume | Glucose
1917 A ce. per cent
September 10..... 660 0.3 | Bread and soup diet
September 11 . ..| 38.7 450 0.4
September 12. .. 510 | Trace | At 10:45 a.m. injected 0.1 cc. per
10:00 a.m. . .| 38.8 kilo of broth culture of Welch
1:15 p.m.....| 39.7 bacillus intramuscularly right
3:30 p.m.....| 40.7 thigh. Marked local edema and:
5:30 p.m.....| 40.4 swelling. Dog depressed; ate
nothing
September 13..... Thigh still swollen. Dog unwell
9:00 a.m.....] 39.7 880 2.6 but took entire diet, part forci-
bly
September 14. .. 9:30 a.m., injected 0.3 cc. per kilo
9:00 a.m. ...), 3&3 670 2.8 of broth culture of Welch bacil-
lus intramuscularly left thigh.
Much local swelling. Dog ill
and feverish; refused diet but
ate a little meat
September 15..... 39.8 320 1.4 | Refused all food
September 16..... 425 0.3 | Refused all food. Great edema.
. and crepitation, extending into
scrotum
September 17..... 510 | Trace | Refused food
September 18..... 650 0 Refused food
September 19..... 420 0 Refused food
September 20..... 400 | Trace | Ate a trifle of meat and bread
September 21..... 39.5 450 0 Ate very little of meat
September 22..... 480 | Trace | Ate more meat. Much swelling
and gas in leg
September 28..... 460 0.6 | Acting better. Ate more meat
September 24..... 400 1.2 | Ate some bread and meat
September 25... . 500 2.1 | Ate full diet
September 26. ... 700 2.9 | Ate full diet
September 27..... 630 3.4 | Ate full diet
September 28..... 610 3.1 | Ate full diet ‘
September 29..... 38.8 1200 2.2 | Both thighs have discharged ne-
crotic material, leaving granu-
lating ulcers. Dog lively and
vigorous

EXPERIMENTAL STUDIES IN DIABETES 385

the glycosuria from meat alone on September 22 and 23, and the heavier
glycosuria thereafter on the regular bread diet.

Dog E5-89. Male; mongrel; age 3 or 4 years; good condition; weight 14 kilos.

- August 24, 1917, removal of pancreatic tissue weighing 25 grams. Remnant
~ about main duct estimated at 1.6 gram (5 to 7+). Severe diabetes being thus
produced, the glycosuria was raised to a maximum by a diet of bread and soup
with 100 grams of glucose daily. .

September 7, at a weight of 12.6 kilos, 0.25 gram additional pancreatic tissue
was removed for microscopic examination.

September 14, at the same weight, 0.1 cc. broth culture of Welch bacillus per
kilo was injected intraperitoneally, in order to test whether under these condi-
tions of maximum glycosuria and hyperglycemia infection would be possible.
The rectal temperature rose within an hour to 39.4°C. After 6 hours it was
39.5°, and the next morning 39.6°. It then subsided, and after one day of slight
malaise the dog continued to eat his diet. The glycosuria continued unchanged
except for a diminution on the one day of anorexia.

September 24, an injection of 0.3.cc. of broth culture per kilo was given intra-
muscularly in one thigh. The usual local and general symptoms occurred in
intense form. September 29, with very large swelling and gas formation present
in the leg, a blood ‘culture was taken and proved negative. The dog regained a
little appetite, taking small amounts of meat and bread daily, but great anemia
was shown by blood examinations, the corpuscle volume being only 10 to 12 per
cent. Death occurred October 5. Glycosuria remained heavy throughout,
including the autopsy urine. The gross autopsy showed no visceral changes sug-
gestive of gas bacillus invasion. Cultures of blood and tissues were also negative
for this organism. !

The pancreas remnant, normal in appearance and consistency, weighed 1.7
grams. Microscopically, the tissue removed August 7 showed a very early stage
of vacuolation of islands. The remnant at autopsy showed a late stage of the
process; islands were scarce and small, and the great majority of the cells (proba-
bly all of the beta cells) were maximally vacuolated.

In this experiment the production of a general infection with the
gas bacillus proved impossible notwithstanding the severe diabetes
and intense glycosuria. The intraperitoneal injection failed entirely.

The intramuscular injection caused extensive sloughing which destroyed
most of the musculature of the limb, but death resulted only from the
immediate and subsequent toxic effects and not from systemic invasion.

Dog E5-90. Male; mongrel, age 3 or 4 years; medium nutrition; weight 10.25
kilos. August 24, 1917, removal of pancreatic tissue weighing 20.3 grams. Rem-
nant about main duct estimated at 1.6 gram (3's to 74). On bread diet there was
a diminishing glycosuria which ceased August 31, probably because of hypertro-
phy of the pancreas remnant, which subsequently at autopsy was found to weigh.
5.4 grams. Beginning September 8 the addition of 100 grams glucose restored a
heavy glycosuria, and the tolerance was brought down so as to produce a perma-.

386 MARY B. WISHART AND IDA W. PRITCHETT

nent mild diabetes. During October sugar freedom was maintained on a diet
of 500 grams lung and 100 grams suet, except for occasional days on which it was
proved that bread and soup diet would promptly bring back a mild glycosuria.

October 25, an intravenous glucose tolerance test was performed, by injection
of 25 ec. of 10 per cent solution of Merck anhydrous glucose every 15 minutes
(1 gram per kilo per hour, on 10 kilos weight) for 3 hours, according to the method
described elsewhere (3). Catheterization was performed and blood samples
taken before the first injection and at pouty intervals thereafter as shown in
table 2.

October 26, 4 cc. of a heavy broth culture of the Welch bacillus were injected
intramuscularly in the right thigh. The rectal temperature rose to 41.1°C. that
evening and was 39.6° the next morning. The dog refused food and there was no
_ glycosuria. By October 31 there was partial recovery and part of the diet was

eaten. The weight had fallen from 10 kilos to 9.75.

i

TABLE 2
BLOOD URINE
Plasma sugar ; Volume Glucose nial

No- No- No- |
Qetes vember] pce) cto; |vember| pers | eras (vember! Denis
per cent |per cent|per cent| cc. ce. ce. grams grams | grams
0.164 | 0.183] 0.122 0 0 0 | Before injection
0.333 | 0.400) 0.384 5 41 | 14 0.034 | 0.450) 0.320) End of Ist hour
0.323 | 0.400} 0.500) 14 42 | 50 0.080 | 1.150} 2.000) End of 2nd hour
0.213 | 0.216} 0.455) 95 14 | 26 | Faint | 0.360) 0.680) End of 3rd hour
0.081 400 | 150 | 30 | Slight | 0.690) 0 Next morning

November 1, an intravenous glucose test was performed, identical with the
dosage on October 25. A lowering of tolerance was indicated by both the blood
and urine analyses.

November 14, 4 cc. of the gas bacillus culture were injected in the other thigh.
Local edema, gas formation and necrosis occurred as before, but the general
symptoms were less. The temperature on the morning of November 15 was
38.8. The dog ate well and showed a spontaneous glycosuria of 2.15 per cent in
340 cc. urine. The following day it was 0.48 per cent in 530 cc. urine, and then ©
disappeared.

Later the dog was unwell and ate poorly, probably on account of secondary
infection of the sloughing area in the leg. No further glycosuria developed, and
by December 18 the animal was again in good general health at a weight of 10
kilos, though a large open ulcer was still present. t

December 18, the animal was given the same intravenous glucose injections as
before. A reduced tolerance was still indicated, either because of the ulcer or
because the lowering due to infection was permanent, as it is in many human
cases.

EXPERIMENTAL STUDIES IN DIABETES 387

An accidental or spontaneous fall of tolerance is probably excluded by the
fact that the dog was kept on the lung and suet diet till March 27 without glyco-
suria. He was then used for other experiments, and no further tolerance test
was made.

CONCLUSIONS

1. Intramuscular injections of pure cultures of B. aerogenes capsu-
latus produced local necrosis and gas formation in partially depan-
creatized diabetic dogs. Systemic or peritoneal infection was not
obtained. The observations failed to indicate any lowering of resistance
in these animals due either to the diabetes itself or to the excess of
sugar in the body fluids. The latter point is further emphasized by
the fact that the reactions were essentially similar in the first dog with
mild glycosuria, in the second dog with heavy glycosuria, and in the
third dog free from glycosuria. These results agree with the general
experience that such animals ordinarily bear operations well and their
wounds heal normally.

2. A lowering of tolerance by infection was demonstrable both by
feeding and by intravenous glucose tests. Though this influence is
less in animals than in human patients, the difference seems to be one
of degree rather than of kind.

BIBLIOGRAPHY

(1) Atuen: Journ. Exper. Med., 1920, xxxi, 394.
(2) Butt AND PritcHett: Journ. Exper. Med., 1917, xxvi, 119.
(3) ALLEN AND WisHART: Journ. Biol. Chem., 1920, xlii, 415.

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK

I. Tar Basat METABOLISM ©

JOSEPH C. AUB |
WITH THE TECHNICAL ASSISTANCE OF K. SANDIFORD

From the Laboratory, of Physiology in the Harvard Medical School

Received for publication August 12, 1920

The marked loss of body temperature is one of the most striking
features of traumatic shock. To study this, as well_as to investigate
the relationships of metabolism and ventilation to the sudden changes
in blood pressure, was the purpose of these experiments. The problem
was approached through the gaseous exahange.

The literature upon this subject is very meager. Guthrie (1),
reported no consistent findings in either O. absorption or CO, output
with animals under ether anesthesia. Henderson, Prince and Hag-
gard (2), in a preliminary note, mention a marked drop in metabolism
in two dogs in shock, but give no details of experiments. Roaf (3),
_working on decerebrate cats, states that his experiments tend to show
that fall of blood pressure does not markedly reduce the production of
CO..

Methods. Cats were used that had not eaten for 24 hours. They
were anesthetized by urethane given by mouth, 8 cc. of a 25 per cent.
solution per kilo of body weight, and only when fully anesthetized
were they stretched out on an animal board. The temperature was
recorded through a rectal thermometer graduated to tenths, and was
kept as nearly constant as possible by means of an electric heating pad.

The operation consisted of inserting a trachea cannula and cannulae
in two arteries, usually both carotids, and also usually one in the
external jugular vein. One carotid cannula was then attached to
a mercury manometer and blood pressure tracings begun. In some
experiments 10 cc. of arterial blood were now removed; in most cases
blood was taken only after several respiration samples had been
obtained. : |

388 .

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK 389

The inspired air was room air. The samples of expired air were
obtained in two 8-liter copper spirometers. The valves used were
Tissot valves, attached directly on the T-shaped glass tracheal can-
nula. The air sample was promptly withdrawn from the spirometer
and preserved under pressure in the usual type of glass sampling tube.
Gas analyses were made in the Haldane apparatus, and careful checks
of room air were made before samples were analyzed. Urinary nitro-
gen determinations were not made (4).

Rebpirafory ( Melbbolism
Trauma hock

Exp ¥XXVI
>

‘han

R.Q. Temp. Vent.

Temp. ¢

‘3
é

3
! a
r 2
0 :
: AAA
3 4 + Bn gaa. enh,
maT we 3
Sahe” aa = q we
o Q =— =_ Nn
3 Ne
3 { ‘
[ A i
3 f
3 i
he . \!
5” >
$ .. 2 \ h
i BE %
a on

Ec:

fet

Fig. 1. Vent. = Ventilation volume per minute in 100 ce.
Cal. = Total calories per hour.
R. Q. = Respiratory quotient.

Hours affer Anaeslhelizalion

om

To produce shock the thigh muscles of both hind legs were thor-
oughly crushed (5). When the blood pressure fell below 70 mm. Hg.
* systolic and stayed below that level; the animal was considered to be
in a state of shock. No attempt was made to measure blood flow, as
all’ extra manipulation was rigidly avoided.

390 JOSEPH C. AUB

The experiments may be grouped as follows:

I. Normal controls. Table 1.

II. The simple traumatizing of muscle tissue and the study of respi-
ratory metabolism before and after the resulting drop in blood pres-
sure. Tables 3 and 4.

III. The production of traumatic shock and the subsequent raising
of the blood pressure by transfusion with cat’s blood. ‘Table 5.

IV. The effect on metabolism of hemorrhage when it alone caused a
marked fall of pressure. Table 2 | |

V. The production of a low Biebd pressure without shock by increas-
ing pericardial pressure. Table 6.

Discussion. Table 1 shows that the metabolism following urethane
anesthesia when given by mouth remains quite constant for 44 hours
at least. It may then fall to a lower level. Raeder (6) kept rabbits
alive for over 3 days by administering urethane subcutaneously. He
came to the conclusion that the total metabolism fell only about 2
_per cent an jhour, and that it’ was a satisfactory anesthetic to use in
studying respiratory metabolism.

The level of the basal metabolism during shock has fallen in all
but one case below the value found before the muscles were crushed.
In six cases of mild shock (table 3) the average reduction in calories
was —19 per cent, and in eight cases of severe experimental shock
(tables 4 and 5) the average fall was —30 per cent. This average
does not include experiment LIII in which the metabolism rose. Like-
wise in five experiments in which pericardial pressure was increased
(table 6) and the blood pressure so reduced, there was a prompt drop
(fig. 2) to an average of —31 per cent below the former height. In
general the “critical level” of blood pressure for the metabolism, as
with the development of diminished alkaline reserve, is at 75 or 80 mm. —
Hg. At that level the metabolism may be within normal limits or it -
may be considerably reduced. Usually when a normal value is found,
the blood pressure has been stationary or rising; but when the meta-
bolism figures are reduced the blood pressure is falling. With a pres-
sure below 75 mm. Hg., the calorie production has, with but one excep-
tion, been reduced.

While the reduced blood pressure “undoubtedly has a great deal to
do with the fall of metabolism, it is probably not the whole story.”
Experiment XX XIV, table 2, shows that a low blood pressure, follow-
ing hemorrhage alone, may be associated with only a slight drop in
metabolism. With a blood pressure of 55 to 62 mm. Hg. immediately

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK . SBE

after bleeding the metabolism was only —10 per cent; and in the third
period, although the rising blood pressure had been below 80 mm. Hg.
for 45 minutes, the metabolism was only —1 per cent. So also in the

last period after the pressure had been 50 mm. Hg. for 20 minutes,
but was rapidly rising at the time the period was taken, the metabolism
was only —7 per cent.

J
BLProGat fressurer
R.@
Vel
GOL
98 a 2
‘a
See
wl 7
i? te
60 [
F Stace
:
Ci i Ht
| a
ct T
tt . t
iz i
it 1
tt i
i a i
. T
r
tt
ime
Cr
; ae tt | if ait
Cy
rH ry a!
m tt 4
! i
+ t
{ - Cg we! 4 rT L 4
: Ceo Ht :
forte th Ceterct 1B a

Fig. 2. Vent. = Ventilation volume per minute. The figure should be
multiplied by ten.

There are also examples of blood pressures above the critical level
associated with reduced metabolism. In experiment X XXIII, table
2, the second period shows a sudden drop of 22 per cent in metabolism
20 minutes after bleeding 15 cc., while later a return to normal limits
occurred even though muscle injury was done. This bleeding only
caused an immediate drop in blood pressure from 110 to 90 mm. Hg.,

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and at the time of observation the blood pressure was 95. It was,
however, not rising. In experiment XXX, table 3, where blood pres-
sure fell from 170 to 108 mm. Hg. after severe trauma, the metabolism
still fell 14 per cent and 29 per cent; and in experiment XX XI, table 3,
even though blood pressure after trauma was 85, the heat production ~
was reduced —21 per cent and —24 per cent. The temperature in these
last two experiments, however, was not satisfactory. Likewise in
experiment XXXII, the metabolism fell 23 per cent even though the
blood pressure was 100. -In experiment XXXVI, table 4, fourth —
period, though the blood pressure was 80-75 in an animal rapidly
developing shock, the metabolism was —26 per cent of the normal
level. The explanation of these differences is obscure. After hemor-
rhage the blood pressure may be low for a time without marked drop
in metabolism. After muscle injury the metabolism may be reduced |
before a great fall in blood pressure has occurred. Possibly the obser-
vations of Gesell (7) will satisfactorily account for these facts. He
found that in the early stages of shock from tissue abuse there is usu-
ally a marked reduction of the “volume flow’’ of blood in peripheral
organs, although blood pressure is only little changed, and le reports
one instance of increased volume flow after hemorrhage though the
blood pressure was falling. The volume flow of blood determines the
oxygen delivery to the tissues, and this may vary to some degree with-
out corresponding variation in blood pressure.

The reduction of blood pressure by increasing pericardial pressure,
as described by Cannon (8), is due to mechanical venous obstruction,
and is similar in its action to the methods described by Janeway and
Jackson (9) and by Erlanger and Gasser (10). When the blood pres-
sure is reduced by this procedure, the metabolism, as calculated from
the respiratory gases, shows a marked prompt reduction to the level
found with similar blood pressures in experimental shock (table 6).
The respiratory quotient is also similar in that it rises. The prompt
reduction of metabolism by this method of merely hindering the venous
return to the heart is evidence that some mechanical factor such as —
retarded blood flow is the cause of the reduced metabolism. This is
emphasized by the rapidity of the appearance of the reduction, which
by this method seems to occur without delay.

The rapid appearance of diminished alkaline reserve in shook as
shown by the lowering of the blood CO. combining capacity, should
not reduce metabolism but, if anything, should tend to raise it slightly,
as shown by studies (11) in conditions where a similar drop in the
reserve May occur.

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK 397

A feature of the results is the rather low average respiratory quotient.
In forty-seven observations in Raeder’s publication (6) and in sixty-
two normal observations in this series, the average respiratory quotient
was the same—0.75. Wilenko (12) in ten periods with cats partly
anesthetized by 1 gram urethane per kilo by mouth, had an average
respiratory quotient of 0.79 in his controls. This rather low value,
as also the work of Underhill (13), and the presence of a hyperglycemia
(paper III), all suggest a decreased oxidation of carbohydrate under
urethane anesthesia. This point will be more fully discussed in a
future publication. Here it is sufficient to say that the control experi-
ments demonstrate that the height of the basal metabolism remains
constant under urethane for 44 hours. Inasmuch as we are dealing
with relative changes in each animal, the low respiratory quotient
in the control periods does not affect the eventual conclusions. The
tendency as shock develops has been for the respiratory quotients to
rise—the average for twenty-one observations being 0.81, an effect,
probably, of increased ventilation and the resulting pumping out of
COs. ;

Having the animal anesthetized makes the determination of the
basal metabolism a great deal simpler, for voluntary movements and
emotional excitement are removed as factors which might raise the
metabolism. The abnormality of some of the respiratory quotients
reported here could practically all be traced to irregularities in breath-
ing just prior to or during the observation. A period of hyperpnea
previous to the observation gave a low respiratory quotient, and with
hyperpnea during the period, the respiratory quotient was always
elevated. In cats the breathing under urethane is apt to vary in
quantity rather markedly and, as a result, no great stress may be laid
upon respiratory quotients. Then, too, there is the factor of rapidly
decreasing alkaline reserve with the marked fall of. blood pressure, as
shown by Cannon (14), and whether this is due to an accumulation of
lactic acid or to a disappearance of alkali into the tissyes, the effect
would probably be a temporary pumping out of extra CO. into the
expired air. The effect of this change in balance might well affect the
dissociation curves of hemoglobin for oxygen and for CO, (15). With
the low blood pressure found in shock, the effect. on the exchange of
substances between blood and tissue fluids may be considerably dis-
turbed, and these factors might distort the relationship between the
OQ, absorbed and.the CO, given off in the lungs. As a result, little stress
may be laid on the respiratory quotients obtained, because of the

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406 JOSEPH C. AUB ;
extremely complicating factors which may be influencing them. How-
ever, the oxygen absorption from the lungs must represent the amount
of oxygen available for metabolic uses in the tissues, inasmuch as the
blood leaves the heart normally saturated with oxygen (16). That this
absorbed oxygen represents the amount used by the body seems highly
probable, as otherwise there would be an accumulation of oxygen in
the tissues, a condition which seems decidedly unlikely. In all these
observations, therefore, the oxygen absorption has been used as the
basis of comparison, and the CO, assumes a relatively unimportant
role through its influence on the respiratory quotient.

The reduced metabolism affords an explanation for the marked
reduction of body temperature in shock, which may go as low as 87.8°,
or even 76.1° in cervical spine lesions, according to Weil (18) and to
Volkmann (17). This investigation, however, does not indicate that
the reduction is usually a forerunner of the onset of shock, or that
it is a causative factor in the production. In fact, in two experiments
the metabolism just before the onset of shock was slightly elevated
above the normal level.

The volume of respiration per minute has likewise been studied.
The average ventilation per minute of the control observations was
557 cc. in twenty-one experiments; the average after crushing the
muscles and before the onset of a shock blood pressure level was 860

cc. in ten experiments, (a variation of +54 per cent from the control

observations), and after the onset of shock it was 635 cc. in thirteen
experiments. This variation is hardly enough to account for the onset
of shock, in these muscle trauma experiments, by the acapnia theory
advanced by Henderson and Haggard (19), (20). Besides, rapid
breathing with a higher ventilation rate per minute than in shock has
been repeatedly seen under urethane anesthesia without the onset of
spontaneous shock. These data also show that the fall of the meta-
bolic rate was not due to changes in. either the volume or exertion
involved in respiration.

With the metabolism so much reduced by shock, it naturally became
of interest to know the effect of transfusion of a sufficient amount of
blood to bring about recovery. Table 5 shows five such experiments
in three of which the metabolism returned to normal limits, while in
experiment XLI the metabolism remained low. In experiment
XLVIII, the figures for the first period after transfusion were above
the normal determinations. Experiment LIV in table 6 shows the
effect of reducing the pressure to shock level by pericardial pressure.

a

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK 407

Periods III and IV were taken directly after releasing the pressure,
and showed a normal rate. It therefore seems that making the circu-
lation adequate causes the low metabolism of shock to disappear
Coogan

CONCLUSIONS

1. Ethyl carbamate (urethane) is a satisfactory anesthetic for the
study of gaseous metabolism in animals over short periods of time.

2. Experimental traumatic shock causes a marked fall in the rate
of basal metabolism to 70 per cent of the original level. The degree
of fall is dependent upon the severity of the shock produced.

3. A similar fall of the metabolic rate may be rapidly accomplished
by interfering with the circulation by increased pericardial pressure.

4. The effect of hemorrhage is not constant. It may temporarily
lower, or have no immediate effect on the metabolic rate.

5. Recovery from shock after blood transfusion is usually associated
with a prompt return of the metabolic rate to a normal level.

BIBLIOGRAPHY

(1) Gururie: Journ. Amer. Med. Assoc., 1917, Ixix, 1394.
(2) HENDERSON, PRINCE AND Haaearp: Ibid., 965.
(8) Roar: Quart. Journ. Exper. Physiol., 1912, v, 31.
(4) BENEDICT AND CARPENTER: Carnegie Inst. of Wash., no. 261, 203.
(5) CANNON AND Baytiss: Rept. of Shock Committee, English Medical
Research Committee, 1919, no. 26, 19.
(6) Rapper: Biochem. Zeitschr., 1915, lxix, 257.
(7) Geseiu: This Journal, 1918, xlvii, 468.
# (8) Cannon: Comp. rend. d. 1. soc. d. biol., 1918, Ixxxi, 850.
' (9) JANEWAyY AND Jackson: Soc. Exper. Biol. and Med., 1915, xii, 193.
(10) ERLANGER AND GassER: This Journal, 1919, xlix, 151.
(11) Arkrnson AND Lusk: Journ. Biol. Chem., 1919, xl, 79.
Avs AND DuBors: Arch. Int. Med., 1917, xix, 865.
(12) Witenxko: Biochem. Zeitschr., 1912, xlii, 44.
(13) UnpERuILL: Journ. Biol. Chem., 1911, ix, 13.
(14) Cannon: Rept. of Shock Committee, English Medical Research Com-
mittee, 1917, no. 25, 85; Journ. Amer. Med. Assoc., wiki Ixx, 531.
(15) Henperson, L. J.: Journ. Biol. Chem., 1920, xli, 401. i
(16) AUB AND Be teat as: This Journal, 1920, liv, 408.
(17) VoLKMANN-ZwicHav: Miinchener. Med. Wochenschr., 1917, Ixiv, 1215.
(18) Wert: Ibid., 338.
(19) HenpEerson: This Journal, 1910, xxvii, 152.
(20) HenpEeRson AND Haaaarp: Journ. Biol. Chem., 1918, xxxiii, 365.

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK

Il. Tar OxyGEen CONTENT OF THE BLOOD

JOSEPH C. AUB ann T. DONALD CUNNINGHAM

From the Laboratory of Physiology in the Harvard Medical School and the Medical
Clinic of the Peter Bent Brigham Hospital!

Received for publication August 12, 1920

In studying the basal metabolism of experimental traumatic shock it
became clear that the reduction of the calories utilized was dependent
upon some undetermined factor. This was suggested by the fact that
the fall in metabolic rate did not always coincide with the fall in blood
pressure. Other observers have noted that there were marked changes in
the circulation before a shock level of blood pressure was approached.
Gesell (1) showed aslowing of blood flow through the salivary gland before
a fallin pressure had developed. Yandell Henderson, (2) while working
with shock induced by intestinal trauma in dogs, found a markedly de-
creased O2 content in the venous blood, which he thought followed the
failure of the venopressor mechanism and demonstrated a true anoxemia.

These observations suggested that the determination of the oxygen
of the arterial and venous blood, as well as the blood flow during the
development and recovery from traumatic shock, might give evidence
as to the cause of the fall of metabolism (8).

Method. The animals used were cats, and the methods used for
inducing traumatic shock and determining metabolism were similar —
in all respects to those previously described (3). The values for blood
oxygen were obtained by the methods of Van Slyke (4).

The blood was withdrawn in two ways: a, by inserting a needle in a
branch of the femoral artery and vein, and so entering the larger ves-
sels without causing any stasis; b, the more satisfactory way, by insert-
ing thin catheters down the right carotid artery and right superficial
jugular vein until they reached the heart. The blood was collected in
glass syringes, under paraffine oil, and put into tubes under oil (5). —

1 This is study no. X of a series on the physiology and pathology of the blood
from the Harvard Medical School and allied hospitals.

408

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK 409

By this method blood could easily be obtained without exposure to
air. In order not to disturb the blood volume in the experimental
animal, the amount removed for analysis was promptly replaced by an
equal amount of citrated cat’s blood. Thus fairly large samples of
arterial and venous blood could be obtained without permanently
affecting the blood pressure or blood volume.

XirssSgasw

WANS
SSG F

SSS

Fig. 1. Normal = Animal under urethane anesthesia.
Before shock = After muscle trauma, but before a true shock level of
blood pressure had been established.
Asphyxia = Clamping off trachea completely for 4 or 5 minutes.

After shock had been established for about 30 minutes, as shown by a
fall of blood pressure to 60-70 mm. Hg., the blood samples were taken
and were immediately followed by a large transfusion of blood, as much
as 100 cc. being given in an attempt to relieve the shock. In one case
this accomplished a permanent recovery of blood pressure to its orig-
inal level. In cases where a few minutes’ delay followed the taking
of the blood samples from the shocked animals, only slight recovery of
blood pressure followed the transfusion.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 2

410 JOSEPH C. AUB AND T. DONALD CUNNINGHAM

Discussion. Figure 1 shows graphically the more important changes
which occur in the oxygen of the blood in traumatic shock. The most
striking effect is seen in the oxygen content of the venous blood, which
falls very markedly in shock—not only in actual content of oxygen
but in percentage of saturation.2. This is well shown in all the experi-
ments in table 2, but possibly most notably in experiments LIII and
LV, and is also present in a control experiment LIV, table 1, where a
low pressure was obtained by mechanically interfering with the circu-
lation. The changes observed in profound shock are also present
as the shock is developing, and to lesser degree after the improvement
which follows large transfusions.

The oxygen capacity of the blood in shock has invariably become
less than in the normal sample. This fall in capacity does not agree
with the observations of Henderson (2), who found in four experiments
that the oxygen of the arterial blood rose 1.5 volume per cent after
shock. This he interpréted as demonstrating a concentration of the
blood. The fall here reported may, however, be explained by the
accumulation of red blood corpuscles in the capillaries, as observed by
Cannon, Fraser and Hooper (6), and therefore a relative reduction of
corpuscles in venous blood, and not necessarily a dilution of the plasma.

The percentage saturation of hemoglobin in arterial blood has not
varied markedly in the various conditions of the experiments. The
ventilation is at least adequate throughout, so that when the blood
leaves the heart and reaches the tissues it is as well saturated with
oxygen during shock as normally. The fall from the normal level of
oxygen takes place in the venous blood, which confirms Henderson’s
observations, and this occurs before shock as well as during shock.
In experiment LVI, while the blood pressure was, and had been, 104—94
mm. Hg. for 14 hours after the muscle injury, the oxygen content of
the venous blood had fallen from 12.27 to 4.55 volumes per cent. So
also in experiment LVII, although the blood pressure was 84 mm. Hg.
after the muscle injury, the oxygen content of the venous blood had
fallen from 13.68 to 6.64 volumes per cent.

_ A similar, though less marked, decrease in the oxygen content of
the venous blood is seen after recovery by transfusion as seen in experi-
ment LIII, table 2. That this is sufficient to indicate a true asphyxia
in the tissues cannot now be proved because the head of oxygen pres-
sure necessary for normal oxidation is not yet definitely known. How-

* The percentage of saturation as used in this paper, represents the oxygen
content of venous blood divided by the oxygen capacity of the arterial blood.

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK All

ever, the very small amount of oxygen present (with the attendant low
partial pressure) makes a true anoxemia possible. |
It is true, however, that the venous blood may be as low in oxygen
in severe anemia as it is in shock, but in this condition the oxygen of ©
the arterial blood is likewise reduced. Morawitz and Rohmer (7)
found that in three human cases of very severe anemia, where the
oxygen-carrying capacity of the blood was 4.5 per cent or less, the
venous blood had an oxygen content as low as 0.67 per cent, and they
assumed an increased blood flow to explain the normal metabolism
which is still found in these cases. Lundsgaard (8), also studying
patients with anemia, found the venous blood contained as low as
1.16 volume per cent of O» in a case with an oxygen-carrying capacity
of 5.93 per cent. He concludes that the tissues extract oxygen from
the blood with equal readiness whether there is a large oxygen reserve
in the blood, ‘‘or practically no reserve,-as in anemia.” These cases,
however, had very low oxygen capacities, and therefore the low O»
content of the venous blood meant a less complete dissociation’ of
hemoglobin and O, than would a similar figure in a normal blood.
It is the amount (percentage) of this dissociation which must influence
the partial pressure of the dissolved oxygen, and this latter is the
important factor in the migration of oxygen into the tissues. The
oxygen content of normal blood may, therefore, be three or four times
that of anemic under the same partial pressure of Os. As a result,
under similar conditions, one would expect to find a much larger figure
for the total venous content of oxygen in these shock experiments than
would be found in anemia, because anemic blood has less hemoglobin.
In fact, the percentage saturation of the venous blood in these anemia
cases, (16 per cent and 20 per cent), is about the same as im the cases of
severe shock, in spite of the lower venous content.
A more direct control of the value of the oxygen content found in
these experiments are the figures obtained in animals after 4 or 5 min-
utes of complete asphyxia, for here, just before death, the oxygen
value of the venous blood was not very much lower than that found
in shock. This is of course indirect evidence, for the matter of greatest
importance to the tissue is the oxygen content of the arterial blood.
Still, the oxygen content of the venous blood must give a satisfactory

8 This dissociation is approximately represented by the ‘‘per cent of satura-
tion’’ column in tables 1 and 2. It is approximate, because the small amount
of oxygen dissolved in the plasma would be about the same in anemic and nornial
bloods.

412 JOSEPH C. AUB AND T. DONALD CUNNINGHAM

indication of conditions in the venous end of the capillaries, and in the
asphyxia experiments must surely indicate an oxygen content which is
entirely inadequate for the use of the tissues.

The explanation of this marked anoxemia lies most probably in a
slowed blood flow which has been demonstrated in shock, and which
can be very well demonstrated in these experiments by the method
used by Means and Newburgh (9). In brief it is based on the formula:

cc. O2 absorbed through lungs

Valais outpa Sat Renreiper Daa Volume per cent oxygen utilization of blood
Using this formula we can calculate the blood flow of several of
our cases.. The results are shown in tables 1 and 2. It is clear
that the blood flow becomes markedly slowed before the onset
of a shock level of blood pressure, and that this slowing precedes
the fall in metabolism, demonstrated in paper I.4 This is demon-
strated in experiments LVI and LVII. It may therefore be assumed
that the fall in metabolism is a secondary manifestation of the decreased
blood flow, and of the markedly reduced oxygen content of the venous
blood, and is probably due to a true anoxemia. This suggestion is
further borne out by the similar findings following increased pericardial
tension (exper. LIV), for this must suddenly decrease the rapidity of
blood flow. There is a much increased oxygen utilization in the blood
and there is also a very prompt fall in the level of the basal metab-
olism (8), although the only disturbing factor is in the circulation, and
no toxic effect from tissue injury can be involved. Verzar (10) has also
shown that when perfused muscles are given inadequate oxygen sup-
ply the height of their metabolism falls.

Under these conditions we may add another factor to the vicious
cycles described by Cannon (11). Krogh (12) has shown that as an
oxygen want develops in contracting muscles, many empty capillaries
fill with blood, a change which reduces the distance necessary for the
diffusion of gases into the tissues. Thus, as anoxemia develops, the -
capillary bed would increase in volume. This would further decrease
the already slowed blood flow, and the slower the flow the greater
would probably become the oxygen consumption per. cubic centimeter -
of blood, and hence the decrease in oxygen of venous blood.

4 Experiment LIII does not show so striking a drop as the other two. It was
abnormal, however, in being the only observation which showed a rise in metab-
olism during shock, instead of a fall. This was probably due to the intense
dyspnea.

413

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK

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STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK 415

CONCLUSIONS

1. There is a markedly diminished oxygen content of the venous
blood in experimental traumatic shock. This change occurs before
the blood pressure falls to a shock level, and is still present after appar-
ent recovery from shock. |

2. The blood flow is also greatly decreased in the development of,
during and after shock.

3. The resulting anoxemia of the tissues may be the cause of the
decreased metabolism.

4. The sequence of these events in traumatic shock is discussed.

BIBLIOGRAPHY

(1) Gesetu: This Journal, 1918, xlvii, 468.
(2) Henperson: fbid., 1910, xxvii, 152.
(8) Aus: This Journal, 1920, liv, 388.
(4) Van Styxe: Journ. Biol. Chem., 1918, xxxiii, 127.
(5) Stapiz: Journ. Exper. Med., 1919, xxx, 215.
_ (6) CANNON, FRASER AND Rootes: Report of Shock Committee, English Medi-
cal Research Committee, 1917, no. 25, 76; Journ. Amer. Med. Assoc.,
1918, Ixx, 526.
(7) Morawitz AND R6umeER: Deutsch. Arch. f. klin. Med., 1908, xciv, 529.
(8) LunpseaarpD: Journ. Exper. Med., 1919, xxx, 147.
(9) Means AnD NEWBURGH: Trans. ages Amer. Phys., 1915, xxx, 51; Journ.
Pharm. Exper. Therap., 1915, vii, 449.
(10) Verzar: Journ. Physiol., 1912, xlv, 39.
(11) Cannon: Journ. Amer. Med. Assoc., 1918, Ixx, 616.
(12) Kroau: Journ. Physiol., 1919, lii, 457.

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK

III. CuemicaAL CHANGES IN THE BLOOD

JOSEPH C. AUB anp HSIEN WU
From the Laboratories of Physiology and Biochemistry in Harvard Medical School

Received for publication August 12, 1920

In experimental traumatic shock there is a marked decrease in the
rate of blood flow and of general metabolism. Decreased blood flow
and blood pressure result in a diminished secretion by the kidney (1).
It is of interest to know what are the effects of such acute abnormal
conditions upon the chemical constituents of blood, especially as a
chemical cause of shock is now being seriously considered.

Some work in this field has been done by French investigators.
Duval and Grigaut (2) studied the non-protein nitrogen of the blood
in shock and concluded from their results that in the war-wounded
there was an increase in the non-protein nitrogen of the blood, which
started promptly after the wounding, was at its height during the sec-
ond day and then gradually returned to normal. This increase was
slight in unshocked cases, whether the wounds were infected or not.
The retention differed from that found in nephritis in that the nitrogen
increase in blood occurred not markedly in the urea portion but in the
remainder of the non-protein nitrogen.

Whipple and his collaborators (3) studied the blood in the intoxica-
tion following intestinal obstruction, and after injection of the toxic
proteoses which developed in obstructed intestines or in closed intes-
tinal loops. The response to these injections was one which in many
ways resembled traumatic shock,—with a fall in temperature and

blood pressure. The injection was followed by a large increase (40

per cent or more) in the non-protein nitrogen of the blood, but this
increase was found chiefly in the blood urea nitrogen, although the
amino- and peptid-nitrogen also showed slight increases.

It is thus seen that the conclusions of the French and of the American
investigators are somewhat contradictory, though it may be conceded
that the response obtained by proteose injection is not a true shock.

416

PP ee Tm

ae ee ee a

ere Se ee a a Ee ee sas

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK 417

At any rate, neither of these investigators have found any chemical
change in blood other than rise in non-protein nitrogen which may be
regarded as characteristic of traumatic shock.

The method of analysis used in our experiments was that of Folin
and Wu (4). The urea was always determined by means of urease and
aeration, as hydrolysis with the autoclave would also decompose the

urethane used as an anesthetic. The figures obtained by the latter

method were 10 to 15 mgm. too high, and it may therefore be con-
cluded that the blood in our experiments contained about this amount
of urethane N per 100 cc. The urethane contributed but little to the
non-protein nitrogen as actually determined. It is so volatile that
most of it is expelled in the course of the digestion. Experiments with
a pure urethane solution containing 10 to 15 mgm. N per liter (.e., as
much urethane N as the blood filtrate might contain) have shown

that only 3 to 4 mgm. N were fixed by the acid digestion mixture. The
values of the total non-protein N obtained are, therefore, only 3 to 4

mgm. higher than the actual total non-protein nitrogen minus urethane

nitrogen. |
The creatin N represents as usual the difference between the total

creatinine and the preformed creatinine multiplied by 0.37. In the

first few experiments both the total creatinine and the preformed

creatinine were determined, but as the latter showed no appreciable
variation during anesthesia,—averaging 2 mgm. creatinine per 100 cc.
blood, in control as well as in shocked animals'—its separate deter-
mination was discontinued in later experiments in order to economize
the blood filtrate. Its average value was used for the calculation of
the creatin. _ :

In tables 1 and 2 are shown the results of our experiments. It is
clear that in the control experiments there was no marked rise in any
of the chemical constituents studied. This is true even in the experi-
ments where the blood pressure and the blood flow were reduced by
mechanical means but without muscle trauma (exper. LIV). In the
animals in shock, however, there was usually a marked increase of all
the constituents which we determined, over what was present. before

1—In a control experiment, 1 hour and 10 minutes after anesthesia the blood
contained 5.2 mgm. total creatinine and 2.1 preformed creatinine per 100 cc.
blood. Five hours later the total creatinine was again 5.2 mgm. and the pre-
formed creatinine was 1.8 mgm. In another experiment (2 hours after trauma-
tization) the blood contained 15 mgm. total creatinine and only 2.3 mgm.
preformed creatinine.

418

JOSEPH C. AUB AND HSIEN WU

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4.22 JOSEPH C. AUB AND HSIEN WU

trauma. This increase was not marked in traumatized animals which

did not go into shock,—and, in fact, the rise tended to run parallel _

with the severity of the shock.

The most significant result of the blood analysis is that in connection
with the creatin. In control animals the creatin content of blood
remained practically unchanged during the five hours of the experi-
ment. In animals which were traumatized but had not gone into
shock the blood creatin showed an unmistakable increase. When
- shock developed, the creatin figure rose frequently to three times the
normal. The source of the increased creatin in blood is the injured
muscle. This is not only in accord with the known facts but is also
shown in experiment XLI. The blood from the femoral vein con-
tained distinctly more creatin than did.the carotid blood. According
to the views of Folin and Denis (5), creatin does not exist as such in
the intact muscle and it is a post-mortem product set free by the dying
tissue. While these investigators based their view on indirect though
convincing data, the results of our experiments seem to afford direct
— evidence.

The parallelism between blood creatin and the severity of the shock
does not of course indicate that creatin itself is responsible for this
condition. Creatin is innocuous even in large doses. Simultaneously
with the liberation of creatin from the autolyzing muscle there is
probably formed a large number of other nitrogenous substances—
possibly histamine and the like, and to these substances may possibly
be ascribed the cause of shock. The increased blood creatin is merely
an index of the extent of the necrosis which the injured muscle has
undergone. Since creatin is derived solely from the injured muscle,
the results of our experiment seem to afford suggestive chemical evi-
dence in support of the view already prevalent that the shock is pro-
duced by some substance coming from the injured muscle (6), (7).

Although an increase in the total non-protein nitrogen always
attended the development of shock, the increase was slight except in
cases where the- shock was profound. ‘This relatively slight increase
of total nitrogen is an excellent check on the increase of creatin, for it
shows that, the accumulation is due to increased production and not —
simply to kidney inefficiency, which would probably cause a parallel
rise of all constituents. We are inclined not to attach much signifi-
cance to the variation of the total nitrogen (and the percentage of
urea N) on account of the complication by the urethane, but our
results agree rather well with those of Whipple and collaborators (8),

ee ee ee eee ee

STUDIES IN EXPERIMENTAL TRAUMATIC SHOCK 423

and it may be concluded that albumoses, as such, could not have played

any important réle in the development of shock.

Some work has been done on the blood sugar content in traumatic
shock. Cannon (8) reported that there was surely a normal amount
if not a slightly increased blood sugar in wound shock. Fabre, Wert-
heimer and Clogne (9), however, report a reduced blood sugar content
in shock. In our control experiments the blood sugar, while always
high on account of anesthesia, did not rise greatly in the second sample.
In the traumatized cases, however, there was nearly always definite
and. marked rise in the sugar content of the blood. Too much stress
may not be laid upon the extent of the rise, however, because of the
urethane. That this anesthetic may affect the carbohydrate metab-
olism was suggested by the work of Underhill (10), who showed that
adrenalin glycosuria was more readily obtained when urethane was the

‘ anesthetic than otherwise. The rise of sugar values which we have

found, however, is very striking and difficult to explain. Three possi-
bilities suggest themselves: a, It may be the hyperglycemia associated
with activity of the sympathetic nervous system; b, The reduced total
metabolism might be used to explain an accumulation in the blood,—
but the respiratory quotients in traumatic shock (11) suggest that at
least a normal proportion of carbohydrate is being metabolized; c,
finally one may look to the liver for an explanation. Several French

_ observers have ascribed shock phenomena to a liver insufficiency, and

one might assume that this rise in blood sugar is due to a loss from
the glycogen commonly stored there. This might be a direct result of
the toxic alterations which may occur in the liver (12).

CONCLUSIONS

1. Animals with marked muscle trauma but without true shock
showed only slight changes in total non-protein nitrogen, urea, creatin

and sugar in the blood. These constituents, especially the creatin and

the sugar, rose markedly as shock developed. In control animals the
determined constituents showed no appreciable change.

2. The marked rise in creatin is direct evidence of the presence in
the blood of products of muscle necrosis, and is therefore suggestive
evidence for the theory of the chemical cause of traumatic shock.

3. The cause of the rise in blood sugar is briefly discussed.

“at ete TORRE ae AND HSIEN : ee:
‘BIBLIOGRAPHY |
(1) CusHny: The secretion of urine, Lo don, 107, 1,

eee

(4) Foun AnD Wo: Piste Biol. Chem. , 1919, 3 xavili, al
(5) Foun anp Dents: Ibid., 1914, xvii, 493.
(6) CANNON AND Bayuiss: Rept. of Shook Committee, English
Committee, March, 1919, no. 26, 19. _ ;
(7) QueNnv: Revue de Chirur., 1918, lvi, 204. ne
(8) CaNnNoN: Rept. of Shock ae ca aE nah M

om bs 531. 8
(9) FaBRE, WERTHEIMER AND rane ae Bull. soe. shina ai
(10) UnpErurty: Journ. Biol. Chem., 1911, ix, 13. ae

(11) Aus: This Journal, 1920, liv, 388. ,
(12) RICHET AND FLAMENT: Compt. sic a. l’acad. r a,

ee a ee ee a a agen

THE AMERICAN

: J OURNAL OF PHYSIOLOGY

‘VOL. 54 JANUARY 1, 1921 | No. 3

EXPERIMENTAL STUDIES IN DIABETES

Series Il. Tue INTERNAL PANCREATIC FUNCTION IN RELATION TO
Bopy Mass AND METABOLISM

7. The Influence of Cold

FREDERICK M. ALLEN
From the Hospital of The Rockefeller Institute for Medical Research, New York

Received for publication August 10, 1920

According to some earlier literature (1), cold causes hyperglycemia
and glycosuria in both warm- and cold-blooded animals, and increases
the glycosuria of diabetic dogs. The use of cold and shivering to drive

out glycogen from phlorizinized animals was familiarized by Lusk.

A single mild test of cold environment in a human patient gave a
negative result (2).

A few words may be devoted to the theory of the subject. In a
totally phlorizinized dog, it is obvious that a release of carbohydrate
which cannot be utilized will result in a temporary rise of glycosuria
and of the D:N ratio. <A similar mobilization of stored carbohydrate,
together with a possible diuretic action of cold, might produce a similar
result in a totally depancreatized animal. The extra heat required to
maintain body temperature would supposedly be furnished by fat,
and there is no reason to expect any increased output of sugar derived
from protein, either in the sense of any appreciable increase of protein
decomposition or any genuine alteration of the D:N ratio. Ina
partially depancreatized animal, the increased carbohydrate mobili-
zation or increased total metabolism may impose an additional burden
upon the pancreas remnant, and any lasting increase of glycosuria:
must be interpreted in the sense of a true aggravation of the diabetes.
At the same time complicating factors come into play. If the animal

425

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 3

426 FREDERICK M. ALLEN

fasts, the extra sugar loss and fat combustion impose a sharper under- .
nutrition treatment in a cold than in a warm environment. Likewise
a diet which is adequate in warmth becomes under-nutrition in the
cold. On the other hand an increase of diet to meet the increased
requirement complicates the problem still further. Shivering is a form
of muscular exercise, and may possibly have a metabolic effect opposed
to the effect of cold per se. ,

Normal dogs can live without artificial heat in the indies winter
weather of New York if merely sheltered, but their metabolism evi-
dently is considerably higher than that of dogs kept in warm rooms.
‘The first experiments were therefore planned to determine whether
there is any practical difference in the ease of producing diabetes in
dogs kept in outdoor cages in winter and in others kept in a room
specially warmed to a high summer temperature. This method was_
considered better for the purpose than the plan of using severely diabetic
dogs and following the variations in their glycosuria, because it was
important to distinguish a tendency to the production of mere glycosuria
and hyperglycemia (such as cold may ‘excite even in normal animals, —
which are certainly not diabetic from this cause) froma tendency to
the production of actual diabetes. If cold has any diabetogenic action,
equivalent to the removal of a small fraction of a gram of aa ica
tissue, this action should be demonstrable in such tests. °

Tce hve dogs: were used for this investigation, with removal of
such portions of pancreas as were known to produce a close approach
to diabetes.! These experiments included fasting and fixed diets, also
single and repeated operations, the latter as usual removing successive —
bits of tissue till diabetes resulted. Comparisons were made between
dogs kept in the warmth and others kept in the cold, and also in the
same animals by sudden changes from one environment to the other.
The animals chosen ranged from small short-haired dogs which were
highly sensitive to the cold and might sometimes be unduly depressed —
by it, to large woolly dogs which scarcely shivered in the winter weather.
The general technic of such experiments is sufficiently clear from the
preceding papers, so that brevity may be served by omitting protocols.
- In two instances the change from warmth to cold seemed responsible
for a definite but transitory glycosuria. Otherwise the results were
negative, and the conclusion was established positively that there is
no demonstrable difference in the amount of pancreatic tissue that

1 All operations were performed under ether anesthesia.

ee ee ee eee ee

EXPERIMENTAL STUDIES IN DIABETES 427

must be removed to produce diabetes in dogs in warm or cold environ-
ment. An effect of cold upon the islands of Langerhans was also not
observable.

Although cold has no diabetogenic influence whatever in the sense
of this test, it thus merely conforms to the rule that the most powerful
functional influences avail little in comparison with the smallest fraction
of a gram of healthy pancreas tissue. As agencies which are negative .
in this respect sometimes appreciably influence the course of an existing
diabetes, some experiments concerning hyperglycemia and glycosuria
were performed upon dogs with various grades of carbohydrate tolerance.
For this purpose the animals’ cages were transferred from a comfortably
warm room to a refrigerator room kept at approximately freezing
temperature. The observations are arranged in a series chiefly accord-
ing to descending assimilative capacity.

Dog C3-36
TIME hss opie REMARKS
per cent
August 6
10:00 a.m........ 0.128 | At summer temperature
o-4U0 P.M... ..:;, 0.116 | Immediately after this bleeding, transferred to
ice room —
moo P.M... oa... 0.098
August 7
10:00 a.m..... ...| 0.118
20 DMs. 0.100 | Immediately after this bleeding, transferred
from ice room to summer temperature
Bee DMs eb 0.095
Piao Pit. ee 8: 465 |

The normal dog C3-36, weighing 17 kilos, was placed in the ice room after the
bleeding at 3:40 p.m. on August 6, and left there until after the bleeding at 4 p.m.
on August 7., The usual diet of bread and soup was fed each evening after the

final blood sample was taken. The dog was in excellent condition and powerfully

muscled, but very short haired, and shivered continuously in the cold. The
plasma sugar seemed to be affected very slightly if at ap At any rate, no eleva-
tion by cold was observed.

Dog B2-00, mentioned several times in previous papers, in August,
1915 was close to the verge of diabetes, but had been free from glyco-
suria on fixed bread diet for several months. Exercise had proved able
to modify the blood sugar and the glucose assimilation considerably,

428 FREDERICK M. ALLEN

and an experiment with cold was therefore performed under similar
conditions. The diet was fed each evening after the last blood test.
There was no glycosuria.
low temperature.

4 a ie
ae ee se ee ee ee ee

The plasma sugar was slightly lower at the

TIME ee REMARKS
per cent
August 5
ALSO BaRietas ies 0.111
Oe Dies a Gd 0.117 | At summer temperature
August 6 ; :
C550: BAe es 0.110
3330 Pal ak 0.098 | Immediately after this hsb: transferred to
. ice room
SAO ORs ia os 0.085
August 7
0:30 G58, Ss. 0.098
Big0 Dials. s sc. A 0.107 | Immediately after this bleeding, transferred
from ice room to summer temperature
S00: 0 ra oss 0.125
OSE DIN. . oc e 0.112
Dog B2-01
PLASMA SUGAR
« TIME
ea September 29, | October 6, Exercise
per cant per cent per cent
0.116 0.106 0.105 Blood before feeding
0.192 0.256 0.147 1 hour after feeding
0.106 0.147 0.148 3 hours after feeding ©
0.131 0.103 6 hours after feeding

Dog B2-01, likewise near the verge of diabetes, received 56 grams Merck anhy-

drous glucose (4 grams per kilo) in 30 per cent solution by stomach tube on three
days, respectively in the cold, in warmth at rest, and with treadmill running.
The control day showed: the highest plasma sugar curve, while both cold and
exercise seemed to depress it.

EXPERIMENTAL STUDIES IN DIABETES

429

Dog B2-02

PLASMA SUGAR URINE SUGAR
July 20, | Septem- | October rewonn i eee
Contr ol eri Exercise ulegaevealbe 28, Cold"
per cent per cent per cent per cent per cent
0.144 | 0.118 | 0.114 0 0 Before feeding
0.270 | 0.228 | 0.111 | 4.50 (13 cc.) | Slight | 1 hour after feeding
0.149 0.115 | 0.100 | 1.85 (84 cc.) 0 3 hours after feeding
0.112 | 0.100 0 0 6 hours after feeding

Dog B2-02 was known to have very mild latent diabetes, but was continuously
free from glycosuria on bread diet. Tests similar to those of dog B2-01 were
carried out with the giving of 30.5 grams Merck glucose (8 grams per kilo) by
stomach tube. Cold seemed to reduce hyperglycemia and glycosuria as compared
- with the control day. On the exercise day there was by far the lowest plasma
sugar and no glycosuria.

Dog D4-29
PLASMA SUGAR
TIME
October 4, Control October 9, Cold October 10, Control
per cent per cent per cent
0.093 0.120 0.147 Blood. before feeding
0.307 0.357 0.256 © 1 hour after feeding
0.307 0.455 0.202 2 hours after feeding
0.322 0.400 0.235 3 hours after feeding
0.307 0.416 0.222 4 hours after feeding
0.313 0.364 0.250 5 hours after feeding
0.322 0.206 8 hours after feeding
0.125 0.200 13 hours after feeding

Dog D4-29. Female; mongrel; age 2 years; good condition; weight 11.5 kilos.
September 28, 1916, removal of pancreatic tissue weighing 26.9 grams. Remnant
about main duct estimated at 3.8 grams (+). The removal of 0.65 gram additional
tissue on October 16 was necessary to produce diabetes. In the interval the tests
shown in the table were performed, with the feeding of 200 grams bread and 150
grams glucose on each occasion (together with 200 grams talcum powder as a pre-
caution against diarrhea). On October 9 the dog was transferred to the cold
room immediately after feeding. The plasma sugar curve ran noticeably higher
on this day than on the control days, but yet was lower at the end of 13 hours, as
if more of the available carbohydrate had either been excreted or consumed by
shivering. An accident prevented comparison of the glycosuria.

Dog B2-86. The history of this animal was given in paper 2 of the preceding
series (3). The animal had been on the verge of diabetes with an unusually large
pancreas remnant, and enormous quantities of bread and glucose had been neces-
sary to keep up glycosuria, but by July 21 this was disappearing and the appe-
tite was failing. Therefore the expedient was adopted of raising the animal’s

430 FREDERICK M. ALLEN

metabolism to the highest level possible, by exercising him on the treadmill to
the limit of his strength daily, and keeping him in the ice room all the rest of the
time, in the endeavor to bring on diabetes either by stimulation of appetite or by
any direct influence upon the pancreas. July 22 was occupied in this manner.
July 23, exercise was deferred until a feeding experiment with 400 grams bread
and 500 grams glucose could be carried out in the ice room in comparison with the
- one at summer temperature on July 21.

PLASMA SUGAR
TIME
July 21, at summer|July 23, at freezing

temperature temperature
per cent. per cent ~

Beldra Tesditian iss <<. css vk wrk ee Bb civ ew hia 0.124 ~ 0.100

2: Hhoure- QICOh eos a ace ecs «CLES oe cee 0.147 0.128
ZT noure afleR bs eee Nes ee ts see ae 0.141 : O.14b-.

The lower sugar curve on July 23 may have been partly the result of the pre-.
ceding day’s exercise, but at least augured failure for the undertaking. The pro-
gram of combined exercise and cold was continued daily, with addition of as much
as 600 grams of glucose to the bread diet, up to the time of the second operation
on August 7. Thestrong animal merely thrived on the program, and such diabetic
tendency as had seemed to be present disappeared.

Dog B2-71. June 3, 1914, at a normal weight of 14.7 kilos, nine-tenths of the
pancreas were removed. In July, 1915 the dog was still in a state of moderate dia-
betes, kept sugar-free on meat diet, at a weight of 12.5 kilos, and was used for exer-
cise and other experiments. Some tests were then performed with feeding 50
grams of bread, following only the urine without blood analyses. Theregular
lung diet was given each evening after completion of the test.

URINE
TIME : REMARKS
Volume Glucose
ce. per cent
July 28
2330. pam i io. os 0 Fed 50 grams bread
5:00 pam it. os 38 0.28
July, 29
S200 Dike. ew, 0 Fed 50 grams bread and placed in ice
room .
+O PR MN Es 47 0 Returned to summer temperature
July 30 . .
10:00 a.m........ 0 Fed 50 grams bread and placed in ice
room ie
5:00 p.m. ...... 214 Trace | Returned to summer temperature
July 31
10:00 amiuciks 0 Fed 50 grams bread
6:00: pws Vi 275 0.4

ej

~

¢

EXPERIMENTAL STUDIES IN DIABETES 431

In this experiment there was distinctly greater glycosuria on July 28 and 31
in warm summer weather than on July 29 and 30 at freezing temperature.

Dog C3-00. Female; fox terrier mongrel; white and brown; age 5 years; good
condition; weight 4.5 kilos. May 6, 1915, removal of pancreatic tissue weighing
11 grams. Remnant about main duct estimated at 1.25 grams (;5). As some-
times happens in small dogs, glycosuria was absent on full meat diet even with
this small pancreas remnant. On May 15 a change to bread and soup promptly
brought heavy glycosuria, which ceased with a return to meat diet on May 21.
A fixed diet of 750 grams lung was then given daily, but was not always eaten com-
pletely. After continuous absence of glycosuria, the dog was transferred to the
ice room on May 29, and in the following 24 hours excreted 0.45 per cent sugar in
375 cc. urine. Glycosuria then continued absent on the same diet as before, until
on June 7 the dog was removed from the ice room. At summer temperature a
return to bread and soup diet produced immediate heavy glycosuria. Accord-
ingly, in this experiment cold failed to maintain glycosuria on meat diet in a dog
which was demonstrably diabetic as proved by glycosuria on bread diet.

In August the same dog was tested with the aid of plasma sugar analyses on a
regular diet of 500 grams lung and 50 grams suet. There was no glycosuria.

bye rears BEMABES
per cent
August 10
Res ee AI ea 0.135 | Immediately after this bleeding, transferred to
ice room ‘ -
12:00 noon....... 0.122
2-4) P.M.,...-%; 0.141
August 15
Tate0 PAM... ose: 0.182
August 16
WET A eee 0.098
4:45 p.m........ 0.112 | Immediately after this bleeding, transferred
from ice room to summer temperature
August 18
12:00 noon....... 0.167 | Summer temperature
Seed Pls. swabs 0.143
Dog B2-88
PLASMA SUGAR
November Neve ati na oveet November eatngen
- Control | Control Cold Cold Exercise
per cent per cent per cent per cent per cent
0.128 0.189 0.131 0.122 0.162 | Blood before feeding
0.250 0.222 0.263 | 3 hour after feeding
0.257 | 0 233 0.189 | 2 hours after feeding
0.286 0.271 4 hours after feeding

432 FREDERICK M. ALLEN

Dog B2-88, with mild diabetes, received test meals of 200 grams bread and 100
grams beef lung. The plasma sugar curve seemed to vary chiefly according to the
initial figure, without much influence of any of the special measures employed.
Thus, both figures on November 15 are a trifle higher than on November 23 in the
refrigerator. Also the differences between November 18, 20 and 26 seemed to be
governed chiefly by the level on which the plasma sugar started. Cold at least
did not elevate the plasma sugar.

Dog B2-58. Female; mongrel; yellow; age 3 years; good condition; weight 11.8
kilos. Fasting was begun May 4, 1914, and by May 21 the weight was reduced to
9.1 kilos. It was thus possible to remove +4 of the pancreas on this date with
only a moderate degree of diabetes resulting. After various tests, the tolerance
was spared by a low protein diet, which could be gradually increased by October
20 to 1 kilo of beef lung daily, without glycosuria. The weight gradually rose by
December 24 to 12.3 kilos, on which date the first glycosuria appeared. About —
the same time the dog began to leave part of the diet uneaten, and the weight thus
fluctuated and in general fell. The partial checking of glycosuria, and its daily
variations, are thus accounted for. Beginning January 6, 1915, the dog’s cage was
kept outdoors, in order to test the effect of cold upon the diabetes either directly, ©
through stimulation of appetite or in any other way. The dog had time to be-
come acclimated because the weather was fairly mild at first, but after January 20
it turned decidedly colder, and there were several days with considerable snow
and ice. As the dog was short-haired, she was shivering practically continuously
while outdoors, but passed through even the coldest weather without actual
impairment of health. The rectal temperature remained normal. Contrary to
expectation, the amount of food eaten was not appreciably different in the cold
environment. After February 12 the glycosuria was checked by fasting. The
experiment seems to indicate a slight increase of glycosuria by cold, but the influ-
ence certainly was not great. :

Dog B2-58
URINE
DATE WEIGHT
Volume Glucose
kgm. ce. per cent
December 24 320 1.0
PO v's. -* 585 "6.5
26 12.8 470 Faint
27 385 0.2
28 950, Faint
29 355 0
30 12.7 384 Q%
January 1 360 0
2 500 0
3 632 0
4 650 Faint

See ee ae ee ee

EXPERIMENTAL STUDIES IN DIABETES

Dog B2-58—Concluded

433

URINE
DATE © WEIGHT :
Volume Glucose
kgm. - ce. per cent
January 5 480 0
6 12.7 330 0
7 12.4 502 0
Today placed| in cage outdoors
8 431 0
9 307 ree
10 mys) 0
11 11.1 713, 0
12 766 0
13 11.2 646 0
14 954 0.
15 10.9 698 0
16 382 0
17 Not} collected
18 : 1849 0
19 988 0.6
20 11.3 995 - 0
21 965 0.7
22 11.1 781 0.9
23 11.2 609 © 0.5
24 740 0.9
25 10.9 700 Faint
26 0 I 749 0
27 11.0 900 V. Faint
28 11.3 731 1.5
29 21.6 630 1.0
30 1}..5 156 2.0.
31 820 bis ek
February 1 11.3 1036 1.0
2 11.3 1300 0.8
3 11.0 950 2.0
As 11.1 785 1.8
5 11.4 1157 2.0
6 11.8 605 2.5
Today moved into} animal room
7 : 650 1.0
8 136 973 0.9
9 11.5 649 0.4
10 41,7 733 0.7
11 11.5 815 Faint
12 600 Faint

434 FREDERICK M. ALLEN

Dog B2-89. This female mongrel, weighing 13 kilos, underwent partial pan-
createctomy on April 12, 1915, leaving a remnant of 7;. Under regulated diets,
the condition by the end of June was such that glycosuria was absent at a weight
of 10.3. kilos on a diet of 1 kilo of lung, but substitution of 250 grams lung by 50
grams bread (making 750 grams lung and 50 grams bread) caused glycosuria of
0.75 per cent in 462 cc. urine on June 30 and 0.71 per cent in 542 cc. urine on
July 1. On the latter date the diet of 1 kilo of lung without bread was resumed,
and glycosuria immediately ceased (4). On July 2 the dog was transferred to
the ice room, in order to test whether the influence of cold would amount-to as
much as the above difference between carbohydrate and protein. The dog was
left in the ice room until July 26. Traces of glycosuria were present on most days
during this time, but only twice reached titratable amounts (0.33 per cent on
July 14, 0.25 per cent on July 21). A single day of exercise on July 22 abolished
the glycosuria, which returned on July 24. It was present also on July 25 and 26,
and then was continuously absent during a control period up to August 5 at sum-
mer temperature. As the weight fell to 9.7 kilos during the period in the refrigera-
tor, the experiment seems to indicate a slight increase of diabetic tendency due to
cold. The difference due to temperature, however, was evidently less than the
difference between the preformed carbohydrate of 50 grams of bread and its
approximate equivalent of potential carbohydrate in protein. The tolerance had
fallen somewhat, for the giving of 50 grams of bread on August 15 resulted in a
glycosuria of 1.4 per cent.

Glycosuria remained absent on the lung diet to September 17. On that day

at 9:30 a.m. the plasma sugar was 0.148 per cent, the rectal temperature 101.4° F.
The dog was then placed in the ice room fasting. At 4 p.m. the plasma sugar
was 0.151 per cent, the rectal temperature 100.7°F. The usual lung diet was then
fed and the dog left in the ice room. The next morning there was a glycosuria »
of 0.42 per cent in 567 cc. urine, and the plasma sugar at 9:30 a.m. was 0.164 per
cent. The urine of the next 24 hours was 492 cc., with 1.9 per cent sugar. At
9:30. a.m. on September 19 the rectal temperature was 101.5°F. Glycosuria then
ceased abruptly, as though the reserve of extra carbohydrate had been exhausted.
At 9:30 a.m. on September 20 the plasma sugar was 0.146 per cent. The dog was |
then transferred to the warm animal room, and at 9:30 a.m. on September 21 the
plasma sugar was 0.133 per cent, the rectal temperature 101.7°F.
- During the following days the weather turned colder. The night of September
23-24 was particularly sharp, and the door of the animal room blew open, so that
the room was cold and the dogs all shivering. This dog was one of five potentially
diabetic animals (out of about twenty) which had been kept sugar-free on regu-
lated diets and which showed sudden glycosuria on this night.

Subsequently, at a body weight of 15 kilos and correspondingly reduced tol-
erance, a comparison was made in this dog of the feeding of 1 kilo of meat in a
warm room and in the ice room.

@

EXPERIMENTAL STUDIES IN DIABETES 435

PLASMA SUGAR
TIME
November 26, December 3,
(warm room) (ice room)
per cent per cent
ER PEG TE i a a 0.200 0.208
pmnGECOY TOCGING. |... ce cl i. ec e le 0.218 0.278
mimeewmrcer feeding. 3. i. 5. ye es ek 0.208 0.313
<myeosuria for the period......4.......3....... 0 4.08 grams

The later observations showed a decided influence of cold for the production

of hyperglycemia and glycosuria.

as the diabetes became more severe.

It is possible that this effect became greater

Dog B2-79
PLASMA SUGAR URINE SUGAR ®
TIME
November 23, | November 30, | November 23, | November 30, ;
Warm Cold Warm Cold
per cent per cent per cent per cent
0.133 0.250 0 Faint Blood before feeding
0.213 0.416 Faint 5.00 1 hour after feeding
0.159 0.525 Faint 5.90 4 hours after feeding
0.189 0.435 0 3.53 6 hours after feeding

Dog B2-79 was an animal which had long been kept in a stage of diabetes such
that there was marked hyperglycemia and occasional glycosuria on a diet of 1
kilo of beef lung, the condition being held in check by fasting when necessary.
The above record shows a strong contrast in hyperglycemia and glycosuria on
days spent in a warm room and in the ice room respectively. The rise of blood
sugar after eating the usual kilo of lung was somewhat similar on the two days,
but the entire curve was on a much higher level in the cold environment. It was
not established that this difference was due entirely to the temperature, because

no comparison was made of the blood sugar before and after moving into the

refrigerator on November 30. Therefore on December 2 the plasma sugar was
determined fasting at 11:40 a.m. and found to be 0.164 per cent. The dog was
then moved into the cold room, and at 5:40 p.m. the plasma sugar (still fasting)
was found to be 0.185 per cent.
Likewise dog C3-19, perehing 13.8 pase was partially depancreatized on fue
23, 1915, leaving a remnant of ;'; to js. In August at a weight of 11 kilos, the
degree of diabetes was such that a diet of 500 grams lung was slightly in excess of
the tolerance. On August 7 the fasting plasma sugar at 10:30 a.m. was 0.156
per cent and at 4:30 p.m. 0.120 per cent. The dog, still fasting, was then moved
to the ice room, where the plasma sugar was found to be 0.167 per cent at 7 p.m.
and 0.133 per cent at 9:45 p.m. Here a falling blood sugar due to fasting was
evidently raised by cold, and then continued to fall slightly.

27) alae FREDERICK M. ALLEN

DISCUSSION

The elevation of blood sugar by cold is so generally accepted as a

truism that it was 4 surprise to encounter instances in which the sugar
was little changed or actually lower in a cold environment. It was not
feasible to extend the investigation further into the physiological
reaction to cold. Supposedly the blood sugar is governed by two
factors, namely the mobilization and the disposal of carbohydrate.
It is conceivable that in a perfectly smoothly working reaction the two
may exactly balance. Under some conditions shivering may perhaps
reduce the blood sugar like other forms of muscular activity. Under
other’ conditions hyperglycemia may occur, particularly when the cold
stimulus is sufficiently violent, as for example in the case of plunging
into ice water. Here the stimulation is so powerful, sometimes to a
pathological degree, that a correspondingly excessive sugar discharge
may be expected, and in the most extreme cases possibly the utilization
of sugar suffers somewhat. Hyperglycemia and glycosuria may be
more readily produced when the utilization of sugar is specifically
impaired, as in the more severe grades of diabetes. In a similar way
exercise sometimes raises the blood sugar instead of lowering it. |

Violent or pathological stimulation by cold was not applicable in
experiments designed to produce diabetes, because the animals’ health
would suffer and ill health would be the surest way to spoil the result
and prevent diabetes. A more powerful temporary discharge of sugar
might occur, but it would cease as soon as the immediate store was
exhausted. Such discharge and cessation was actually seen in certain
of these experiments, without any lasting diabetogenic effect. As
usual, clear thinking requires a distinction between diabetes, which is
deficiency of the pancreatic function, and mere glycosuria. ‘The mere
excessive discharge of sugar from the glycogen depots is not diabetes,
for the power of utilization may remain unimpaired. This has been
abundantly proved, for example, in such a condition as epinephrin
glycosuria (5). It is also not diabetes if the utilization of sugar is
depressed by any extraneous mechanism, such as the chilling of the
muscles or their nervous supply, but only if the impairment of utili-
zation is due to impairment of the pancreatic function. If the glyco-

suria produced by cold is regarded as a diabetes, cold must be a very —

powerful diabetogenic agent to cause even a temporary diabetes in a
normal animal, for it must thus temporarily paralyze about nine-tenths
of the function of the dog’s pancreas. ‘Trial of this agent in animals

' EXPERIMENTAL STUDIES IN DIABETES 437

depancreatized almost to the point of diabetes proves that it possesses
- no such power; and as the effect in these animals is not greatly different
from that in normal animals, cold evidently does not act by direct
depression of the pancreatic function. Its indirect influence upon
diabetes through increasing metabolism is a much more delicate point
to demonstrate, and the question is answered only somewhat doubtfully
in the affirmative by some of the above experiments.

CONCLUSIONS

1. Cold environment, such as did not lower the rectal temperature
to any important extent, in some instances failed to affect the plasma
sugar of dogs or slightly lowered it, but in the majority of experiments
_ produced hyperglycemia and-sometimes glycosuria. These were pro-
duced more easily and in higher degree in proportion as the power of
sugar utilization was impaired, i.e., as the diabetes was more severe.

2. The power to produce glycosuria is to be distinguished from the
power to produce diabetes. There is no demonstrable difference in
the proportion of pancreatic tissue that must be removed to produce
diabetes in dogs in warm or cold environment, and it was proved by
successive operations upon the same animals that the influence of cold
is not equivalent to the removal of the smallest fraction of a gram of
pancreatic tissue. In animals already diabetic, the course of the
diabetes in a few instances seemed to be influenced slightly for the worse,
so as perhaps to warrant the conclusion that cold imposes an increased
burden upon the pancreatic function by increasing metabolism. But
the slightness of this influence is emphasized by control experiments;
for example, it amounts to less than the difference between the pre-
formed carbohydrate of 50 grams of bread and the approximate equiva-
lent of potential carbohydrate in protein.

3. The impression that diabetic patients do worse in cold weather
is probably explainable by the discomfort of chilliness when they are
undernourished, the tendency to take more food, and sometimes by
the limitation of exercise. These may be important sometimes from
a practical standpoint, but any direct influence of climate upon diabetes
- must be very slight if it exists.

(2) iviae STILLMAN AND Pere: Roel
(3) Auten: Journ. Exper. Med., 1920,
(4) For the greater glycosuric effect of ‘pref

_ protein in mild diabetes. sia some

30740 B89. <a
(5) Lusk: Proce. Soc.. Exper. Biol.
of this series.

EXPERIMENTAL STUDIES IN DIABETES

Series II. Toe INTERNAL PANCREATIC FUNCTION IN RELATION TO
Bopy Mass AND METABOLISM ;

8. The Influence of Extremes of Age upon the Production of Diabetes

FREDERICK M. ALLEN
From the Hospital of The Rockefeller Institute for Medical Research, New York

Received for publication August 10, 1920

An influence upon the production of diabetes may conceivably be
expected from the conditions of extreme age or youth.

Senility. The relation of senility to experimental diabetes may have
interest from at least three standpoints: a, The total metabolism is
known to be slightly lowered in old age; b, diabetes in elderly patients
is generally characterized by a mild and prolonged course; c, the increas-
ing incidence of diabetes with the advance of age suggests the possi-
bility of functional or organic impairment of the pancreas.

Toothless decrepit senility is familiar in dogs, and there is reason
to expect as great metabolic changes as in aged human beings. Obesity
is also common in such animals, so that a number of them had to be
included in a previous paper (1). A comparison of the relation of
pancreas weight to body weight was made in fourteen dogs, which
showed extreme senility together with an average nutritive state.

From a comparison of table 1 with a similar study of normal adult
dogs (2), it may be inferred that there is no gross change, in particular
no atrophy of the pancreas accompanying senility.

The susceptibility of obese senile dogs to diabetes could seldom be
determined, owing to the sudden death to which they are subject
following pancreas operations, as previously mentioned (8). Such
operations were performed without accidents in senile dogs without
obesity, and these were used for diabetic experiments in the same way.
as younger dogs. No experiments were performed to determine the
precise proportion of pancreas that must be removed to produce diabetes,
but the incidental observations were sufficient to exclude any marked
differences from average adult dogs. The senile animals were some-

439

440 FREDERICK M. ALLEN

what weaker and more subject to loss of appetite and cachexia. Other-
wise their diabetes ran a course indistinguishable from that of younger
animals. ‘The experimental answers may therefore be stated in the
following form, corresponding to the questions raised above.

TABLE 1

Relation of pancreas weight to body weight in senile dogs

NUMBER BODY WEIGHT PANCREAS WEIGHT cee Out wa

kgm. grams grams
1 4.0 15.0 3.75
Z 4.4 7.0 1.59
3 5.8 9.5 1.64
4 8.8 23.1 2.62

5 11.3 19.6 1.73 ©
6 11.2 24.4 2.16
7 11.5 25.6 2.23
8 11.7 32.1 2.74
9 14.2 53.6 3.78

10 15.0 33.6 2.24
11 16.0 20.2 1.26
12 21.3 46 .4 2.18
13 21.8 : 39:6 1.81
14 ys Oy Rs ' 41.4 1.65

a. The lowered metabolism characteristic of senility does not render
dogs less susceptible to diabetes from pancreatic resection. As the
reduction of metabolism with age is slight, and the method of judging
susceptibility by the size of the pancreas remnant with which diabetes
occurs is a crude one according to former observations, too much stress
need not be laid upon these negative findings.

6. A more decisive observation is that diabetes does not run any
slower course in senile animals, but follows the same rapid progress
which is generally characteristic a diabetes in dogs. |

c. If there were any anatomic or functional deterioration of the
pancreas with age, a remnant of a given size might be expected to be
less efficient in preventing diabetes than in younger animals. An
‘increased susceptibility to. diabetes in this sense is excluded by the
observations. ‘The microscopic study, as reported in subsequent papers,
showed no visible abnormalities resulting from simple senility. Pan-
creatitis is much rarer in dogs than in human beings, and is apparently —
due to causes independent of age. Also there is no such arteriosclerosis

EXPERIMENTAL STUDIES IN DIABETES 441

in senile dogs as in man, and it can by no means be said that a dog
‘fs as old as his arteries.’ Also, as previously mentioned (2), the
pancreas remnant of a senile dog may possess as great power of hyper-
trophy as that of any younger animal.

Numerous glucose tolerance tests have been performed indiscrimi-
nately upon old and young adult dogs, and no indications of alteration
of assimilation with age have been found.

In general these observations, made upon a species practically free
from the pancreatic changes to which man is subject with advancing
years, indicate that the rising incidence and special characteristics of -
diabetes in older persons are due to the changes in question and not
to senility per se. 3
- Youth. The outstanding feature of experimental interest is the
characteristically rapid and fatal course of diabetes in children. This
has often been attributed hypothetically to their high metabolism, -
which imposes a heavier burden upon the pancreatic function. There
is also good evidence that this rapid downward progress indicates a
susceptibility of the islands of Langerhans to rapid destruction by
hydropic degeneration. As there are occasional cases of acute and
severe diabetes in the aged and of mild and prolonged diabetes in
children, there is no absolute distinction on the basis of either the level
of metabolism or the island changes.

Diabetes is also less frequent in children than in older persons. In
seeking possible reasons, it might be imagined that the youthful pan-
creas is larger in proportion to the body, that it is anatomically richer |
in islands or that these have stronger functional power, or that the
capacity for regeneration after injuries is greater. According to work
previously reviewed (4), especially that of Bensley, the pancreas at
birth probably contains as many islands as the adult organ, but during
early life there is probably a loss followed by a gradual new formation
of islands. Observations on the gross relations of the pancreas in
‘puppies are contained in tables 2 to 6. They indicate, in comparison
with those on adult dogs (2), that the ratio of pancreas weight to body
weight in puppies is not large but rather small in proportion to what
might be expected from the small size of the animals; that the tendency
to regeneration is often marked but yet not in excess of that often
found in adults; and that the tendency to diabetes is at least no greater
and often is distinctly less than in adult dogs. 7

In qualification of this statement, it should be noticed that the
observations do not exclude possible alterations of the ratio of pancreas

442 FREDERICK M. ALLEN
; TABLE 2
Litter of black and tan mongrel pups. Weight of mother, 17 kilos
PANCREAS
NUMBER gEBx AGE BODY WEIGHT | AeA WEIGHT FER
kgm. gram grams
1 Male Newborn 0.3 0.6 2.0
2 Male Newborn 0.37 0.6 2 Oe
3 Female ._| Newborn 0.2 0.4 2.0
4 Female Newborn 0.3 0.5 1.67
5 Male Newborn 0.3 0.6 2.0
6 Female Newborn - 0.3 0.7 2.33
7 Male ‘ Newborn 0.4 0.8 2.0
8 Female Newborn | 0.3 0.9 3.0
9 Male Newborn 0.3 0.6 2.0
10 Male Newborn 0.3 0.8 2.66
11 Female Newborn 0.3 0.6 2.0
TABLE 3
Litter of harrier mongrel pups. Weight of mother.13 kilos
PANCREAS
NUMBER — SEX AGE BODY WEIGHT pf sath ranae belie bye ‘
kgm. gram grams
1 Female 1 day 0.2 0.3 1.5
2 Male 1 day 0.2 0.3 1.5
3 Male 1 day 0.3 0.6 2.0
4 Male 1 day 0.3 0.4 1.33
5 Male 1 day 0.3 0.7 2.33 .
6 Male 1 day 0.2 0.7 3.50
TABLE 4
Litter of spaniel mongrel pups. Weight of mother, 16 kilos”
NUMBER AGE BODY WEIGHT eee: , WRIGHT PER
kgm grams grams
1 Slightly premature 0.2 0.7 3.5
2 Slightly premature 0.2 0.6 3.0
3 ‘Slightly premature 0.3 0.8 2.67
4 Slightly premature — 0.3 0.6 2.0°
5 2 days 0.3 0.9 3.0
6 3 weeks 0.6 2.1 3.5

EXPERIMENTAL STUDIES IN DIABETES

443

weight to body weight with age. In other words, there is no proof

that a puppy having a certain ratio will maintain this same ratio up

to adult age. In tables 4 and 5 no great change was noticed in the

TABLE 5

Litter of yellow and brown mongrel pups. Weight of mother, 21.6 kilos

NUMBER AGE BODY WEIGHT | PANCREAS wercut | >“) URS

kgm. grams grams.

1 % week 0.3 1.7 3.67

2 % week 0.4 1.7 4.25

3 + week 0.4 1.3 3.26

4 2 weeks 0.6 2.6 4.33

5 2 weeks 0.7 2.8 4.00

TABLE 6
Relation of pancreas weight to body weight in puppies
PANCREAS -~
NUMBER AGE BODY Starr ianatd spehnaie ah pono lta

kgm. grams grams

1 1 week premature 0.1 0.2 2.0

2 1 week premature 0.1 0.2 2.0
3 1 month 0.7 2.2 3.14
_ 4 1 month 1.5 él 4.74
5 + months 2.1 4.1 1.95
6 % months 1.3 2.6 2.00
a 2 months 0.9 3.5 3.89
8 2 months 2.3 5.1 aise
9 2 months 2.0 5.9 2.95
10 2 months 1.8 5.8 3.22
11 2 months 4.3 8.4 1.95
12 2 months 2.3 7.4 8.21
13 23 months 1.8 7.4 4.10
14 24 months 2.5 5.7 2.28
15 3 months 2.5 12.2 4.89
16 3 months 2.3 5.2 2.26
17 5 months 3.9 13.8 3.54
18 7 months 2:5 11.4 4.55

ratio up to 2 or 3 weeks of age, using other pups of the same litter as
controls. The ratios varied widely among the different pups in table
6. They were. sometimes larger in the smaller breeds, as found for
adult dogs (2), but the rule was not uniform. The state of nutrition

ee
444 — ne | FREDERICK M. ALLEN —
Partial pancreatectomies in puppies
rai. ae 21°
CREAS ; a
NUM- BODY PAN- WEIGHT] ¥ @IGHT oso
per | “SP \wmicnn|weromr| PER |°xant | TION
| GRAM ‘
‘months | kgm. | grams | grams | grams
1 1 3.0] 18.2 14.4 | 0.8 | fe-zy
2 » a gs Wa GS og pO Se
Fis 4 es al ee,
3 2 AE S962) OF i xe-we Cachexia. W
Sag : SS aa kgm. at
pie: Z _ |. Probably m:
4 3 | 2.5] 11.0|4.4 | 0.5 | a |0.5-0.6 | Cachexia.
goa Baio 8b 1 | Bey ae
6:|° 3] 80 | 178) 46°) Behe y
7: {BR 1 2.07) 180 18.0.1 2:7) eas
8 3452.17 °S3 13.8 OR wae :
9 4 13.7/158/43 | 15]. Ze
10 | 4 | 8.4)27.6/3.3 |} 46] | | Removal of
“7 aan on ae additional t
- operations
aD he Remnant at
ae - was then 1.1 g
11} 8 | 1.9) 77.84.11 80.65) $e ee ea
12 53 | 5.8|25.8/49 | 44] % [5.48.4] Nodiabetes —
13 | 6 | 8.0] 22.5/2.8 | 9.3] #4 | |
14°") "6; | °8.8}--8.5.] 2:2 |°1.04) e# 2a
15 7}. 6.9 720.1 | 34 (°R7 tT & _ | Peritonitis, no g
pe ire es
16 7 5.91 15.0 | 2.54] 1.6] $25] — Diabetes stopp
7 distemper _
17 7 3.454 9.3.) B74 le LG t Cachexia, no gh
18 1d 444.829.) 2.02.) 1.0.1 - Pe | Diabetes, mild,
; . “cy fi SOORY oh
“19 8 0-8 AB.8 1.2.75 1 1.8 bale oe .

EXPERIMENTAL STUDIES IN DIABETES 445

TABLE 7— Concluded

PAN-

PAN- nega WEIGHT] SIZE OF

NUM- BODY

WEIGHT HYPER- ;
AGE CREAS GT|OF REM-| FRAC- REMARKS
BER WHIGHT| Saar! PER |"xanr | tion | TROPHY
KILO-
GRAM
Me months| kgm. | grams | grams | grams grams
20 9 §.8 | 21:5.}.3.71 | -1.3°| Diabetes prevented by

: preliminary fast
Peritonitis, no diabetes
Severe diabetes, even

during fasting
23 9 7.1 | 26.2 | 3.69 | 3.0] §. |3.0-1.95) Severe diabetes checked

. by cachexia. Emaci-

ated to 5.3 kgm.

2.7 |. 10.5 | 3.90} 1
22 9°| 7.4 | 24.8 | 3.386] 2.

- 24 |. 9 | 10.3} 30.9 | 3.00] 3.1] % | Consid-
: erable
25 | Wl | 4.1 | 14.3) 3.50}) 1.1 | zs | 1.1-2.2) Cachexia, no diabetes

or rate of growth is probably an important factor. The differences
found among animals of the same litter at the same age in tables 2
and 3 interfere seriously with studies based on any method of controls.

Partially depancreatized puppies are especially liable to cachexia
from respiratory infections or diarrhea, so that diabetes is often thus
suppressed and the experiments spoiled. The above statement con-
cerning the relatively slight disposition to diabetes is based on animals
which remained vigorous and thriving after operation. It is the more
striking in view of the fact that the sugar tolerance. of the young is
distinctly less than that of adult normal animals (5). <A series of
observations was made upon thé question whether partial pancreatec-
- tomy short of diabetes interferes with the growth or health of puppies,
with negative results except for the temporary backset due to the
operation, and a possible specific inhibition of development of sexual
and other adult characters. Anything in the nature of a masked dia-
betes or other fatal metabolic deficiency seemed to be excluded.

As explained in a subsequent publication on acidosis, the expectation
that puppies might prove more susceptible than adult dogs to diabetic
acidosis was entirely disappointed. Acidosis was absent throughout
the reported experiments except where specially mentioned.

As puppies approach adult age, they approach more closely to the
adult behavior respecting diabetes. After about the ninth month they
generally react like adults except for susceptibility to distemper.
Nevertheless in pup 18 in table 7 it is noticeable that diabetes was

446 FREDERICK M. ALLEN

transitory with a pancreas remnant of only ;'5. Some typical records
of younger puppies are the following.

No. G7-49. Male; mongrel; black and white; age 1 month; well nourished;
weight 3 kilos. June 25, 1918, removal of pancreatic tissue weighing 12.4 grams,
Remnant about main duct estimated at 0.8 gram (75 to #y). Following operation
there was diarrhea and loss of weight but no glycosuria, though the pup lived on
bread and milk. July 4, 0.32 gram additional pancreatic tissue was removed.
The condition was still cachexia without glycosuria, up to death on July 11.
The pancreas remnant weighed 0.7 gram, and was too badly autolyzed for micro-
scopic study. In the tissue removed on July 4, the acini were normal and well
filled; islands were present in normal number and size but showed slight distinct
vacuolation. This hydropic change makes it probable that hyperglycemia was
present, and possibly only the animal’s weakness prevented a frank diabetes.
Acidosis was absent as usual. .

No. D4-85. Female; mongrel; age 3 months; brown and white; well nourished;
weight 2.5 kilos. November 9, 1916, removal of pancreatic tissue weighing 10.5
grams. Remnant about main duct estimated at 0.5 gram (ss). Glycosuria was
brought on by the feeding of 20 grams meat and 20 cc. milk, but ceased readily
with fasting. Death occurred from weakness, notwithstanding a low meat diet,
on November 20. The pancreas remnant weighed 0.6 gram, and was not examined
microscopically. The pup obviously had a potentially severe diabetes, but dif-
fered from an adult dog in that glycosuria did not begin spontaneously and was
promptly checked by fasting and low diet, with such a very small pancreas
remnant.

No. D4-34 similarly underwent removal of 8.2 grams pancreatic tissue, leaving
a remnant estimated at 0.65 gram (7/3 to #z). Glycosuria could readily be checked
by fasting, but on full meat diet was as high as 1.2 per cent. Notwithstanding
the liveliness following operation and the meat diet, the weight and strength
failed rapidly. The animal was killed when moribund on the tenth day after
operation, when the plasma sugar was 0.05 per cent. The rapid cachexia was the
chief point of difference from an adult dog.

No. G7-25. Female; Boston terrier; brindle; age 6 weeks; good nutrition;
weight 3.8 kilos. June 17, 1918, removal of pancreatic tissue weighing 13.1 grams.
Remnant about main duct estimated at 1.1 gram (75). Glycosuria remained
absent, first on beef lung, then on bread and soup diet, then with addition of 50
grams glucose. June 25, at a weight of 3.5 kilos, 1 gram additional pancreatic
tissue was removed, the remnant having undergone marked hypertrophy. Gly-
cosuria remained absent on bread and milk diet, though weight was lost. July 4,
0.95 gram additional pancreatic tissue was removed. The animal was lively and
ate considerable lung on July 5, then gradually lost appetite and weight while
retaining spirits up to death on July 9. Glycosuria remained absent. The
plasma sugar on June 17 was 0.148 per cent, on July 8, 0.162 percent. The pan-
creas remnant weighed 0.8 gram. In the various specimens of pancreas from
June 17 and July 4 and 9, inflammation and fibrosis were limited to the peripheral
areas. In the central portions the acini were well filled, sometimes large and

1 All operations were performed under ether anesthesia.

‘

EXPERIMENTAL STUDIES IN DIABETES 447

irregular. Islands were normal in number and free from vacuolation. Here the
persistent absence of diabetes is perhaps attributable to the hypertrophy of the
pancreas remnant.

No. E5-40. Male; sictaitel, part Newfoundland; age 3} months; good nutri-
tion; weight 8.4 kilos. May 14, 1917, removal of pancreatic tissue weighing 23
grams. Remnant about main duct estimated at 4.6 grams (4). Glycosuria was
absent on bread and soup diet, and only transitory with addition of 100 grams
glucose.

May 24, additional pancreatic tissue weighing 0.9 gram was removed. There
was obvious hypertrophy of the remnant in all three dimensions. Thereafter
glycosuria was absent on bread diet, and could be maintained only by increasing ~
additions of glucose, first 50, then 100, then 200 grams daily. The dosage of 200
grams was continued from June 6 to 27 with only traces of glycosuria and with a
gain of weight to 9.3 kilos.

June 27, 0.6 gram additional tissue was removed from the pancreas remnant.
which again was obviously hypertrophic. Thereafter heavy but transitory gly-
cosuria resulted first from 100 and then from 200 grams glucose added to the bread
diet.

July 12, an additional 0.35 gram of pancreatic tissue was removed, the weight
then being 9.8 kilos. ‘Glycosuria was them heavy with addition of 200 grams glu-
cose to the bread diet, but ceased July 19.

July 20, an additional 0.4 gram of pancreatic tissue was removed. Glycosuria
was then heavy on bread and soup diet, next with addition of glucose up to 200
grams, but was absent after July 29.

August 31, at a weight of 10.6 kilos, glycosuria being still absent on the bread
diet with 200 grams glucose, an additional 0.52 gram of pancreatic tissue was
removed. Glycosuria was again transitory.

September 7, an additional 0.3 gram of pancreatic tissue was removed with a
similar result, glycosuria ceasing September 18.

September 28, an additional 0.55 gram of pancreatic tissue was removed.
Glycosuria was then-continuous, first with glucose, then on plain bread and soup,
till stopped by a change to meat diet on October 22. It may be noted that the
downward progress was slow rather than rapid, for the glycosuria of many adult
dogs after 3 weeks reaches a point where it cannot be stopped by fasting.

The pup did not thrive on carbohydrate-free diet, suffered from indigestion,
diarrhea and loss of weight, and died in cachexia November 16 at a weight of 7
kilos. The blood sugar was low during this period, and there was the usual
absence of acidosis.

The pancreas remnant weighed 1.1 gram. Nothing significant was found in
the gross autopsy, or in the microscopic examination of the liver, kidneys, adre-
nals, thyroid and parathyroids. Sections from the pancreas at all the opera-
tions and at autopsy showed normal parenchyma and absence of hydropic
degeneration. Though the hypertrophy may have gone far toward preventing
diabetes, it is evident from the repeated operations and the prolonged carbo-
hydrate excess that this puppy was difficult rather than easy to make diabetic.

No. D4-21. Male; mongrel; age 7 months; good nutrition; weight 6.7 kilos.
September 15, 1916, removal of pancreatic tissue weighing 24 grams. Remnant
about main duct estimated at 2.9 grams (4). Glycosuria at first was absent on

448 FREDERICK M. ALLEN

moderate quantities of milk, but was heavy on bread feeding September 18. It
was then checked by fasting, and kept absent on a diet of lung, suet and 100 grams
bread. The animal thrived and maintained a weight of about 7 kilos.

December 5, a full bread diet was resumed, and heavy glycosuria returned
promptly. After 4 days this was stopped as before. The animal grew to adult
life on a diet of 400 grams lung, 100 grams suet, and part of the time 100 grams
bread or 100 cc. milk. Yeast was added part of the time, with the idea that it and
the milk might supply needed vitamines; also an admixture of bonemeal assured
adequate salts. The animal became plump and strong at a weight of 8.7 kilos,
but remained always undeveloped in mentality, in the size of the sexual organs
and the absence of any apparent sexual function; urination was performed squat-
ting, and the puppy contour of the body and puppy-like behavior toward other
dogs were retained. Pratt (6) has made similar observations in young dogs with

pancreatic atrophy.

_- November 16, 1917, it was found that the addition of 100 grams bread to the
diet produced a glycosuria of 1.3 per cent in 820 cc. of urine. The tolerance was
thus evidently lower than before, and thereafter the diet was kept carbohydrate-
free (400 to 600 grams of lung and 100 grams of suet). April 6, 1918, the fasting —
plasma sugar was found to be 0.250 per cent, indicating that hyperglycemia was
now continuous. The weight at this time was 9.15 kilos. Traces of glycosuria —
began in July, at first intermittently, but by July 30 they were continuous and
increasing, so that fasting had to be used. The weight at this time had reached
its maximum of 9.5 kilos. The animal appeared not obese but in excellent
nutrition. : 3

The diet was then gradually built up to 200 grams lung and suet adlibitum,
without glycosuria. This diet continued while the writer was in military service.
At a visit on September 16, 1918, the animal was found in excellent strength and
spirits, weighing 8.3 kilos, with intense glycosuria and hyperglycemia and heavy
nitroprusside reactions in urine and plasma. Notwithstanding the fat-rich diet
and acidosis, there was no visible lipemia. The condition was ideal for the
development of coma, but under the circumstances there was nothing to do but
to kill the animal for autopsy.

The body appeared in excellent condition and retained much fat. Except for
a very fatty liver and the puppy-like characters noted above, the gross autopsy
was negative. The pancreas remnantweighed 3.1 grams. The practical absence
of hypertrophy may be noticed. —

Microscopically, the liver was crammed with fat even to the periphery of the
lobules. Stains with Best’s carmine showed absolutely no glycogen except in
occasional leukocytes, which stood out prominently in the capillaries by reason.
of their stuffing of red granules.

The kidneys were normal except for maximal Armanni vacuolation, as seen in
routine Zenker specimens stained with methylene blue and eosin. Best’s carmine
applied to the alcohol fixed specimens showed only a sparse sprinkling of glyco-
gen, so that the vacuoles evidently represented chiefly fat deposit, as demon-
‘strated by fat stains in some other animals but not in this one.

The adrenals showed no more than the average lipoid vacuolation in the cor-
tex, and were normal in other respects.

The thyroid and parathyroids were normal, with average colloid content in
the former.

EXPERIMENTAL STUDIES IN DIABETES 449

The pancreas remnant was normal in structure and fullness of acini and
number and size of islands, but a minority of island cells were maximally
swollen and vacuolated. _ :

This puppy, in respect to “spontaneous” downward progress and
other features, behaved practically like an adult animal, and most
of the history actually represented an adult period of life. Many
long experiments of this character were begun for the purpose of testing
whether a partial pancreatectomy which did not suffice to produce
immediate diabetes in a puppy might result in diabetes after a certain
stage of growth had been reached. In other words, the.problem was
whether a damaged pancreas might lag behind in development so as
finally to become inadequate for the demands of a growing body. The
best observations would have been those upon much younger animals,
but all these failed for one cause or another. It is evident that in many
cases the hypertrophy of the pancreas remnant fully compensates for
any bodily growth. It is also very doubtful whether an animal which
is actually non-diabetic will become diabetic by simple growth. But
when the injury results in such a mild diabetes that there are no symp-
toms on whatever dietary régime is followed, it is entirely possible
that diabetes may develop openly at a much later period under the
strain of growth and gain of weight. Such a result is exemplified in
this experiment, which covers a rather long period in proportion to a
dog’s life. “Such experimental evidence makes it easily comprehensible
that diabetic symptoms may follow either soon or late after an injury
of the human pancreas; in particular, that glycosuria may be present
immediately after an infection, or may be delayed to a time when there
is no plain clinical connection with a long antecedent infection which
may have been the real cause.

CONCLUSIONS

1. An influence of senility upon carbohydrate assimilation or diabetes
is conceivable from a quantitative reduction or other alterations of
metabolism, or from functional er anatomic changes in the pancreas.
The observations upon senile dogs failed to show any departures from
the normal in the glucose tolerance, the ratio of pancreas weight to
body weight, the microscopic structure of the pancreas, the size of the
pancreatic remnant with which diabetes occurs, the capacity of such
a remnant for hypertrophy, or the clinical course of the diabetes.

450 FREDERICK M. ALLEN

2. An investigation was also made of the possible influence of the

elevated metabolism or the pancreatic peculiarities of youth. Previous
work had indicated that the glucose tolerance of puppies is less than —
that of adult dogs. No exact studies were made of the most important
point in the microscopic anatomy, namely the richness in islands, but
no striking departure from the adult average was noticeable in numerous
routine observations. The ratio of pancreas weight to body weight
was somewhat irregular, and the question of possible. changes in an
individual during growth could not be accurately decided, but the
general average in puppies did not differ appreciably from that in adults,
particularly with consideration of the small body weight. The remnant
- left after partial pancreatectomy generally grows considerably in pup-
pies; the hypertrophy on the whole is greater than in adult dogs, but
does not surpass what is found in occasional mature or adult animals,
and may be slight or absent especially in older puppies. The tendency
to diabetes is distinctly less in puppies than in adult dogs, partly on
account of weakness and cachexia, partly because of hypertrophy of
the pancreas remnant, and perhaps sometimes because of a high fune-
tional efficiency of a small remnant. There is no specific tendency to
rapidity of downward progress or to diabetic acidosis in puppies.
“ 3. An example is given of long delayed onset of diabetic symptoms
on a fixed diet after gain of weight, this gain being largely growth instead
of mere obesity. With a still milder diabetic tendency it is readily
conceivable that the delay might be longer and might occur on an
ordinary diet, also that the diet might in large measure determine the
onset of symptoms. In this way it is possible that childhood infections
injuring the pancreas may be responsible for some cases of diabetes
which make their appearance at a much later period.

BIBLIOGRAPHY

(1) AuLeN: Amer. Journ. Med. Sci., paper 2 of this series (in press).
(2) AtLeN: Journ. Exper. Med., 1920, xxxi, 363.

(3) Auten: Amer. Journ. Med. Sci., paper 2 of this:series (in press).
(4) ALLEN: Studies concerning glycosuria and diabetes, 1913, 905.

(5) ALLEN: Studies concerning glycosuria and diabetes, 1913, 34.

(6) Pratt: Journ. Amer. Med. Asse., 1912, lix, 322.

EXPERIMENTAL STUDIES IN DIABETES

Series I]. Tue InTERNAL PaNcREATIC. FUNCTION IN RELATION TO
Bopy Mass anpD METABOLISM

9. The Influence of Pregnancy upon Experimental Diabetes

FREDERICK M. ALLEN
From the Hospital of The Rockefeller Institute for Medical Research, New York

Received for publication August 10, 1920

Diabetes may conceivably be aggravated by the increased metabolism
of pregnancy, which involves both additional food assimilation and the
formation of considerable new tissue. Unknown toxic or metabolic
factors may possibly have an influence. An opposite possibility is
suggested especially by the work of Carlson and collaborators (1),
- namely, that diabetes in the latter part of pregnancy may be prevented
by the internal secretory activity of the fetal pancreas. The chief
question here is whether internal secretions pass in appreciable quanti-
ties through the placenta. In clinical practice pregnancy has long
been regarded as decidedly injurious, to such an extent that therapeutic
abortion was commonly recommended in women suffering from. any
serious grade of diabetes. The most comprehensive and recent survey
of clinical experience is by Joslin (2), who considers that the supposed
injurious effects are largely explained by the higher diet taken. One
case of apparent gain of tolerance during pregnancy (3) is inconclusive,
partly because the blood sugar rose during gestation and partly because
it is uncertain whether the changes observed were due strictly to the
pregnancy.

Partially depancreatized animals offer the most accurate obtainable
conditions for studying the effect of pregnancy upon the internal
pancreatic function. Before proceeding to the principal experiments,
it is desirable to mention briefly some controls and unsuccessful attempts
relating to this problem.

One control to be thought of is the possible effect of changes i in the
sexual organs themselves upon the pancreas or its function. This is

45]

452 FREDERICK M. ALLEN

part of the larger question of the interrelation of the sex glands with
the pancreas and diabetes. A few ‘extirpation experiments. were per-
formed as follows.!

Dog B2-89, a female mongrel aged 4 years, had been close to the verge of dia- .
betes-since the first operation on April 12, 1915, so that the removal of 0.25 gram

additional pancreatic tissue on March 16, 1916 brought on mild diabetes. By
repeated tests up to May 18 it was proved that glycosuria was regularly absent
on a measured bread and soup diet, and was present to the amount of about 5
grams daily with the addition of 75 grams glucose. May 18, both ovaries were
removed, together with the tubes and part of the uterine horns. A trace of gly-
cosuria followed the operation, and another trace when the regular bread diet
was fed. The addition of 75 grams glucose resulted in glycosuria of 12.5 grams
one day and 13 grams another day. By May 27 glycosuria was again absent on
the plain bread and soup diet and the glycosuria from glucose was no higher than
before operation. The absence of any perceptible influence upon the diabetes,
other than the transient effects of the trauma, was also confirmed by the animal’s
later history.

Dog D4-62. Female; mongrel; long haired; age 6 to 8 years; well nourished;
weight 19.2 kilos. December 16, 1916, removal of pancreatic tissue weighing
27.5 grams. Remnant about main duct estimated at 3.2 grams (345). After fast-
ing, glycosuria was found consistently absent on a diet of 1 kilo of beef lung, and
moderate (0.5 to 1 per cent) with addition of 50 grams bread. January 17, 1917,
both ovaries were removed. A trace of glycosuria followed the operation, but

ceased on the lung diet. With addition of 50 grams bread it was distinctly greater —

than before (2.8 to 4.8 per cent, in larger urine volumes), but ceased when the
bread was omitted,on January 26. Thereafter the diabetic tendency was no
greater than before the odphorectomy. |

Dog C3-67. Male; Dalmatian; age 1} years; in excellent nutrition; weight
17.6 kilos. March 14, 1916, castration, and removal of pancreatic tissue weighing
28.5 grams. Remnant about main duct estimated at 2.6 grams (;'z to 7/5). The
dog was used for a few tests (especially feeding of pure fat or talcum powder, as
described elsewhere) which had no influence upon the glycosuria. Otherwise no
food was given, but heavy glycosuria continued until death occurred from weak-
ness on April 1, at a weight of 11 kilos. The pancreas remnant, weighing 3.1
grams, was normal except for the usual vacuolation of island cells. The bladder
urine contained 0.61 per cent of sugar, and the autopsy otherwise was negative.
Such irrepressible glycosuria with a pancreas remnant of this size is so unusual
that the possibility of an aggravating influence of the castration was suggested.

Dog D4-63. Male; mongrel; long haired; age 3 years; medium nutrition; weight
15.25 kilos. After removal of pancreatic tissue on February 14 and March 7,
1917, glycosuria was maintained almost continuously on bread and soup diet with
addition of 200 grams glucose to March 28, after which it was absent. Such a

duration of glycosuria is evidence that an animal is very close to diabetes, which -

will be produced by the removal of a trifle more pancreatic tissue. The actual
absence of diabetes was confirmed by blood sugar tests, the plasma sugar being

1 All operations were performed under ether anesthesia.

ee ea ee ee ee

EXPERIMENTAL STUDIES IN DIABETES 453

0.087 per cent before feeding and 0.145 per cent at the highest point afterward, and
down to 0.122 per cent by evening even on the bread and glucose diet. March 28,
castration was performed. Glycosuria remained absent on bread diet to April 2,
and therafter also with addition of 200 grams glucose. The attempts to induce
diabetes by diet were continued through May, with merely the usual slight gain
in tolerance. The removal of 0.5 gram pancreatic tissue on May 8 proved inade-
quate, but mild diabetes followed the removal of 0.65. gram additional on June 1.

This single experiment with dog D4-63 probably suffices to prove
that removal of the testes has no influence for or against the production
of diabetes. Aggravation of an existing diabetes, which is a separate
question, was. not produced by removal of the ovaries in dogs B2-89
and D4-62. The apparent aggravation after removal of the testes in
dog C3-67 may have been purely accidental, and any specific influence
remains improbable. Circumstances prevented further experiments

~ of this sort.

Numerous attempts were made to determine the size of pancreas
remnant with which diabetes occurs in pregnant animals, with uniform
failure on account of abortion or death. Efforts to immunize by one
or several preliminary subcutaneous or intraperitoneal injections of
aqueous extract of fresh sterile dog pancreas gave no protection. ‘Trials
were made with removal of five-sixths to nine-tenths of the pancreas
in single operations, performed as quickly and easily as possible so as
to be over in half an hour or less, but the animals always became unwell
and aborted within a few days or at most. a week. Such an interval,
especially when glycosuria is prevented by malaise and refusal of food,
can decide nothing with the pancreas remnants mentioned, though
permitting some observations after total pancreatectomy as in Carlson’s
experiments. Other attempts were made with successive operations,
in the hope that each might be so easy as to avoid abortion, also by
leaving a duodenal remnant and a subcutaneous graft early in preg-
nancy, with the idea that diabetes might be brought on later in preg-
nancy by simple removal of the graft. All such attempts failed for
one cause or another. It is sometimes possible to remove considerable
portions of pancreas, even in the later stages of pregnancy, without
accident, just as other abdominal operations are often feasible; but
any pancreatic resection to the point of diabetes seems absolutely
incompatible with continuance of gestation. The possibility that
this disturbance may be due to the sudden deficit of the internal pan-
creatic function was excluded by two sets of controls; first, the results
are just as bad when the entire uncinate process is left as a subcutaneous
graft in addition to the usual pancreatic remnant; second, pregnant

4.54 FREDERICK M. ALLEN

dogs ordinarily survive a week of fasting and phlorizination without
abortion, though the glycosuria and acidosis apparently represent a
greater disturbance of carbohydrate metabolism than that following
the pancreatic operations mentioned.

An attempt to test the influence of the increased food requirement
and lactose formation of the lactation period was made as follows.

Dog C3-90, mongrel, in excellent condition, weighing 20.5 kilos, was found preg-
nant in an operation on May 25, 1916, when the splenic process and body of the
pancreas down to the main duct were removed, with the idea that diabetes might
later be produced by a very easy operation for removal of the uncinate process.
The pregnancy continued uneventfully, but by mistake too long an interval was
allowed, and on June 22 the dog gave birth to eight healthy puppies. She was an
excellent mother and the puppies all thrived until, on June 29, the uncinate proc-
ess was removed in an operation requiring only afew minutes. There was quick
recovery from the brief etherization, and the dog showed the usual devotion and
nursed the puppies immediately on being returned to the cage. She acted well
and lively but ate very little on the following days, during which time the pups
continued to nurse though the mother paid less attention to them. By July 4
her appetite was fully restored, but she refused to have anything further to do
with the pups, and even injured them if they approached to nurse. Part of the
trouble may have been due to tenderness about the abdominal wound, but there —
seemed also to be a genuine breaking up of the physiology and psychology of
lactation by the operation. Milk rapidly disappeared from the breasts. Diabe-
tes remained absent notwithstanding glucose feeding. Whether the disturbances
' observed were peculiar to the pancreatic operation or might follow any other
abdominal interference, the experience showed that this method was not applica-
ble for studying the influence of lactation upon diabetes.

Numerous microscopic examinations have been made of the pancreas
of dogs in various stages of pregnancy and of a few during lactation,
without the finding of any departure from the normal in any respect.

It was evident that a satisfactory study was possible only through
the occurrence of pregnancy in animals already diabetic, so as to repro-
duce exactly the conditions encountered in diabetic women. Properly
prepared animals with potential diabetes are normal in their entire
behavior, and only the unfavorable laboratory conditions made the
experiment difficult. It was hoped that cats might be particularly
suitable for the purpose, but in the only instanee in which a partially
depancreatized cat became pregnant the experiment ended in failure,
as follows. |

Cat A1l-84. Female; strong adult; weight 3.5 kilos. December 20, 1913, pan-
creatic tissue was removed, not quite to the point of producing diabetes. The |
cat remained continuously free from glycosuria on meat, bread and milk diets

I ee ee a we ee

EXPERIMENTAL STUDIES IN DIABETES 455

‘thereafter, but had a permanently lowered tolerance, as shown by the production

of glycosuria in subcutaneous glucose tests by doses between 1 and 1.5 grams per
kilo. Impregnation occurred February 18, 1914. Thereafter the diet was meat
and milk, mixed with as much lactose as the animal could be induced to take.
Considerable carbohydrate could thus be given, though cats usually object
strongly to the sweetness of glucose or saccharose. About the middle of March
the animal was noticed to be weak and unwell. Abortion occurred March 16
and death March 18. There was no diabetes and the pancreas remnant was nor-
mal. The fatal outcome was almost certainly a sugar intoxication (4), with no
specific relation to the partial pancreatectomy or to diabetes. Cats may possibly
prove to be well suited to pregnancy experiments in diabetes, but they are as a
rule an unfavorable species for carbohydrate over-feeding.

Far more numerous attempts were made with partially depancreat-
ized dogs during three years, but all failed owing to the unfavorable |
environment. Finally a successful experiment became possible in dog
B2-00, which was particularly valuable for the purpose because of the

long previous records which had established the tolerance accurately.

These observations have been reported in previous papers, especially
no. 3 of series I (5) and no. 1 of series II (6).

A series of operations beginning in 1913 had made the dog nearly diabetic, but
after the removal of an additional 0.8 gram of pancreatic tissue on September 6,
1916, it was still impossible to maintain glycosuria with the heaviest bread and
glucose feeding. Pregnancy then became evident, though the exact time of its
beginning was unknown. ‘Tests of the tolerance were performed by feeding, as
described below. As diabetes remained absent with advancing pregnancy, there
was danger that the entire result would be negative.

Accordingly, on December 16, 1916, 0.1 gram pancreatic tissue was removed,
in an operation so short and easy that the dog did not even lose appetite for the
day. Diabetes resulted, as proved by the fact that plain bread and soup feeding
now maintained slight glycosuria (0.4 to 0.8 per cent, in 400 to 680 ec. urine).
December 26 on this diet the plasma sugar was 0.091 per cent before feeding, 0.182
per cent four hours after. The special feeding test was repeated in late pregnancy
on December 30.

As an additional experiment, it was attempted to learn whether the pregnant
dog with latent diabetes had any special tendency to acidosis. Therefore nothing
was fed on December 31, and only 300 grams suet daily on January 1,2 and 3 (1917).
Abortion then occurred, though the dog appeared only slightly unwell and re-
mained free from acetone bodies in urine and blood, both before and afterward.
The plasma sugar was constantly normal (0.089 to 0.105 per cent) after omission
of carbohydrate, both before and after abortion. The carbohydrate tests had
seemed harmless, and it may be possible that the fat feeding was responsible for
the abortion even without acidosis.

January 27, the special feeding test was repeated as a control in the non-preg-
nant condition. The existence of diabetes was unmistakable. The diet was then
chariged to meat to prevent downward progress, and the dog was left most of the

456

FREDERICK M. ALLEN

Dog B2-00. Tests with feeding 100 grams beef lung, 200 grams bread and 150
grams glucose

DATE weiguT | PLASMA! Dh ee REMARKS
1 SUGAR :
Volume}Glucose
1916 kgm. | per cent |per cent cc. |per cent
November 21| 13.4 | 0.106 | 101 18 0 | Before feeding
0.123 97 16 O | 2 hours after feeding
0.156 98 24 | Faint| 4 hours after feeding
| 0.128 96 38 | Faint] 6 hours after feeding
November 23 0.092 95 0 | Before feeding same diet as
Nov. 21
0.095 88 13 | Faint] 2 hours after feeding
0.139 92 | No |urine | 4 hours after feeding
0.130 93 | 105 | Faint) 6 hours after feeding

December 16

Removed 0.1 gm. pancreatic tissue. Dog pregnant

December 30

1917

January 7

February 23

May 17

July 11

July 19

15.6

12.9

15.6

14.9

0.085
0.345
0.455
0.500

0.125

0.400
0.400
0.333
0.116
0.238
0.244
0.232
0.130

0.346
0.435
0.384
0.106

0.322
0.304
0.312
0.077

0.200
0.218
0.232

212
192
68

f

25
35
38

18
17
22

23
16
13

0
3.45
6.46
5.89

j=)

oon

oS
SSAIBS-SBS

Before feeding

2 hours after feeding
4 hours after feeding
6 hours after feeding

Before feeding. After abor-
tion :

2 hours after feeding

4 hours after feeding —

6 hours after feeding

Before feeding

2 hours after feeding

4 hours after feeding

6 hours after feeding

Before feeding. (Early preg-
nancy)

2 hours after feeding

4 hours after feeding

6 hours after feeding

Before feeding. (Late preg-
nancy.)

2 hours after feeding

4 hours after feeding

6 hours after feeding ©

Before feeding. (After deliv-
ery) :

2 hours after feeding

4 hours after feeding

6 hours after feeding

EXPERIMENTAL STUDIES IN DIABETES 457

DATE WEIGHT PRABMA Hb eral REMARKS
SUGAR ms
Volume|Glucose
1917 kgm. ‘per cent |per cent} cc. |per cent
August 9 - 12.9 | 0.081 0 | Before feeding
| 0.164 18 | Very | 2 hours after feeding
faint
0.109 36 | Faint| 4 hours after feeding
. 0.135 17 | Faint} 6 hours after feeding
October 5 13.5 | 0.109 0 | Before feeding
0.159 |. 17 0 | 2 hours after feeding
0.145 | . 36 | Faint| 4 hours after feeding
0 152 46 | 0.31 | 6 hours after feeding
November 22| 14.0 | 0.109 . 0 | Before feeding
: 0.204 24 | Faint] 2 hours after feeding
0.238 | 36 | 0.39 | 4 hours after feeding
0.125 24 | 0.44 | 6 hours after feeding

time with a male in the hope of a second pregnancy. The plasma sugar meanwhile
remained normal (0.087 to 0.113 per cent) on the carbohydrate-free diet. The
special feeding test was repeated on February 23 and May 17.

Pregnancy then became manifest, and the feeding test was repeated in this
condition on July 11. June 28 and July 2, the plasma sugar on the meat diet was
normal (between 0.075 per cent before fhading and 0.105 per cent after feeding).

July 12, less than 0.1 gram of pancreatic tissue was removed without appreci-
able disturbance. :

Three pups were born on July 16. They were probably 2 or 3 days premature,
and were all dead by July 19 owing to failure to nurse properly. This result
may have been independent of the experimental conditions, for the dog was of a
type which often has troubles with puppies.

The special feeding test was repeated on August 9, October 5 and November 22.

Owing to the perfect manner in which the dog bore the experiments
through two pregnancies, the questions at issue received very satis-
factory answers as follows:

a. In the first instance, pregnancy failed to produce diabetes in an
animal which was so close to the verge that diabetes resulted from the
removal of 0.1 gram additional pancreatic tissue. Against an assump-
tion that the diabetes here was due largely to the pregnancy and_only
partially to the pancreas operation is the fact that diabetes persisted
after termination of the pregnancy. Also in the second instance, when
the dog was known to have mild diabetes but was free from symptoms
on meat diet, pregnancy failed to produce either glycosuria or hyper-
glycemia on this diet.

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. 54, NO. 3

458 FREDERICK M. ALLEN

b. The removal of a bit. of pancreatic tissue in the latter part of
each pregnancy supplemented the observations of the negative effects
of gestation and especially demonstrated the absence of vacuolation

of the islands first when the animal was not quite diabetic and second

when she was mildly diabetic. The absence of such hydropic changes
with latent diabetes corresponds fully to the sta in non-pregnant
* animals.

c. The passage of any appreciable quantity of pancreatic internal
secretion from the fetuses to the mother is disproved by two facts.
First, the occurrence of diabetes following the operation of December,
16, 1916, was not prevented; in other words, there was no transfer of

pancreatic hormone sufficient to compensate for the loss of 0.1 gram of —

maternal pancreatic tissue. Second, the feeding tests demonstrated
an actual aggravation of the diabetes during pregnancy. Test meals

were used instead of intravenous glucose injections because according —
to previous experience (7) they afford a more accurate index of the ~

diabetic condition and also because they seemed to promise less danger
of abortion or other accidents. Attention may be called to. the results
of these tests as shown in the table.

The test meal consisted of 100 grams beef lung, 200 grams bread
and 150 grams glucose. Only slight hyperglycemia and faint glycosuria
resulted from this diet on November 21 and 23, when the dog was non-
diabetic. The removal of 0.1 gram pancreatic tissue on December 16,
produced a radical change, so that the hyperglycemia and glycosuria
in the test of December 30 were of plainly diabetic character. The
tolerance was fully as low on January 7, after termination of pregnancy,
but the dog was still unwell from the recent abortion. The test of
February 23 showed a considerably better tolerance as judged by both
blood and urine.

May 17, early in the second pregnancy, the test showed a well-
marked fall in the assimilation, and the result was not greatly different
in the late stage-of pregnancy on July 11. The bit of pancreas removed
on July 12 was so tiny that. it apparently had little effect upon the
tolerance. At any rate, in spite of this operation, the assimilation on
July 19, three days-after normal delivery, showed a decided improve-
ment. This improvement was still greater on August 9, after all
puerperal disturbance had subsided. It was also maintained up to
October 5, the length of the experiment and the uniformity of results
being thus sufficient to exclude accidental fluctuations of tolerance.

nme

EXPERIMENTAL STUDIES IN DIABETES 459

CONCLUSIONS

1. No positive influence of the sex glands upon diabetes was demon-
strable by extirpation experiments. Also no anatomic changes in the
pancreas were perceptible with pregnancy or lactation.

2. Observations. upon a partially depancreatized dog during preg-
nancy are opposed to the view that any appreciable quantity of internal
pancreatic secretion passes from the fetus to the mother.

3. A distinct lowering of carbohydrate assimilation was shown during
pregnancy. This was not clearly associated with the increase of
metabolism, in the sense either of increased food requirement or new
tissue formation, for it seemed approximately the same in early and
late pregnancy and was also evident during the illness following abortion.
It may therefore be regarded chiefly as a toxic manifestation and thus
classifiable with the influence of infection. The effect is relatively
slight, because pregnancy failed to produce diabetes in a dog where the
removal of 0.1 gram pancreatic tissue sufficed for the purpose, and also
after the dog was demonstrably diabetic on carbohydrate diet pregnancy
gave rise to neither glycosuria nor hyperglycemia on carbohydrate-free
diet.

The tests with partial pancreatectomy, which affords the most exact
method of study, suggest that Carlson’s results in totally depancreatized
dogs are to be interpreted as cachexia. Clearer information of the
influence of pregnancy upon the internal pancreatic function is also
afforded by the freedom from the variables which enter into clinical
cases. The slight tendency to aggravation of the diabetes and the
ready control by diet support Joslin’s experience of the feasibility of
completion of pregnancy by diabetic women under suitable conditions
of treatment. If the toxic factor is the principal one, as suggested, the
_ possibility remains that the injurious action in some women may be
considerably greater than in dogs and accordingly may require more
radical,measures.

BIBLIOGRAPHY

(1) Cartson AND Drennan: This Journal, 1911, xxviii, 391.
~ CARLSON AND Ginspoura: Ibid., 1914, xxxvi, 217.

(2) Josuin: Treatment of diabetes mellitus, 1917, 448.

(3) Josuin: Treatment of diabetes mellitus, 1917, 456.

(4) Auuen: Journ. Exper. Med., 1920, xxxi, 393.

(5) AtLtEeN: Journ. Exper. Med., 1920, xxxi, 563.

(6) Auten: Amer. Journ. Med. Sci., 1920, elx, 781.
(7) ALLEN AND WisHArT: Journ. Biol. Chem., 1920, xlii, 415.

RHYTHMICITY OF THE PYLORIC SPHINCTER

HOMER WHEELON anv J. EARL THOMAS

From the Department of Physiology of the St. Louis University School of Medicine,
St. Louis, Missouri

Received for publication August 19, 1920

The functions of the pyloric sphincter are usually considered depend-
ent upon the physical condition and relative degree of acidity of the
gastric content. Such an explanation fails in great part to account for
certain phenomena associated with the processes of gastric evacuation
both in health and disease. It also fails to consider the purpose of
gastric motility save for mixing the gastric contents and discharging
them into the duodenum at irregular periods of opening of the sphincter.
However, recent work tends to show that the opening and closing of
the sphincter is related, not only to the degree of fluidity and acidity
of the gastric content, but also to peristalsis and the degree of tonicity
demonstrated in the stomach.

Our present interest in the physiology of the sphincter was stimulated
by our inability to explain, upon accepted theories, radiographic obser-
vations made upon the human stomach. We therefore undertook to
study, upon laboratory animals under easily controllable conditions,
the motility of the pyloric sphincter. In this study the term pyloric
sphincter means that narrow band of muscle constituting the last
portion of the stomach.

METHODS

Forty-four experiments were performed upon dogs in this series.
All operative procedures were carried out under ether anesthesia.
Unanesthetized dogs were studied radiographically. In 14 experiments
records were obtained immediately following operation and while the
animal was under the influence of a light anesthesia. Six dogs were
studied from 5 to 18 hours after operation, no anesthesia being used
during the taking of records.. One animal was studied 3 days after
operation, another after an interval of 2 weeks. Four animals were

460

pe

RHYTHMICITY OF PYLORIC SPHINCTER . 461

operated under ether and morphine (10 mgm. per kilo) and studied at
once, ether being discontinued during the observations. This pro-

cedure gave a quiet animal with a very active stomach. Although the

type of sphincter action under this anesthesia was similar to that
obtained under other anesthesias, it was discontinued because of the
known action of morphine upon the gastro-intestinal tract. The most
constant graphic results were obtained with only sufficient ether to
abolish voluntary movements. Under such an anesthesia the behavior
of the sphincter is more constant and uniform than in the unanesthetized
animal under experimental conditions.

The operations in most cases consisted in opening the abdomen
by a midline incision, entering the fundic portion of the stomach and
sewing the recording apparatus in place. The gastric incision was then
closed with a pursestring suture about the rubber tube leading from

‘the anchored balloon. The opening in the abdomen was closed with

a running suture. Aseptic precautions were observed in all cases in
which the animal was permitted to survive 12 hours or longer. In
3 animals the muscle on either side of the pyloric sphincter was cut,
thus confining action upon the apparatus to the sphincter proper.

All apparatus except the recording instruments was designed and
constructed especially for this work. The apparatus is the equivalent
of a balloon and is so constructed that it can be placed and maintained
within the lumen of the pyloric sphincter. These balloons, or as we
have termed them, pylorographs, are of three types: a, flexible, closed
pylorograph; 6, rigid, closed pylorograph, and c, open, flexible pyloro-
graph.

The closed flexible pylorograph. (fig. 1, a) consists of a short section
of finger cot stretched over two cones of hard rubber the apices of which
face each other. One cone is perforated by a small hole which leads
out through a nipple on its base for the attachment of a rubber tube.
When in position the rubber is pressed down upon the sloping surfaces
of the cones by the tonus of the sphincter to such an extent that the
recording apparatus is affected by the contraction of a width of muscle
not exceeding 5 mm. This device when placed in position tends to
remain there because of the V-shaped surfaces offered to the pyloric
ring. Complete fixation of the pylorograph was obtained by passing
a ligature around the base of each cone and securing it in the muscle.

The rigid pylorograph is similar to the one described above save that
the two cones remain connected (fig. 1, b).

462 HOMER WHEELON AND J. EARL THOMAS

The open, flexible pylorograph (fig. 1, c) is constructed on the same
principle as the flexible, closed pylorograph except that an_open tunnel
4 mm. in diameter runs throughout its entire length. The apices of
the two cones are connected by means of a piece of rubberdam tubing.

oT tone Sth tlzem.

1em. ——»

hr:

Fig. 1. Illustrations of the various types of pylorographs. A, Closed flexible
pylorograph. B, Closed rigid pylorograph. C, Open flexible pylorograph. V,
Air transmission vent for tube connection with water manometer. F.C., Flexible
wall of pylorograph made of finger cot. 7'., Opening connecting stomach with
duodenum. R&.D., Tube of rubber dam composing inner wall of air chamber.
Dotted lines indicate positions of the rubber surfaces during contraction of the
sphincter.

The bases are connected by a section of finger cot. This device, the
equivalent of a tunnelled cylindrical balloon, permits the sphincter to
demonstrate its motility and at the same time permits material to
leave the stomach.

-RHYTHMICITY OF. PYLORIC. SPHINCTER. . 463

Graphic records were obtained with tambours, and piston and.bellows

- recorders. The pylorograph was connected with the recording appa-

_ratus through a water manometer. The manometer was adjusted to
a pressure (3 to 30 cm.) which gave best results in any given case.
Air transmission was used throughout.

RESULTS

_ The pyloric sphincter demonstrates two types of motility; a, active
_ rhythmic contractions (figs. 2 and 3), and b, tone waves (figs. 5 and 6).
The characteristics of these two types of action are as follows.

a. Rhythmic contractions. The rhythmic movements of the sphincter -

are characterized by contractions and relaxations each followed by a |
quiescent period or pause (fig. 3). These contractions occur at the
rate of from 3 to 5 per minute; that is, each cycle is completed i in an
interval of from 12 to 20 seconds. The phase of contraction is 4 to 5
seconds, the phase of relaxation 3 to 7 seconds; the quiescent phase
plus the period of inhibition prior to contraction occupy the remainder
of the cycle. These figures are necessarily only relative because of the.
_ different rates of contraction shown by various animals, the depth of ©

the anesthesia, and also because of the general motility of the stomach

and small intestines. During periods of rhythmic action of the sphinc-

_ter the contractions and relaxations are uniform in degree; that is, the
movements are initiated and consummated from a constant level (figs.
2 and 3). There is usually a definite degree of relaxation (inhibition)
of tone immediately preceding a contraction. At times relaxation
continues from the completion of contraction until the beginning of
a second contraction, no definite phase of quiesence or inhibition being
shown. Similar results were obtained both from the filled and recently
- emptied stomach.

The cycles of the sphincter are definitely altered under various con-
ditions; also, periods of increased activity may occur; however, as will
be shown in a later communication, such alterations bear a definite
relation to anesthetics, trauma and the activities of the - Eee oma
canal.

Rhythmicity of the sphincter is not lost following denervation of the
stomach (fig 4).

b. Tonicity of the sphincter. The tonicity of the pyloric sphincter
is gained or lost during a series of rhythmic contractions of which the |
heights of the individual contractions vary in proportion to the degree

HOMER WHEELON AND J. EARL THOMAS

ExPWo./%

7] Tune 1 see

FME TSEC

Fig. 2. Pylorograms obtained from four different experiments; A, experiment
4; B, experiment 19; C, experiment 42; D, experiment 26. Animals under ether
anesthesia. Graph A obtained with tambour, B and C with piston recorder, D
with bellows recorder. Time, 23 seconds in trace A, 1 second in B, C and D.

Trace A shows periods of inactivity followed by rhythmic contractions and
alterations in tonicity. Traces B, C and D demonstrate periods of constant
rhythmic activity. Note the variation in the height of contractions in traces A
and D. The four graphs are typical of the entire series of experiments.

465

RHYTHMICITY OF PYLORIC SPHINCTER

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466 HOMER WHEELON AND J. EARL THOMAS

of tonicity gained (figs. 5 and 6). On the other hand, periods of.
increased or decreased rhythmic contractions may appear at a time of
constant tonicity (fig. 5, trace C). Waves of increased tonicity appear
to result because of a lengthening in the time required for complete
relaxation; that is, a second wave of contraction appears during the
relaxation phase of the previous cycle. A reduction in the degree of
tonicity accompanies an increased degree of lengthening of the relax-
ation phase of the rhythmic cycles, the rate of the rhythmic contrac-
tions remaining constant.

TIME TSEC -

es Oe We eae eee eee Ai it

Fig. 4. Experiment 8; January 17, 1920. Rhythmic contractions of the pyloric
sphincter following denervation of the stomach. Ether anesthesia. Time in
seconds.

The rhythmicity and type of sphincter action are not altered by
increasing or decreasing, within limits, the pressure within the pyloro-
graph. However, if the pressure is greatly increased the height of the
individual contractions is decreased. Longer excursions of the writing
lever are recorded when the pressure in the pylorograph is low (3 to
10 cm..of water). In other words, the greater the degree of resistance
offered by the pylorograph to the force’ of the sphincter’s action the
smaller becomes the excursion of the muscle while acting. The primary

a

RHYTHMICITY OF PYLORIC SPHINCTER 467

effect of distention of the balloon in the pyloric canal is to excite the
sphincter to rhythmic action. Following this there usually appears
a gradual loss in tone for several moments after which the rhythmic
contractions appear from a constant level. The rapidity with which
the sphincter adapts its tonicity to an alteration in resistance is remark-

ExP No 2¢

|

|
|

rene

4
ay MEAL

Time 2h SEC.

Fig. 5. Experiment 24; February 9, 1920. Pylorograms obtained by use of
open pylorograph. Animal operated under ether. Records begun 18 hours later.
Piston recorder used for registration. Experiment checked by radidgraphic rec-
ords. . A, Band C, Sphincter action. C-C, Administration of 8 ounces of barium
mixture by stomach tube. 1, 2,3 and 4, Tone waves carrying contraction waves.
Trace C shows three active and four reduced phases of activity.

able. Apparently, the normal tonicity is only sufficient to close the
sphincter or to approximate its surfaces against those of a body in its
lumen. Non-resisting materials: permit of complete occlusion of the
lumen during the positive phase of the sphincteric cycle. Tonicity

468 HOMER WHEELON AND J. EARL. THOMAS

of the sphincter appears to be ‘unusually high immediately following
the ingestion of food; either normally or by means of the stomach tube.
Tonicity is also high immediately following the opening of the abdomi-
nal cavity. Doubtless'this is reflexly the result of peritoneal irritation:
Tonicity is not lost by reason of operative procedures on the stomach:

EXPNo0.13

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WAN Pa SW UA A A

Fig. 6. Experiment 13; January 9, 1920. Ether anesthesia for operation.
Double flexible enterograph. Records obtained 3 days after operation upon the
conscious animal. A, Sphincter contractions. B, Duodenal contractions.
Note periods of heightened tone of the sphincter at times of pronounced activity
on the part of the duodenum (3 and 4). X, Synchronous points. Time in
seconds.

RHYTHMICITY OF PYLORIC SPHINCTER 469

The approach of the pylorograph or examining finger to the antral
region causes marked contraction of the pars pylorica. Only under the
influence of a deep or*surgical anesthesia does the sphincter lose tone
completely.

SPECIAL EXPERIMENTS

In dog 25 in which a permanent. gastric fistula had been established,
it was possible to insert the finger into the antrum and even through
the pyloric canal into the duodenum without placing the animal under
ether or causing it much discomfort. The primary effect of the finger
in the antrum was to excite a state of violent and maintained contrac-
tion. Under a constantly applied pressure contracture passed off and
the finger was finally permitted to pass through the sphincteric canal.
With the finger in the canal one could distinctly feel pressure applied
rhythmically during the positive phases of the sphincteric cycles. The
- sphincter never relaxed completely nor drew away from the finger
during the relaxation phase or the period of quiescence. The obser-
vation that an initial irritation of the antrum causes a heightened
degree of tonicity in the pars pylorica corroborates the generally
accepted views as to the functions of these fee in relation to solid
objects in the stomach. - :

In an effort to throw more light on. re finelion of the sphihioter the
following experiment was performed. An open-pylorograph (fig. 1, ¢),
as described above, was anchored in the pyloric canal. Graphit records
were begun 18 hours following operation (fig. 5). At this time the
sphincter demonstrated powerful contractions which occurred irregu-
larly. The animal was then placed on the radiographic table and given
an 8-ounce meal of barium sulphate and milk by means of the stomach
tube. Immediately following the withdrawal of the tube a marked
tone wave appeared on the graphic tracing, superimposed upon which
was a.series of contractions of varying degree (fig. 5, A-1).. This was
followed by a series of smaller waves each of which lasted from 3 to
7 minutes. Four such tone waves are shown in tracings A. and B of
‘the same figure. As previously stated, these tone waves are built
up during a series of rhythmic contractions and lost through a series
of gradual and prolonged relaxations. At X in figure 5 a series of
relaxations permitted the recording lever to drop below the tone level
held by the sphincter prior to the administration of the meal. At
points marked Y and Y-1 there is a total absence of rhythmic contrac-
tion although the tonicity of the sphincter at Y-/ is markedly above

470 HOMER WHEELON AND J. EARL THOMAS

that at Y where the level is lower than the pre-meal normal. A radio-
gram taken at the height of a rhythmic contraction (P.1), and at a
time of relatively high tonicity, showed the antrum in a state of high
tonicity with the pyloric canal closed. As time went on these severe
tonic contractions gradually gave way to a constant tone level and
rhythmic contractions of varying height (fig. 5, c). The stomach
emptied itself of the barium mixture in less than 80 minutes. At the
end of this time a second meal was given. Rapid distention of the
operated stomach easily produces vomiting. This difficulty was over-
come by using a small catheter as a stomach tube. The type of sphinc-
ter action shown in tracing C was continued for 4 hours, at the end
of which time the stomach was completely free from the second meal.

EXPWNo. 27

Mf, 7" i" e a
ha fh iA ‘al vy A f fe hh / "A ra rs fe we » } ‘
4 |]

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i yy

Fig. 7. Experiment 27; February 19, 1920. Ether used throughout experiment.
Records begun immediately following operative procedures. Tambour myo-
graph and piston recorder used to obtain graph. Because of construction of the
myograph the contraction phase is recorded as the downstroke; relaxation the
upstroke, Note the regularity of contraction and the tone level changes.

This experiment seems of special interest because it demonstrates
that a foreign body in the pyloric canal through which the gastric
contents may pass does not alter the normal progress of gastric evacu-
ation. Further, it supplies information from both the experimental
and the radiographic standpoint which indicates that the sphincter is
rhythmical in its activities. The development of tone waves in this
case does not differ in character from those observed by other types
of recording apparatus through which the gastric contents may not
pass.

A third type of experiment was performed to check the graphs
obtained by means of the closed and open types of pylorographs. In
these experiments, three in number, a tambour myograph was attached
to the exterior of the sphincter, the animal being immersed in a tank
of warm saline solution. The type of rhythmic contractions and tone

RHYTHMICITY OF PYLORIC SPHINCTER ' 471

waves shown in figure 7 are common to the three experiments, and they
show no fundamental variation from tracings obtained with other
types of apparatus. The animal (no. 27) from which this figure was
obtained had been fed about. an hour before the experiment started.
Often a gurgling noise was audible as material was forced from the
antrum into the duodenum, these occurring during a wave of contrac-
tion of the pars pylorica.

The results obtained from the various types of experiments and
radiographic studies are similar to each other, therefore the movements
described for the pyloric sphincter may be considered as representative
of normal functioning of this organ.

DISCUSSION

The closure of the pyloric sphincter, according to the prevailing
theories, results because of: a, the presence of solid masses in the antrum
which mechanically excite the mticosa of this region; b, an insufficient
acidification of the gastric content; and c, the presence of acid chyme
in the duodenum. Following liquefaction and acidification of the
_ gastric contents these mechanical and chemical stimulants cease and
the sphincter relaxes to permit the ejection of chyme into the duodenum.

The theory of an “‘acid control of the pylorus” (Cannon) does not
account for the rapid discharge of water and solutions of neutral egg
white from the stomach, neither does it explain the rapid clearance of
the stomach in certain pathological conditions. In part the theory of
fluidity accounts for the rapid evacuation of the stomach following the
ingestion of fluid masses. However, this does not explain the processes
involved in the control of the sphincter. Granting that the two theories
do explain a certain number of facts relative to the control of the pylorus,
we still have to account for pyloric activity when functioning in the
absence of acid. That is, something more than fluidity and acid is
necessary to open the sphincter to permit the passage of material into
the duodenum. Granting that the sphincter is open, all theories agree
that peristaltic contractions in the stomach are directly responsible
for the discharge of gastric contents. Hence it may be assumed that
intragastric pressure and peristalsis bear some definite relation to the
activities of the sphincter. That posture and peristalsis do act in such
a manner as to facilitate the emptying of the stomach has been pretty
definitely shown.

4:72" HOMER WHEELON AND J. EARL THOMAS

Neilson and Lipsitz (5) have shown that posture to a great extent
determines the time of retention of water in the stomach. Individuals
lying on their right side retain less of a given amount of water at the
end of a stipulated time than individuals assuming other positions.
Cole (2), from a study of serial radiograms of the human stomach, has
shown that the activities of the sphincter bear a definite relationship
to the activities of the antrum in that the amount of contraction of the
‘sphincter is in proportion to the activity of the gastric waves. He has
‘also shown that during the active phase (contraction) of every gastric —
cycle the pyloric ring is open and a small portion of the gastric contents —
is propelled through its lumen into the reservoir cap, The terminal
- wave (peristaltic) which has meanwhile been advancing toward the
sphincter, upon attaining it, effects its closure. The recent article by
Luckhardt, Phillips and Carlson (4) clearly demonstrates that both
in man and dogs the pyloric sphincter opens for the ejection of chyme
with the arrival at the sphincter of powerful advancing rings of contrac-
tion and a general increase in the tone'of the musculature of the stomach
‘as a whole. Their observations demonstrate that a more definite
relation exists between the muscular activity and the opening of the
- sphincter than between the opening of the sphincter and the reaction
of the gastric contents. Ivy (3) made the suggestion prior to the appear- —
ance of the paper by Luckhardt, Phillips and Carlson, that the rhythmic
discharge of water from the dog’s stomach is such that it could very
ares correspond to peristaltic activity.

. Such ‘observations clearly demonstrate that the functions of the

dilasin sphincter are dependent, in part at least, upon gastric motility.
‘Our observations also tend to show that the sphincter because of its
rhythmic type of motility acts in such a manner as to supplement
gastric motility. Further observations to be reported later also show
that the rhythmic contractions of the sphincter bear a definite and
‘constant relation to the motility of the stomach (fig. 3).
a | SUMMARY AND CONCLUSION

1. A method is described for recording the motility of the pyloric
sphincter.

2. The pyloric sphincter of the dog. demenbetates rhythmic activity
‘or cycles of motility which occur at the rate of from 3 to 5 per minute. _

. 3. A single cycle of motility is characterized by a phase of contrac-
tion, relaxation and quiescence followed by a definite phase of inhibition
prior to a subsequent contraction.

RHYTHMICITY OF PYLORIC SPHINCTER . 473

4. The sphincter demonstrates tone changes, such changes being
gained or lost because of shortening or lengthening of the relaxation
phase of the rhythmic cycles.

5. The observations here reported, therefore, show that the pyloric
sphincter of the dog possesses the property of rhythmic contractility,
_ the degree of which is influenced because of changes in tonicity.

- BIBLIOGRAPHY |

(1) Cannon: This Journal, 1898, i, 359; The mechanical factors of digestion,
Longmans, Green and Co., New York, 1911; This Journal, 1907, xx, 283.

(2) Coxe: Journ. Amer. Med. Assoc., 1913, lxi, 762; This Journal, 1916, xlii, 618.

(3) Ivy: This Journal, 1918, xlvi, 420.

(4) LuckHarpT, PHILLIPS AND Cartson: This Journal, 1919, 1, 57

(5) Netuson anp Lirsitz: Journ. Amer. Med. Assoc., 1915, lxiv, 1052.

A DIFFERENCE BETWEEN THE MECHANISM OF
HYPERGLYCEMIA PRODUCTION BY ETHER
AND BY CHLOROFORM ,

ELLISON L. ROSS ann L. H. DAVIS

From the Department of Physiology and Pharmacology, Northwestern lake

Medical School

Received for publication August 21, 1920

It has already been reported that there are grounds for believing that
the hyperglycemia produced by ether anesthesia is due chiefly to the
action of ether to reduce the influence of the internal secretion of the
pancreas (1). It is considered that this reduction of the action of the
internal secretion of the pancreas is a reduction of the inhibitor influence
on glycolysis. Since the primary source of blood dextrose is liver glyco-
gen, any injury to the liver should affect the ease with which glycogen
is set free. Whether the injury to the liver cells should make the liber-

ation of dextrose easier or more difficult there is no way to foretell. It -

is well known that chloroform is capable of producing liver pathology.
Davis and Whipple (2) showed that chloroform anesthesia produced
injury to liver cells and this injury could be increased by fasting before
the administration of chloroform. It was thought worth while to com-
pare the hyperglycemia from ether and from chloroform with special
reference to the injury produced in the liver.

Experimental work. A group of five normal undieted dogs was given
ether for half an hour. Anesthesia was induced by inserting the ani-
- mal’s head into a cylinder into which air that had passed through ether
was forced. The animals were bled before anesthesia and after fifteen
minutes of anesthesia. The blood sugar was determined by Benedict’s
method (3). The next day the same procedure was repeated except
that the animals were kept under the anesthetic only fifteen minutes.
The results are given in tables 1 and 2.

A second group of dogs was given chloroform to the surgical anes-

thetic stage and kept at this degree of anesthesia for half an hour and -

the following day the response to fifteen minutes of ether anesthesia was

determined. The bleeding, analyses and administration of the anes-

thetic were carried out as before. The results are given in table 3.
474

se

EFFECT OF CHLOROFORM ON ETHER HYPERGLYCEMIA

Ether hyperglycemia of a group of normal dogs

TABLE 1

GLYCEMIA BEFORE

GLYCEMIA AFTER 15 MIN-

INCREASE

npsaees ETHER UTES ETHER ANESTHESIA
1 0.113 0.143 0.030
2 0.084 0.111 0.027
3 0.116 0.127 0.011
4 0.096 0.141 0.045
5 0.105 0.132 0.027
Average.... 0.1028 0.1308 0.028

Ether hyperglycemia of same group of dogs, day following half an hour ether

TABLE 2

ANIMAL

GLYCEMIA BEFORE

GLYCEMIA AFTER 15 MIN-

INCREASE

ETHER UTES ETHER ANESTHESIA
1 0.100 0.166 0.066
2 0.093 0.106 0.013
Bees: 0.117 0.140 0.023
4 0.097 0.129 0.032
5 0.085 0.144 0.049.
Average.......... 0.1004 0.1370 0.0366

TABLE 3 :
Ether hyperglycemia one day following half hour chloroform anesthesia

ANIMAL

GLYCEMIA BEFORE

GLYCEMIA AFTER 15 MIN-

INCREASE

ETHER UTES ETHER ANESTHESIA
6 0.095 0.106 0.011
7 0.092 0.116 0.024 -
8 0.099 0.120 0.021
9 0.097 0.136 0.039
10 0.097 0.106 0.009
Average. ......<%% 0.0960 0.1168 0.0208

TABLE 4
Ether hyperglycemia following half hour chloroform anesthesia after fast

GLYCEMIA BEFORE

GLYCEMIA AFTER 15 MIN-

ek OP ETHER UTES ETHER ANESTHESIA INCE RAGE
1 0.094 0.097 0.003
12 0.093 0.134 0.041
13 0.088 0.094 0.006
14 0.094 0.116 0.022
15 0.095 0.115 0.020
PO OTSOR: ieee: 0.0928 0.0184

0.1112

476. Lo ELLISON L. ROSS AND L. H. DAVIS

A third group of animals fasted two days. Then these dogs were
put through the.same treatment as the second group. The results are
given in table 4.

Discussion. When a drug i is administered asanghene the later reac-"
tions usually differ in degree from the first one. This may be due to
decreased or increased sensitiveness of nerves, glands or tissue, or
injury to cells. To determine whether or not ether had less power to —
produce hyperglycemia the second day than the first, the first two series
of tests were made.

According to tables 1 and 2, the average glycemia before ether the
first day was 0.1028 and the second day 0.1004 per cent. This small
_ difference is negligible. Half an hour of ether anesthesia did not change
the amount of blood sugar found the following day. This harmonizes
with the general impression that ether anesthesia causes very little if
any injury to the subject. The first day fifteen minutes of ether anes-
thesia brought the glycemia up to 0.1308 per cent and the following ©
day the’same procedure produced an average glycemia of 0.1370 per .
cent. The average increase the first day was 0.028 per cent and the
average increase the next day was 0.0366 per cent. There was appar-
ently an increased tendency for ether to liberate dextrose the day fol-
lowing half an hour of ether anesthesia. The reason for this increase
we do not attempt to give at this time. However, we are able to make
the important deduction that ether did not injure the mechanism in-
volved in the production:of ether hyperglycemia. If such an injury had
- occurred the reaction to ether the second day would have been less than
that of the first day and the normal glycemia before anesthesia the
second day would have been less than that of the first day. —

The effect of chloroform on the glycemia of the following day is shown
in tables 3 and 4. The average blood sugars before ether of the first
and second groups of animals on the day following the chloroform anes-
thesias were 0.096 and 0.0928 per cent respectively. These values com-
pared with those of normal dogs shown in tables 1 and 2—0.1028 and
0.1004 per cent—indicate a decided tendency of chloroform to par-
tially paralyze the mechanism of sugar mobilization. This phase of
the action of chloroform is of great importance. In view of the con-
clusion of Cannon that blood sugar is the most satisfactory source of
energy in emergency, it is of the greatest importance that the mobiliza-
tion of blood dextrose be not interfered with at a time such as that fol- —
lowing an operation when often every vital fie is strained to the
limit in order to sustain life.

EFFECT OF: CHLOROFORM ON ETHER HYPERGLYCEMIA 477

Fifteen minutes of ether anesthesia the day following a chloroform
anesthesia of half an hour produced an average glycemia of 0.1168 -per
cent for the group of non-fasting dogs and’a glycemia of'0:1112-per cent’
for the group of fasting animals. These glycemias comipared with
those of the group of normal dogs (0.1308 per cent) and of the group
which had an ether anesthesia of half an hour the preceding day (0.1370
per cent), show a decided inability of the animals given chloroform the
previous day to develop as great a hyperglycemia as normal animals. |

The increases in blood sugar due to fifteen minutes of ether anesthesia
of the groups of animals: which received a chloroform anesthesia the pre-
ceding day, were 0.0208 and 0.0184 per cent. The average increase due
to ether anesthesia of untreated dogs given in table 1 was 0.028 per cent
and the average increase of a group of 17 dogs, given in a previous pub-
lication (4), was 0.037 per cent. A comparison of these increases from
ether anesthesia obtained the day following a chloroform anesthesia
with the increases of normal dogs shows a considerable decrease in the
power of the animals to mobilize dextrose after chloroform anesthesia.

There were two groups of animals that were given ether the day fol-
lowing chloroform anesthesia of half an hour. These animals differed
in that the second group fasted two days before the tests were made.
The day following chloroform anesthesia the non-fasting animals had
an average glycemia of 0.0960 per cent and the fasted animals a glyce-
mia of 0.0928 per cent. Fifteen minutes of ether anesthesia of unfasted
dogs produced an average increase of blood dextrose of 0.0208 per cent,
making a glycemia of 0.1168 per cent. A similar ether anesthesia of the
fasting animals produced an increase of 0.0184 per cent making the av-
erage blood dextrose value 0.1112 per cent. A comparison of the normal
glycemias, the increases due to fifteen minutes of ether anesthesia, and
the glycemias after the ether anesthesia of the group of fasting animals
and of the group of non-fasting animals, shows greater values for blood
dextrose in every case for the non-fasting group. These differences are
small but constantly in favor of the one group.

SUMMARY AND CONCLUSIONS

A group of dogs was anesthetized with ether for half an hour. The
next day the dogs were anesthetized with the same drug for fifteen
minutes. The blood sugar changes were measured for the first fifteen
minutes of anesthesia both times.

478 ELLISON L. ROSS AND L. H. DAVIS

A second group of dogs was anesthetized with chloroform for half an
hour and the following day each was given fifteen minutes-of ether
anesthesia. The blood dextrose changes the second day were measured.

A third group of animals fasted two days and was then treated the
same as the second ‘group.

Half an hour of ether anesthesia did not alter the glycemia of the fol-
lowing day, and did not decrease the hyperglycemia resulting from
fifteen minutes of ether anesthesia. |

Half an hour of chloroform anesthesia produced on the following
day a glycemia lower than normal, an increase in blood dextrose due to
fifteen minutes of anesthesia less than normal, and a hyperglycemia
from fifteen minutes of ether anesthesia lower than normal.

A fast of two days preceding half an hour of chloroform anesthesia
produced on the following day a still lower glycemia and still less reac-
tion to fifteen minutes of ether anesthesia than occurred in non-fasting
dogs.

These results in conjunction with the conclusions of Davis and Whip-
ple (2) that the liver injury produced by chloroform is increased by a
fast preceding anesthesia leads us to the following conclusions:

1. Ether anesthesia does not produce any injury to the mechanism of
dextrose mobilization that can be detected the following day.

2. The injury to the liver cells produced by chloroform anesthesia
reduces the glycemia of the following day and injures the mechanism
of dextrose mobilization according to the degree of injury...

3. The hyperglycemia due to chloroform anesthesia is not due prima-
rily to the direct action of chloroform on the liver. Probably chloro-
form, like ether, produces hyperglycemia chiefly through its sor
action on the internal secretion of the pancreas.

BIBLIOGRAPHY

(1) Ross anv Davis: This Journal, 1920, liii, 391.

(2) Davis AND Wuippte: Arch. Int. Med., 1919, xxiii, 612.
(3) Benepict: Journ. Biol. Chem., 1918, xxxiv, 203.

(4) Ross: Journ. Pharm. Exper. Therap., 1919, xii, 377.

DIGESTIBILITY OF SOME HYDROGENATED OILS!

ARTHUR D. HOLMES ann HARRY J. DEUEL, Jr.
From the Office of Home Economics, U. S. Department of Agriculture?

Received for publication August 28, 1920

Until quite recent times table and culinary fats used in the United
States were obtained almost wholly from the animal kingdom—dairy
butter being the universal table fat and lard the principal culinary fat.
The constantly decreasing per capita supply of animal fats has caused
a very rapid increase in the use of vegetable oils for food, until olive,
cottonseed, peanut and corn oils are now more or less generally used not
only for salad but also for cooking purposes. Except for salad purposes,
in the past the housewife apparently preferred fats which were very
nearly, if not actually, solid at ordinary temperatures. To meet this
demand for solid fats, vegetable oils were hardened either by removing
a portion of their low melting constituents or by the addition of stearin
or a fat rich in stearin, and fats prepared by one or the other of these
methods came into quite general use under a variety of trade names.
These processes for hardening vegetable oils have now been largely
replaced by the hydrogenation process which is based on the discovery
that hydrogen may be added, under proper. conditions of temperature
and pressure, to the glycerides of the unsaturated fatty acids by means
of a catalytic agent, such as nickel in a finely divided state. When the
process is carefully controlled it is possible to prepare hydrogenated
oils having any desired melting point.

Since it is well known that some metals when taken into the alimen-
tary tract. under certain conditions are toxic, it has been very properly
questioned whether the ingestion of hydrogenated oils containing ap-
preciable amounts of a metallic catalyst might not be followed by harm-
ful physiological disturbances. Recent investigations (1) indicate that
properly prepared hydrogenated oils do not contain sufficient nickel to
produce toxic effects. The Federal Meat Inspection Division (2) shares

1 Published with permission of the Secretary of Agriculture.
* Prepared under the direction of C. F. Langworthy, Chief, Office of Home
Economics.

479

480 ARTHUR D. HOLMES AND HARRY J. DEUEL, JR.

this opinion and permits the sale for edible purposes of those hardened —
oils which do not contain over two parts of the catalyzer per “million
parts of oil—an amount about five times that ordinarily found in normal
hydrogenated oils produced in this country.

The literature contains relatively little information regarding the
digestibility of hydrogenated oils. Thoms and Muller (3) found that
a hardened oil melting below body temperature was more satisfactory
than one in which more complete saturation occurred and a harder fat —
obtained. Smith, Miller and Hawk (4) determined the relative digesti-
bility of lard (melting at 45°C.) and hydrogenated cottonseed oil (melt-
ing at 36°C.) and found the lard to be 94.7 per cent and the hardened
oil 93.4 per cent digested—a difference, which in the opinion of the
authors, is well within the limits of experimental error. Pekelharing
and Schut (5) found that on a diet which contained no fat other than
hydrogenated cottonseed oil, mice maintained a steady body increment.
They also determined the digestibility of some hydrogenated cottonseed
oils in feeding experiments on a dog. On the average the dog digested
about 90 per cent of the hydrogenated oils and increased its weight by
about one-third the original weight. From the results of the four
months’ experiment they found that the digestibility of the hydrogen-
ated oils was inversely proportional to their melting points, that mix-
tures of lard and hardened oils were more completely digested than the
hardened oils alone, that no physiological disturbances occurred, and
that the feces of the hydrogenated oil diets contained more fat and more
fatty acids than those resulting from the lard diet. Erlandsen, Frid-
ricia and Elgstrom (6) report studies of hardened whale oil in which the
digestibility varied from 91.6 per cent to 94.9 per cent for butter and
whale oil; the difference in digestibility of the two fats did not exceed
0.9 per cent.

The present paper reports a series of digestion experiments with. hy-
drogenated cottonseed, peanut and corn oils in which the entire sample
was subjected to the hy drokenetion process. Material is also available
for reporting the results of experiments on the digestibility of blended
hydrogenated oils, in which some hydrogenated oil, hardened to a high
melting point, was mixed with enough untreated oil to give a fat of the
desired hardness. This investigation is a continuation of an extended
_series of digestion experiments which has been conducted by this office
with about fifty more or less common animal and vegetable fats. The
general conclusions from these studies are that edible fats are highly
digestible, that they do not unfavorably influence the digestibility of

DIGESTIBILITY OF SOME HYDROGENATED OILS 481

other constituents of the diet, and that when eaten in normal amounts
they do not cause any pronounced laxative effects.

Experimental. In these experiments the hydrogenated oils under
consideration were included in the diet by being incorporated in the
usual specially prepared cornstarch blancmange (7) or pudding. The
basal ration, which was nearly fat-free, consisted of wheat biscuit, fruit,
sugar and clear tea or coffee. The subjects of the experiments reported
below were young men, apparently in normal health, from 20 to 40
years old, students in local universities, who as a result of their previous
experience in this. type of studies were familiar with the experimental
procedure and who were thoroughly trustworthy. The experimental
methods for separation and collection of feces and analysis of foods and
feces outlined in earlier papers (8) were followed in the experiments
below.

The presence of ether-soluble metabolic products in the feces has
been taken into account and a suitable correction introduced in calcu-
lating results. Since the feces of a given day do not necessarily repre-
sent the residue of the food for the preceding day, it has seemed best
in these experiments as in all the preceding ones to identify the feces of
the experimental period with charcoal or carmine markers and to retain
all the feces belonging to the period for analysis. Since the experimental
procedure has been uniform throughout the tests on the animal and
vegetable fats, the results obtained in these experiments are directly
comparable with each other and with the results of our earlier studies.

Results of studies of the digestibility of common fats and oils indi-
cate that their digestibility varies inversely with their melting points in
the case of those melting above body temperature. In order to study
the relationship between the digestibility and melting points of hydro-
genated oils, those considered here were so chosen that some melted
above and some below the temperature of the body (387°C.).

The majority of the samples of hydrogenated oil were hardened in
the laboratory of Carleton Ellis by one of us (H. J. D.). J. R. Kuhn,
of that laboratory, assisted in their preparation.

The melting points of the hydrogenated cottonseed oil were 35°C.,
38.6°C. and 46°C.; the melting points of hydrogenated peanut oil were
37°C., 39°C., 48°C., 50°C. and 52.4°C.; and the melting points of hydro-
genated corn oil were 33°C., 43°C. and 50°C. The iodine numbers of
the samples are given in table 4.

All the hydrogenated oils included in this study were of a white color,
solid or practically so at ordinary room temperature, and without any

482 ARTHUR D. HOEMES AND HARRY J. DEUEL, JR.

characteristic odor or flavor. When melted they were of a straw yellow
color resembling melted tallow. They were very homogeneous and if
heated sufficiently they boiled without any sputtering and did not
smoke until a relatively high temperature was reached. On cooling, in
some instances, different portions of the resulting mass differed slightly
in physical appearance, quite likely because of a partial apa of
the softer and harder constituents of the hydrogenated oils.

The experiments made with the hydrogenated corn, cottonseed and
peanut oils were carried out under conditions essentially the same as
those of experiments with the same kinds of oil untreated. As earlier
reports show, those oils had coefficients. of digestibility as follows:

corn oil (9), 96.9 per cent; cottonseed oil aie 97. 8 per cent and peanut | |

oil (11), 98.3 per cent.

Digestibility of hydrogenated corn oil. Fifteen digestion experiments
were conducted with hydrogenated corn oil, five each with hardened
fats having a melting point of 33°C., 43°C. and 50°C. These fats were
prepared by one of the authors (H. J. D.) at a large commercial research
laboratory and are believed to be typical of commercial hardened corn
oil.

The same group of subjects assisted in each series of experiments with
hydrogenated corn oils and the usual standardized experimental condi-
tions were employed.

This report of the individual experiments with hyde corn oil
and other oils included in this investigation is somewhat condensed,
but the experimental data in full are on file in the Office of Home Eco-
nomics, States Relations Service, U. 8. Department of Agriculture.

The data which were obtained from the study of hydrognaeies corn
oils are summarized in table 1.

The average amount of hydrogenated corn oil eaten per man per day
was 78 grams for the fat melting at 33°C.; 74 grams for the 43°C. fat,
and 44 grams for the 50°C. fat. The digestibility of the hardened corn
oils studied was for 33°C. oil, 94.7 per cent and for 43°C. oil, 95.4 per
cent; thus from the standpoint of practical dietetics there was no mate-
rial difference in the digestibility of these oils. The digestibility of hy-
drogenated corn oil melting at 50°C. was 88.5 per cent which is identical
with that of mutton fat (12) (88 per cent) melting at 50°C. The coef-
ficients of digestibility obtained in these experiments are somewhat
lower than 96.8 per cent (13) reported in an earlier paper for the digesti-
bility of commercial, edible corn oil.

DIGESTIBILITY OF SOME HYDROGENATED OILS 483

The experimental diet as a whole was well utilized, the carbohydrates
being very completely absorbed, which would indicate that hydrogen-
ated corn oils having melting points a very little higher than body tem-
perature did not have any unfavorable effect on the digestibility of the
other constituents of the diet.

TABLE 1
Summary of digestion experiments with hydrogenated corn oil in a simple mized diet
pee DIGESTIBILITY OF ENTIRE RATION ie ce
oaccen HYDROGEN- SUBJECT | ; HYDROGEN-
wei Peptein | Rat? | gee jaan, come
i 5 per cent per cent per cent per cent
1022 . 33 WwW. V. D 75.3 90.8 97.8 93.5
1023 33 mM. -G. 71.0 93.6 96.8 96.7
1024 33 K. L. M. 69.6 91.5 97.4 94.5
1025 33 G. 8S. M V2.2 87.6 98.1 90.7
1026 33 J.C. W 72.0 95.0 96.9 97.9
NICSE Sd se ea 72.0 91.7 97.4 94.7
1037 42 W. V. D 83.0 92.5 98.3 95.0
1038 42 H. :'G. 81.0 93.8 . 97.1 96.8
1039 42 E. L. M. 70.3 89.4 96.6 93.6
1040 42 G. 8. M 70.5 92.5 ‘97.3 96.9
1041 42 J.C. W 76.6 90.7 95.9 94.6
DME. See eek Ski. ec Gy 76.3 91.8 97.0 95.4
1042 50 W. V.D 65.2 84.8 97.8 90.2
1043 50 H. L. G. 65.9 81.6 95.9 87.9
1044 50 E. L. M. 86.2 91.2 98.3 94.0°
1045 50 G. 8. M 60.7 78.9 97.5 85.6
1046 . 50 J.C. W 69.9 79.4 ae 84.9
Averages. icles. dey bs eeiiieus 69.6 83.2 97.3 88.5

Digestibility of hydrogenated cottonseed oils. The experiments made
with hydrogenated cottonseed oil were conducted under experimental
conditions identical with those employed in the study of the digestibility
of cottonseed oil which was found to be 97.8 per cent digested (14).
The hydrogenated oils which have received attention in this investiga-
tion were not all prepared from the same lot of cottonseed oil; one lot
melting at 35°C. (used in experiment 512) was a well-known commercial
product which was purchased in the open market; the fat melting at
38.6°C. was specially prepared for our studies in the research laboratories

484 ARTHUR D. HOLMES AND HARRY J. DEUEL, JR.

of a concern manufacturing edible hydrogenated oils; the fats melting
at 35°C. (used in experiments 1027-1031) and 46°C. were prepared by
one of us in a large consulting laboratory. While these products were
not all of commercial origin it is believed that they are, nevertheless,
typical of the commercial article.

Table 2 summarizes the data from the experiments with hydrogen-
ated cottonseed oil.

TABLE 2
Summary of digestion experiments with hydrogenated cottonseed oil in a simple
mixed diet
MELTING DIGESTIBILITY OF ENTIRE RATION DIGESTIBIL-
POINT OF ITY OF
ela aaa OU
ala eso rotein at hydrate Baba
°@ per cent per cent per cent per cent
512 35 Pk. 67.0 92.6 96.6 96.2
1027 35 WwW. V. D 84.8 96.9 99.0 98.3
1028 35 H. L. G 61:53 94.4 95.2 98.6
1029 35 E. L. M 70.2 94.4 97.9 97.0
1031 35 aA. We: 63.6 89.7 95.7 93.9
AyGPae, ee tates daw te 69.2 93.6 96.9 — 96.8
459 38.6 eL. § 69.5 92.7 97.3 95.5
1052 46 W. V. D. 75.9 93.8 98.4 96.3
1053 46 H. L. G 69.5 93 .2 96.7 96.4
1054 46 E. L. M 68.7 91.0 97.7 91.9
AVOPIMO 5 Er ad cig Phys be Ud each 71.7 92.7 97 .6 94.9

The average digestibility of the entire ration in the above experi-
ments indicates that this diet. was quite well digested. The daily con-
sumption of hydrogenated oil in these experiments was on the average
about 84 grams. In the last group of experiments with hydrogenated —
cottonseed oil melting at 46°C., 89 grams of the fat were eaten daily
without causing any physiological disturbances, which would indicate
that the limit of tolerance for this fat was in excess of 89 grams. The
coefficients of digestibility, 96.8 per cent for hydrogenated oil having
a melting point of 35°C., 95.5 per cent for hydrogenated oil having a
melting point of 38.6°C. and 94.9 per cent for hydrogenated oil having a
melting point of 46°C., indicate that the hydrogenated cottonseed oils
having these melting points are well utilized by the body.

DIGESTIBILITY OF SOME HYDROGENATED OILS 485

Digestibility of hydrogenated peanut oil. Twenty digestion experi-
ments were made with hydrogenated peanut oils having melting points
of 37°C., 39°C., 48°C., 50°C. and 52.4°C. under the usual uniform
experimental conditions. The peanut oils melting at 39°C. and 52.4°C.
were prepared for us through the courtesy of the chief chemist of a
large manufacturing concern. The oils melting at 37°C., 48°C. and
50°C. were hydrogenated by one of us at a commercial fat and oil
research laboratory. The results of the experiments with hydrogenated
peanut oil are given in table 3.:

The average amounts of hydrogenated peanut oil eaten daily in the
above groups of experiments were: 37°C. fat, 76 grams; 39°C. fat, 78
grams; 43°C. fat, 91 grams; 50°C. fat, 59 grams; and 52.4°C. fat, 62
grams. No instance of intestinal disturbance resulted which indicates
that the above quantities of these hardened oils are well tolerated by the
average adult.

A comparison of the melting points and digestibility of these har-
dened oils is of interest. An increase of 2 degrees in the melting point
(from 37°C. to 39°C.) was accompanied by a 2 per cent decrease in diges-
tibility, an increase of 4 degrees from 39°C. to 48°C. caused no significant
change in digestibility, an increase of 7 degrees from 43°C. to 50°C.
caused a decrease of 4.5 per cent in digestibility, while an increase of
2.4 degrees from 50°C. to 52.4°C. caused a decrease of 13 per cent in
digestibility. The coefficient of digestibility of 79 per cent for peanut
oil melting at 52.4°C. is the lowest value obtained for any fat of
this series; the digestibility (15) of mutton fat (melting point 50°C.)
being 88 per cent and that of oleo stearin (16), 80 per cent. Since the
melting point, 52.4°C., of this hardened oil is considerably higher than
37°C., the temperature of the human body, it is probable that in the
process of digestion saponification takes place only on the exterior of the
particles of hardened oil which decrease in size as the process of digestion
continues. If surface area be thus a factor, then the rate of digestion
and possibly the extent of digestion of a hydrogenated fat having a high
melting point is governed to some extent by the size of the particles of
hydrogenated oil ingested. Additional experiments are necessary to
supply conclusive evidence on this point.

The hydrogenated oil melting at 37°C. was as completely digested
(98.1 per cent) as the untreated peanut oil which was found to be 98.3
per cent absorbed (17) by the body. As a group the hydrogenated
peanut oils were well digested and well tolerated by the subjects of this
investigation. .

a

486 ARTHUR D. HOLMES AND HARRY J. DEUEL, JR.
TABLE 3 ct
Summary of digestion experiments with hydrogenated peanut oil in a simple
mixed diet -
ee ee DIGESTIBILITY OF ENTIRE RATION Oe ae
UM BER | HYDROGEN- SUBJECT 15P HYDROGEN- |
i <auene OIL Protein Fat hydrate cieaten OIL
5; per cent per cent per cent per cent
1012 37 W. V. D. 74.7 95.1 97.5 97.6
1013 37 H. L, G. 78.3 96.9 97.5 99.1
1014 37 E. L. M. 68.3 94.3 97.4 97.4
1015 37 G. S. M. 57.7 94.0 96.3 97.9
1016 Tf J. C. W. 66.7 94.9 95.8 98.6
Averame ai. 65 ieGe bibs 34s cares 69.1 95.0 96.9 98.1
464 39 H. R. G. 74.0 95.4. 98.1 97.8
466 39 | 1 ge 81.3 94.2 97.8 96.3
467 39 Ro: B: 66.7 90.2 96.8 93.7
AVGREBO] Le yee spc twins late 74.0 93.3 97.6 95.9
1032 43 WwW. V.D 87.0 95.9 98.3 97.6
1033 43 H. L. G 71:0 93.7 95.0 97.5
1034 43 EK. L. M 74.6 91.1 97.2 94.1
1035 43 G. 5S. M 51.6 90.1 96.8 94.7
1036 43 J.C. W 84.6 96.5 96.9 98.6
Avebawho.. Gil: bonteila abla. 73.8 93.5 96.8 96.5
1057 50 W. V. D. 70.0 87.0 98.2 90.6
1058 50 H. L. G. 65.4 88.5 96.1 93.1
1059 50 E. L. M. 69.9 90.3 — 97.8 93.6
1060 50 G. 8. M. 69.0 86.6 98.1 90.7
VERE he oa ee OE oe Weee 68.6 88.1 97.6 92.0
472 52.4 D. D. G. 65.0 65.8 96.4 71.6
73 52.4 H. &..G. 61.0 7B. 96.3 79.5
475 52.4 P. ih: 41.7 81.8 98.9 85.9
Avereeer wi BUA) 55.9 73.8 97 .2 79.0
SUMMARY

Three vegetable oils, cottonseed, peanut and corn, have been par-
tially hydrogenated to obtain hardened oils having different melting
points and their digestibility studied. The results are summarized in

table 4.

DIGESTIBILITY OF SOME HYDROGENATED .OILS 487 |

With the exception of peanut oil melting at 52.4°C. and the corn oil
melting at 50°C. all of the hydrogenated oils were 92.0 per cent or more
digested. They did not cause any observed digestive disturbances nor
did they decrease the digestibility of the experimental diet as a whole.
In general, the results showed uniformly that as the melting point of the
oil was increased the coefficient of digestibility was decreased. The

TABLE 4
Summary of digestion experiments with hydrogenated vegetable, oils in a simple
diet
whe Paraben DIGESTIBILITY OF ENTIRE i ocak yl
q< RATION
Tape vecEmasie | Qexuten| youpen | axenic ere
tage mmm | protsin | Tet) | ee ees
Ci per cent per cent per cent per cent
tl 385 89.6 5 69.2 93.6 96.9 96.8
Cottonseed...;| 38.6 my 1 69.5 92.7 97.3 95.5
fe 72.8 3 71.7 92.7 97 .6 94.9
[87 81.3 5 69.1 95.0 96.9 | 98.1
39 ae we 74.0 93.3 97.6 95.9
Peanut.......4| 48 78.8 5 73.8 | 93.5 | 96.8 | 96.5
50 58.5 4 | 68.6 88.1 97.6 92.0
(| 52.4 3 55.9 73.8 97.2 79.0
33 89.0 5 72.0 91.7 97.4 94.7
OMR eT. 48 74.9 5 76.3 91.8 97.0 95.4
50 55.4 5 69.6. 83.2 97.3 88.5

coefficient of digestibility decreased at a much faster rate in the case of
fats with melting points. above 46°. The number of experiments con-
_ ducted with the majority of the hydrogenated oils under consideration,
it is believed, is sufficient to permit of fairly definite conclusions regard-
ing the digestibility of each of the oils in question. 5

The results of these experiments indicate that these hydrogenated
oils are as well utilized as natural fats of corresponding melting points.

BIBLIOGRAPHY

(1) Sursmann: Arch. Hyg., 1915, Ixxxiv, 121.
LACKEY AND SAYRE: Journ. Amer. Pharm. Assoc., 1917, vi, 348.
LEHMANN: Chem. Ztg., 1914, xxxviii, 798.

(2) Kerr: Personal communication.

(3) THoms anp Muuuer: Arch. Hyg., 1915, Ixxxiv, 54.

488 ARTHUR D. HOLMES AND HARRY J. DEUEL, JR.

(4) Smiru, MILLER AND Hawk: Journ. Biol. Chem., 1915, xxiii, 505.

(5) PEKELHARING AND Scout: Pharm. Weeklad., 1916, liii, 769.

(6) ERLANDSEN, FRIDRICIA AND Exestrom: Tidskr. Kem., 1918, xv, 109.

(7) Lancwortuy AND Hotmgs: U.S. Dept. Agric. Bull. 310, 1915.

(8) LanewortTHy AND Ho.tmgs: U. S. Dept. Agric. Bull. 310, 1915.
LANGWoORTHY AND.Hotmes: U. 8S. Dept. Agric. Bull. 505, 1917.
LanewortHy AND Homes: U. S. Dept. Agric. Bull. 507, 1917.
Houmes: U.S. Dept. Agric. Bull. 613, 1919.

Homes: U.S. Dept. Agric. Bull. 630, 1918.

Hoimes: U.S. Dept. Agric. Bull. 687, 1918.

Homes: U. 8. Dept. Agric. Bull. 781, 1919.

DEVEL AND Hotmgs: Journ. Biol. Chem., 1920, xli, 227.

(9) Hotmss: U. 8. Dept. Agric. Bull. 687, 1918, 20.

(10) Lanewortuy AND Hotes: U.S. Dept. Agric. Bull. 505, 1917, 18.
(11) LanewortHy AND Hotes: Ibid.

(12) Lanewortuy AND Houmes: U. S. Dept. Agric. Bull. 310, 1915, 21.
(13) Hotmss: U.S. Dept. Agric. Bull. 687, 1918, 20.

(14) LanawortHy AND Homes: U. S. Dept. Agric. Bull. 505, 1917, 18.
(15) LANGwortuHy AND Hoimgs: U.S. Dept. Agric. Bull. 310, 1915, 21.
(16) Hotmss: U.S. Dept. Agric. Bull. 613, 1919, 16.

(17) LanawortHy AnD Hormes: U. 8. Dept. Agric. Bull. 505, 1917, 18.

INDEX TO VOLUME LIV

CACIA, effect of intravenous injec-
tion of, on blood, 1.

Acid and alkali tolerance in planarians,
138.

_ Adrenalin, effect of, on venous blood
pressure, 96.

——, ——- —— subcutaneous injection
of, 248.

Age, influence of extremes of, on pro-
duction of diabetes, 439.

Alkali tolerance in planarians, acid
and, 138.

Alkaline reserve of blood of insane, 147.

Auten, F. M. Experimental studies
in diabetes. Series II. The inter-
nal pancreatic function in relation
to body mass and metabolism:

5. The influence of fever and in-
toxication, 375.

7. The influence of cold, 425.

8. The influence of extremes of age
upon the production of diabetes, 439.

9. The influence of pregnancy
upon experimental diabetes, 451.

Atropin, effects of, on volume-flow of
blood, 204.

Aus, J. C. Studies in experimental
traumatic shock. I. The basal me-
tabolism, 388.

— and T. D. CunninaHAm. Studies
in experimental traumatic shock.
II. The oxygen content of the blood,
408.

—— and H. Wu. Studies in experi-
mental traumatic shock. III.
Chemical changes in the blood, 416.

BARR, D. P., and J. P. Permrs, Jr.
Studies of the respiratory mech-
anism in cardiac dyspnea. III. The
effective ventilation in cardiac dysp-

- nea, 345.
——,. See Prerprs and Barr, 307, 335.

Blood analyses following acacia-glu-
cose injection, 1.

——, chemical changes in, in experi-
mental traumatic shock, 416.

—— of insane, alkaline reserve of, 147.

——, oxygen content of, in experi-
mental traumatic shock, 408.

—— pressure, arterial, relation of
cerebral hemispheres and thalamus
to, 355.

—— ——.,, venous, effect of adrenalin
on, 96.

——, sugar of, under chloroform and
ether anesthesia, 474.

—., volume-flow of, 166, 185, 204.

Brain stem, studies on, 355.

APILLARIES and venules, func-

tional activity of, 30.

Carbon dioxide, low alveolar, of car-
diac dyspnea, 307.

Cardiac dyspnea, respiratory mech-
anism in, 307, 335, 345.

Carson, A. J., and A. B. Dock icepr.
Studies on a visceral sensory nerv-
ous system:

I. Lung automatism and lung re-
flexes in the frog (R. pipiens and R.
catesbiana), 55.

III. Lung automatism and lung
reflexes in reptilia (turtles: Chry-
semys elegans and Malacoclemmys
lesueurii, Snake: Eutenia elegans),
261.

See LuckHARDT and CARLSON,
122.

Cerebral hemispheres, relation of, to
arterial blood pressure, 355.

Cold, influence of, on diabetes, 425.

Connet, H. The effect of adrenalin
on venous blood pressure, 96.

489

THE AMERICAN JOURNAL OF PHYSIOLOGY, VOL. LIV, NO. 3

490

CrvuicksHank, E. W. H. The distri-
bution and quantitative action of
the vagi as determined by the elec-
trical changes arising in the heart
upon vagus stimulation, 217.

CunnincHamM, T. D. See Avs and
CUNNINGHAM, 408.

DAVIS, L. H. See Ross and Davis,
474,
DrevEh; Hii J. “IR.
Devet, 479. |

See HonMeEs and

Diabetes, experimental studies in, 375,.

- 382, 425, 430, 451.
Digestibility of some hydrogenated
oils, 479.

RLANGER, J. See Wuitr and
ERLANGER, 1.

EVER and intoxication, influence
of, on diabetes, 375.

({ASTRIC hunger contractions, 153.

GESELL, R. Studies on the submaxil-
lary gland:

VI. On the dependence of tissue
activity upon volume-flow of blood
and on the mechanism controlling
the volume-flow of blood, 166.

VII. On the effects of increased
salivary pressure on the electrical
deflections, the volume-flow of blood
and the secretion of the submaxil-
lary gland of the dog, 185.

VIII. On the effects of atropin
upon volume-flow of blood, electri-
cal deflections and oxidations of the
submaxillary gland, 204.

Glucose, effect of intravenous injec-
tion of, on blood, 1.

HEART, vagus action on, 217.

Homes, A. D., and H, J. Devet, Jr.
' Digestibility of some hydrogenated
oils, 479.

INDEX

Hooker, D. R. The functional ac-
tivity of the capillaries and venules,
30. >

Hyperglycemia, production of, by
chloroform and by ether, 474.

LENHART, C. H. See Marine and
LENHART, 248.

Lucxnuarpt, A. B., and A. J. CARLSON,
Studies on the vibecwal sensory nerv-
ous system. II. Lung automatism
and lung reflexes in the salamanders
(necturus, axolotl), 122.

See Caruson and LucKHARDT,
55, 261.

Lung automatism and lung reflexes in
the frog, 55.

— —— —— —— —- in reptilia, 261.

— —~ — — —— in the sala-
manders, 122.

—— volume, effective, in cardiac dysp-
nea, 335.

MacARTHOUR, J. W. Changes in

acid and alkali tolerance with

age in planarians. With a note on
catalase content, 138.

Marine, D., and C. H. LEenuart.
The influence of glands with internal
secretions on the respiratory ex-
change. I. Effect of the subeutane-
ous injection of adrenalin on normal
and thyroidectomized rabbits, 248.

Metabolism, basal, in experimental
traumatic shock, 388.

ERVOUS system, visceral sensory,
55, 122, 261.

OILS, hydrogenated, digestibility of,
479.

PANCREATIC function, internal, in
relation to body massand metabo-
lism, 375, 382, 425, 439, 451.
PatrEeRSON, T. L. Gastric tonus of
the empty stomach of the frog.
Comparative studies IV, 153.

ree
a

INDEX

Peters, J. P., Jr., and D. P. Barr.
Studies of the respiratory mechanism
in cardiac dyspnea:

I. The low alveolar carbon dioxide
of cardiac dyspnea, 307.

II. A note on the effective lung
volume in cardiac dyspnea, 335.

See Barr and Peters, 345.

Planarians, acid and alkali tolerance in,
138.

Pregnancy, influence of, on experi-
mental diabetes, 451.

Pritcuert, I. W. See WisHarT and
PRITCHETT, 382,

Pyloric sphincter, rhythmicity of, 460.

—

RESPIRATORY exchange, influence
of glands with internal secretions
on, 248.

— mechanism in cardiac dyspnea,
307, 335, 345.

Rogers, F. T. Studies on the brain
stem. IV. On the relation of the
cerebral hemispheres and thalamus
to arterial blood pressure, 355.

_ Ross, E. L., and L. H. Davis. A dif-

ference between the mechanism of

hyperglycemia production by ether

and by chloroform, 474.

SALIVARY pressure, increased, ef-
fects of, on volume-flow of blood,
185.
Shock, studies in experimental trau-
matic, 388, 408, 416.
Submaxillary gland, studies on, 166,
185, 204.

491

Suitsu, N. Studies on the alkaline
reserve of the blood of the insane,
147,

(THALAMUS, relation of, to arterial
blood pressure, 355.
Tuomas, J. E. See Wuereton and
Tuomas, 460.

VAGUS action on heart, 217.

Venous blood pressure, effect of ad-
renalin on, 96. :

Ventilation, effective, in cardiac dysp-
nea, 345.

Venules, capillaries and, functional
activity of, 30.

Visceral sensory nervous system, 55,
122, 261.

HEELON, H., and J. E. THomas.
Rhythmicity of the pyloric
sphincter, 460.
Wuite, H. L., and J. Eruancer. The
effect on the composition of the blood
of maintaining an increased blood
volume by the intravenous injection
of a hypertonic solution of gum
acacia and glucose in normal, as-
phyxiated and shocked dogs, 1.
WisHart, M. B., and I. W. PritcHert.
Experimental studies in diabetes.
Series II. The internal pancreatic
function in relation to body mass
- and metabolism. 6. Gas_ bacillus
infections in diabetic dogs, 382.
Wu, H. See Avs and Wu, 416.

QP American Journel of Physiology

- 1920

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