The cytological characters of the areas of Langerhans

Property of
tepartment of

HOL9CY lIBRAtf

LIBRAE

E CYTOLOGICAL CHARACTERS OF THE AR1
OF LANGERHANS

BY

M. A. LANE

From. Hull Laboratory of Anatomy, University of Chicago
WITH 1 PLATE

Reprinted from THB AMBBICAN JOUBNAL OF ANATOMY, Vol. VII, No. 3, pp. 409.423

NOVBMBEB 10, 1907

THE CYTOLOGICAL CHARACTERS'' ME ARP4S OF

BY

M. A. LANE.

.From .HuH Laboratory of Anatomy, University of Chicago.
WITH 1 PLATE.

In the course of a comparative study of the pancreas, begun in the
autumn of 1905, I was struck with a peculiar reaction in certain cells of
the Islets of Langerhans in the pancreas of the guinea pig — one of the
first animals used in the study. This reaction, — to be described presently,
— indicated the existence in the islets of two types of cells, chemically
and morphologically different from each other. A part of the ensuing
investigation is the subject of the present paper, which is to be followed
by a further publication dealing in detail with a comparative study of the
islets which I have carried on side by side with that of the islets in the
guinea pig.

The principal difficulty thus far in dealing with the Islets of Langer-
hans has been the want of a definite method by which to distinguish the
cells of the islets from the cells of the pancreas itself ; for although there
is an apparently constant content of islet tissue in the pancreas, and
although the areas of islet tissue, in sectioned pancreas, stand out in sharp
contrast with the tubules of the pancreas, the physiological distinctness
of the one kind of tissue from the other is the very question upon which
histologists and pathologists have most disagreed. Pancreas cells ex-
hausted by stimulation with alkaloids, and thus thoroughly discharged
of their secretion products, have thus far been indistinguishable — so far
as positive evidence goes — from cells of the islets; so that it has been
impossible to say that exhausted cells which are indisputably pancreas
cells are not essentially the same as islet cells; and, on the other hand,
that cells which are indisputably islet cells are not in reality exhausted
cells of the pancreas. To establish a method of differentiation between
these two orders of cells was a purpose which thrust itself forward very
early in the work, as the establishment of such a method would go far
toward testing the claims of the two leading theories respecting the
meaning of the islets.

AMERICAN JOURNAL OP ANATOMY. — VOL. VII.
32

737105

410 Cytology of the Areas of Langerhans

The adherents of one of these theories have consistently held that the
islets produce a substance which, in one or another way, controls carbo-
hydrate metabolism. This view, so carefully considered and so capably
studied "£f.'0,pie ("J. <£): iiscs; a« particular significance when looked at in
the li^ht of my own. experiments on the chemism of the islets, especially
as reJS»r8S/tlie: J)*reeJjptaJ)iJjty <?f the substances produced by the two
types of cells mentione*d*at)6ve. "What may be called the " sugar function "
of the islets broadly suggests the outright physiological independence of
the islets, and sharply marks off this view from that of the other party,
the adherents of which have long urged the probability that the islets
are merely exhausted acini which, as such, have no active function what-
soever, but are, so to speak, in a state of rest, or obscuration, and, at the
end of the cycle, return to the active state as typical pancreatic acini.
These being the two main interpretations of the islets, a demonstration
that the cells of the islets have a chemical value of their own (and are
not, as a matter of fact, merely exhausted -pancreas cells, but cells which,
whatever may have been their former state, have, as islet cells, a positive
function) would seem to be indirectly confirmatory of the sugar theory,
or confirmatory at least of the broader notion that the islets have an
independent physiological activity of their own. Such confirmatory
evidence, I believe, will be found in the various chemical tests described
below.

* A few words of history, bearing particularly on these considerations, are
necessary here. For a larger historical review the reader is referred to
Oppel (13) and to Sauerbeck (19). The latter has an ample review of the
pathological as well as of the anatomical literature of the islets. *

The structures called the Islets of Langerhans were discovered by
Langerhans (10), who first called attention to them in 1869. The same
year (subsequently to Langerhans' s announcement) the name ' Les Hots
de Langerhans' was applied to them by Laguesse. Kiihne and Lea
afterwards gave them the name of " intertubular cell clumps." They
have been called secondary cell groups (by Harris and Gow), points
folliculaires (by Eenaut), and Islands of Langerhans (by American
anatomists).

The history of the islets from the date of their discovery until 1886
is chiefly interesting for the controversies it contains, and for the opinions
hazarded as to the nature and function of the structures. Langerhans
himself believed them to be the end-apparatus of nerve fibers. Eenaut
(17) described them in a very general way, and was unfortunate in being
misquoted by some earlier writer who, since Kenaut's announcement
in 1879, has been extensively followed throughout the whole of the

M. A. Lane 411

literature — a fact to which Sauerbeck also calls attention. Renaut has
been represented as saying that the islets were lymph structures. This
is not so. He did not say they were lymphoid tissue — a misconception
arising from the title of his paper. He simply made a note of their
existence, at the same time remarking that they had not been described
before. His only reference to lymph tissue in this paragraph of his
paper is to the effect that the islets (called by him points folliculaires)
were of the size of a lymphatic follicle.

Other writers hazarded other notions without, however, coming to any
satisfactory conclusion. The first definite step in that direction was taken
by Lewaschew (11) who, after considerable experiment with mammals,
suggested that the islets were temporarily exhausted acini which, after a
period of rest, resumed the acinous form. This theory would imply a
continuous transformation of acini into islets, together with a dis-
appearance of the lumen of the acinus ; and, again, a continuous trans-
formation of islets into acini, with an accompanying rebuilding of the
lumen, together with the entire complex of changes in the form of the
cell, in the nucleus and its content, in the arrangement of the glomerulus
of the islet capillary system, and in whatever other changes that might be
necessary in this peculiar process.

Lewaschew's theory further implies that these transformations are
continually going on in the entire substance of the pancreas, and he
urges, in point of probability that the islet cells are in continuity with the
cells of the acini. Eennie (18) has studied peculiar structures in fishes
which he identifies with the islets of Langerhans, although these struc-
tures lie remote from the pancreas in the abdominal cavity. Generaliza-
tions, however, concerning the islets in other animals based upon the
existence of these isolated structures in fishes, await the results of Eennie's
experimental work. Lewaschew's description of transitional cells, inter-
mediate between typical islet and the typical acinus cells is very obscure,
and the obscurity is only deepened by the uncolored drawings with which
his paper is illustrated. Lewaschew's views, however, have been widely
accepted and still have a considerable following. Dale (2) urges them
as probable from his experiments on the toad by stimulation with " sec-
retin/' although the embryological studies of Helly (6), Opie (15), and
Pearce (16) seen to point the other way.

Laguesse (7) investigated the islets of Langerhans in vipers from
the histological point of view, and (8) the islets in the sheep from the
histo°:enetic point of view. His work, in these respects, has brought to
the study of these structures much of the most interesting matter thus
far published. Laguesse did not distinguish two types of cells, but he (as

412 Cytology of the Areas of Langerhans

well as others) observed the fact that the cells of the islets contained
granules which could not be considered as artifacts, but were properly
to be regarded as products of the metabolism of the cells themselves. He
argues that the granules are not artifacts because

. (a) The cells are literally crowded with brilliantly stained (safranin
or gentian violet) granules.

(b) In the living structure the granules are also present when examined
in serum.

(c) They are analogous (1) in their arrangement, (2) in their refrac-
tion, (3) in their browTn coloration with osmic acid, and (4) in their vivid
red coloration with safranin, to zymogen granules, and

(d) They are soluble in acetic acid.

Considering these facts in connection with the results of his histo
genetic study of the islets in the pancreas of the sheep, he concludes that
the islet and the acinous cells are transformable one into the other, as
he gathers, also, from his studies of the embryo sheep alone. He believes
that the islets normally furnish an internal secretion to the pancreas, but
have the property of alternation from internal to external secretion, the
former predominating. In the viper there are (1) secondary islets,
scattered throughout the gland; developed from acini they return again
to acini; and (2) permanent islets which, developed directly from the
embryonic pancreatic tubes and not from acini, have no tendency to
transformation into acini. But in the sheep they are atrophied, for the
greater part, at a certain stage in their development, and are eliminated ;
in the viper they tend to persist to maturity. Laguesse finds vestiges of
lumina among the cell-cords of the islets in vipers. From the above it will
be seen that Laguesse coincides (with certain modifications) with the
speculation of Lewaschew.

Flint (5) has studied the islets with a view of demonstrating the
presence of a reticular capsule, and De Witt (3), in the course of an
important experimental study of the islets, has constructed very handsome
models of these structures showing their structural independence, and has
furnished experimental evidence of the presence in the islets of the acti-
vator substance of Cohnheim.

The presence of granules in the islet cells was observed, as we saw
above, by Laguesse; and Diamare (4) called attention to large granular,
deeply staining cells in the islets of the rabbit's pancreas. W. Schulze
(20) called attention to similar cells in the islets of the guinea pig's
pancreas, and Mankowski (12), who repeated Schulze's work, found that
on ligature of the pancreas the epithelial elements disappeared. Man-
kowski, however, found that an injection of silver nitrate disclosed certain

M. A. Lane 413

black specks in the islet cells of the guinea pig; a fact indicating tho
presence in the islet cells of a substance which unites with silver in a
reducible form,, which does not occur — so far as Mankowski's experiments
show — in the pancreas cell itself. These indications, however, were not
pursued farther by any of the above-mentioned observers. Negative
results as to granules were found by Hanseman, Stangl, and Ssobelew,
although Mankowski, however, confirmed Laguesse's observations of gran-
ules in the islets (of the guinea pig's pancreas) after fixation in Flem-
ming's fluid. Eeferences to the publications of these observers will be
found in Sauerbeck's review above mentioned.

TECHNIQUE.

Out of a rather wide range of fixing and staining fluids, three fixations
and one stain were found to be the most valuable in the present investi-
gation. The fixations used were (1) alcohol-chrome-sublimate, a fluid
consisting of equal parts of a solution of potassium bichromate (3.5 per
cent in water) and a saturated alcoholic (95 per cent) solution of mercuric
chloride; (2) 70 per cent alcohol; and (3) Mueller's fluid with 5 per cent
mercuric chloride added — a fluid called here aqueous-chrome-sublimate.
Very small pieces of the pancreas (preferably from the splenic end) are
taken from the living animal and quickly transferred to a generous quan-
tity of fluid. For small pieces two hours (with one change) is sufficient
in the alcohol-chrome-sublimate fluid. Tissues are left in the 70 per cent
alcohol twenty-four hours. In the aqueous-chrome-sublimate from three
to four hours is sufficient. It is of the utmost importance in all this
technique that acetic acid be carefully avoided, as I have found that even
a few drops of this acid, after repeated trials with numerous fluids con-
taining acetic acid, were enough to vitiate the entire work. The tissues,
after fixation, were hardened in the customary graded alcohols, cleared in
bergamot oil, and imbedded in paraffin. Sections were cut three to five
micra thick and were fixed to slides by the water method. Out of a score
of stains I found the most effective to be Bensley's neutral gentian, I. To
a saturated aqueous solution of gentian violet is added a saturated equeous
solution of orange G. The acid dye precipitates the basic one. This is
filtered and thoroughly washed and dried. The precipitate is dissolved
in 25 or 30 cc. of absolute alcohol. For staining purposes enough of this
stock solution is added to 20 per cent alcohol to color the alcohol solution
a deep violet.

The sections were stained 25 hours in this stain, blotted quickly and
thoroughly with thick blotting paper or several sheets of filter paper in

414 Cytology of the Areas of Langerhans

a pad, and two methods of differentiation were used. In the first method
the slide, instantly after blotting, was douched with absolute alcohol from
a medicine dropper to dissolve the excess of the stain, the alcohol quickly
blotted off, and the sections instantly covered with oil of cloves. The
differentiation was then watched under the microscope until the zymogen
granules in the acinous cells were seen to be fairly discrete, the violet
stain being differentiated out of the cytoplasm which, with this method,
retains the brownish-yellow of the orange G. In the second method the
sections were quickly blotted, as before, and the differentiation done with
acetone (dimethylketone). The sections were douched with acetone from
a medicine dropper, quickly placed under the microscope, and when
the zymogen granules appeared, as before, the slide was placed in xylol.
Xylol was also used for the final clearing of the alcohol-differentiated
sections. The sections were then mounted in Canada balsam.

CHEMICAL CHARACTERS.

The first sections examined were those fixed in alcohol-chrome-subli-
mate. This fixation is an admirable precipitant of the zymogen granules
in the pancreas cells, but it has the disadvantage of shrinking the tissues
somewhat. The granules of zymogen in the acinous cells are discrete and
handsomely stained by the dye. The islet cells are somewhat shrunken,
the majority of them taking up the yellow of the orange G. In the center
of the islet, sometimes eccentrically placed, and seldom near the edges,
were seen a number of conspicuous and brilliantly violet cells, apparently
much larger than the remaining cells of the islet and most frequently-
seen in a sharply defined group ( Fig. 1 ) . They seldom appear scattered
or isolated. Examined with powers which distinctly show the individ-
uality of the zymogen granules, the large violet cells of the islet appear
to be of a diffuse color; but when examined under 2 mm. apochr. these
cells are found to be filled with granules very much smaller than the
zymogen granule of the pancreas, but quite distinct none the less. The
nuclei are large and vesicular, and at times surrounded by a very narrow
clear zone in which there is seen occasionally a centrosome. The remain-
ing cells of the islet, considerably more numerous than the cells reacting
to the violet stain, show no granules in the cytoplasm, are smaller than
the granular cells, and present other morphological characters which
differentiate them from the latter. I will recur to these matters presently.

Preparations Fixed in 70 Per Cent Alcohol. — The presence of granules
in certain of the islet cells, simultaneous with the presence of similarly
reacting granules of zymogen in the acinous cells, suggested the query

M. A. Lane 415

whether or not the chromatophile granules of the islet cells were zymo-
genic; whether or not the use of a reagent which would be a solvent for
zymogen would nevertheless act as a precipitant for the granular substance
in the islet cells. This experiment was made with alcohol of 70 per cent
strength. Small pieces of pancreas of the guinea pig were fixed, therefore,
in 70 per cent alcohol and stained with neutral gentian. In sections
treated in this way the acinous cells were quite devoid of zymogen gran-
ules except at the extreme edge of the piece, where a partial fixation of the
granules was obtained, whereas the islets presented the same appearance
as in the sections fixed in the alcohol-chrome-sublimate. The same groups
of violet-granulated cells were present. But, as the alcohol had not dis-
solved out the prozymogen of the acinous cell, the query still remained
whether or not the substance in the islet cell granule partook of the
nature of prozymogen. To check this query I applied MacCallum's iron
reaction on these sections and failed to bring out the slightest trace of
Prussian blue in the suspected cells of the islets, except in the nuclear
chromatin.

Preparations Fixed in Aqutous-Chrome-Sublimate. — The use of this
fluid I found advisable after exhausting the list of desirable acetic fixations
and reducing the quantity of acetic acid to an almost negligible pro-
portion. Sections from tissues fixed with an acetic mixture were invar-
iably blank as to granules in the cells of the islets. But with tissues fixed
in aqueous-chrome-sublimate a most unlooked-for result appeared. The
large cells of the islets which had been filled with violet granules in
sections fixed with the other fluids were quite free from stained granules
in the sections fixed with aqueous-chrome-sublimate, whereas, on the
contrary, the cells which gave no violet reaction with the other fluids,
in this one were filled with granules of a brilliant violet, while now it was
the small groups of large cells that were colored with the yellow-brown
of the orange G (Fig. 2).

From a consideration of these facts several conclusions arise. These
conclusions have to do with the microchemistry of the cells of the Islets
of Langerhans in the guinea pig's pancreas, and they may be stated
somewhat as follows :

1. The Islets of Langerhans in the pancreas of the guinea pig consist
of two types of cells: (a) a type containing a granular substance that is
precipitated by alcohol of a strength of from 50 to 70 per cent; and, (b)
a type, the granular content of which is precipitated by an aqueous-
chrome-sublimate fluid of the general character described.

2. The granular substance that is precipitated by alcohol is dissolved

416 Cytology of the Areas of Langerhans

by the chrome-sublimate fluid, and the substance that is precipitated by
the chrome-sublimate fluid is dissolved by alcohol.

3. Neither of these granular substances is of the same chemical char-
acter as the zymogen granules of the pancreas cell, and

4. Neither of them is of the same chemical character as the prozy-
mogen of the pancreas cell.

To avoid cumbersome periphrase and repetition I will hereafter desig-
nate the cells in which the granules are fixed with alcohol as A cells, and
these in which the granules are fixed with the chrome-sublimate fluid as
(3 cells.

That the chemical nature of the granules in the A cells differs from
that of the granular content of the (3 cells is borne out by the difference
of the reaction of these substances to various fixing reagents non-alcoholic
in character, and for the most part very simple in composition. The
results follow :

Saturated Aqueous Sublimate. — With this fixation the islet cells act in
virtually the same manner as with aqueous-chrome-sublimate, with the
exception, that the tissues are rather shrunken than the reverse. The A
cells remain devoid of basic granules, the /? cells are very well preserved,
and, throughout the whole of their cytoplasm, they are crowded with the
violet granules, which are, however, not as brilliantly stained as in
preparations fixed with aqueous-chrome-sublimate. The zymogen granules
in the acini, on the contrary, are well fixed and react with a brilliant stain
to the neutral gentian.

Saturated Picric Acid. — This fixation is poor, in a general way, and
both types of islet cells are entirely devoid of granules, taking up only
the plasma stain. The zymogen granules, here also, are sharply defined
and stain well.

Nitric Acid, 10 per cent. — The general fixation is poor, but the acid
seeks the A cells much in the same way as does alcohol, and the granules
in them are well preserved. The (3 cells, with this fixation, remain clear
of granules altogether. So far as the A cells are concerned, the picture
here presented is substantially the same as that obtained with the use
of alcohol-chrome-sublimate and 70 per cent alcohol.

Formal, 10 per cent. — This fluid fixes the granules in the A cells, leaving
the f3 cells clear. Although the stain is by no means as brilliant as that
obtained with the three principal fluids, yet the individuality of the
granules in the A cells is capitally preserved. The zymogen granules in
the pancreas cells are broken down and diffused.

Chromic Acid, 1 per cent. — In this fixation the granules in the A and

M. A. Lane 417

/? cells both remain unfixed, and the islet appears uniformly brownish
yellow. The zymogen granules, on the contrary, appear to be well fixed
and react readily to the neutral gentian.

Aqueous-Chrome-Sullimate plus 5 per cent of 10 per cent cent Nitric
Acid. — This fixation gives much the same result as that with aqueous-
chrome-sublimate,, but the chromatic effects are not so clear. The ft
cells are admirably picked out and almost invariably show the entire
cytoplasm crowded with granules. In this fluid the zymogen granules
are well preserved and take the stain fairly well.

The affinity of the two types of cells for certain fixing agents is peculi-
arly brought out in tissues fixed in a combination of these fluids. Thus
in tissues fixed in chrome-sublimate solutions with equal parts of alcohol
added, an islet here and there near the edge of the section shows both
types of cells equally granulated; and the same is true of islets near the
edge of sections from tissue fixed in aqueous-chrome-sublimate to which
has been added 5 per cent of 10 per cent nitric acid.

These various tests, together with those furnished by the use of the
three principal fluids above mentioned, enable us to make certain positive
statements concerning the nature of the two substances contained in the
A and (3 types of cells in the guinea pig's pancreas. That contained in the
A cell is fixed by alcohol, by a 10 per cent solution of nitric acid, and by
a 10 per cent solution of formol. In all these fixations it is stainable with
Bensley's neutral gentian. It is soluble in acetic acid, in saturated
aqueous mercuric chloride, in non-alcoholic chrome-sublimate fluids, in
these last-mentioned fluids also in the presence of nitric acid, in saturated
aqueous picric acid, and in 1 per cent solution of chromic acid. The
substance is chemically different from that in the zymogen granules, for
in all these solutions the zymogen granule is fixed and remains stainable
with neutral gentian, while on the contrary the zymogen granule is
soluble in alcohol in which the A granules are well preserved.

The substance in the granule of the p cell is fixed in aqueous saturated
solution of mercuric chloride, in chrome-sublimate fluids in the presence
of nitric acid, and in non-alcoholic chrome-sublimate fluids. It is
soluble in alcoholic solutions, in acetic acid, in aqueous saturated solution
of picric acid, in 10 per cent solution of formol, and in 1 per cent solution
of chromic acid. And it differs chemically from the zymogen granule of
the pancreas because the latter :s uniformly fixed by the above solutions
with the exception of formol, in which it is not completely dissolved but
only partially preserved.

These considerations go to show in contrast to Laguesse's theory that

418 Cytology of the Areas of Langerhans

the substances contained in the granules of the A and of the /? cells are
chemically different from the substance in the zymogen granule ; and a re
different chemically from each other.

MORPHOLOGICAL CHARACTERS.

Coincidental with these chemical differences are found certain differ-
ences in the morphological characters of the A and (3 cells of the islets
in the guinea pig's pancreas. The A cell is comparatively large and its
nucleus is, for the most part, elliptical, although frequently it is circular
(Fig. 1). It is markedly vesicular, strikingly large and vivid, and its
chromatin content is very small. The chromatin is distributed in a few
small, spherical masses, and this contributes, in section, to the lucid,
vacuous, and prominent appearance of the nucleus of these cells. In
some of the cells the granules are packed together throughout the entire
cytoplasm and seem to lie directly against the nuclear membrane. In
others the granules are determined in a mass bordering closely on the
capillary, while the remainder of the cytoplasm is comparatively or
completely free of them. The cells are polygonal and stand out in high
relief against the lighter and yellowish back-ground formed by the mosaic
of the (3 cells. This is true only when the stain used is the neutral gentian
of Bensley. The A cells can be chromatically distinguished only with
great difficulty when other stains are employed — a fact struck out after
trying a score or so of different basic dyes. Gentian violet, safranin,
licht-griin, and other granule stains gave negative chromatic results,
although the a cells could be recognized by their conspicuous size even
when the section was treated only with a plasma stain.

The /? type of cell appears, as a rule, considerably smaller and is, at
the same time, vastly more numerous in the islet. Entire cords of them,
uninterrupted by the presence of the A cells, appear in the picture, and
almost invariably the cytoplasm of the entire cell is packed with the violet
granules, which are uniformly distributed around the nucleus and which
everywhere border on the capillaries. The nucleus of the (3 cell is invar-
iably centrally placed, is smaller than the nucleus of the A cell, circular,
markedly less vesicular than the nucleus of the A cell, and is also dis-
tinguished from the nucleus of the A cell by the comparatively large
quantity of chromatin it contains. In the nucleus of the ft cell the
chromatin is frequently seen in the form of fine strands forming a net-
work. In some of the islet cells there were found, indifferently as to
either kind, a centrosome and, now and then, a mitotic figure. The cyto-
plasm consists of a delicate network.

M. A. Lane 419

SUMMARY AND CONCLUSIONS.

Eecapitulating the facts above described the following positive state-
ments may be made :

1. In the Islets of Langerhans in the guinea pig's pancreas two types
of cells, morphologically and physiologically distinct, are demonstrable.
These cells show constant reactions to constant chemical tests. I have
called these cells A and ft cells, respectively.

2. The granular content of the A cell differs chemically from that of
the ft cell.

3. The granular contents of the A cell and of the ft cell, while differing
chemically from each other, differ chemically from the granular content
of the pancreas cell, and cannot, therefore, be identical with zymogen.

4. The granular contents of the A and of the ft cell differ chemically
from the prozymogen manufactured by the pancreas cell as the antecedent
of the zymogen granule of the pancreas cell.

5. The chemical and morphological differences between the A and the ft
cell are correlated ; that is, the relations between the anatomical and
physiological characters of both types are found to be constant.

In drawing conclusions from these facts one is led to the conviction that
the Islets of Langerhans are structures which in all probability have the
function of producing a twofold substance which, poured into the blood
stream, has an important effect upon metabolism. That this dual
character of cell in the islet is constant throughout the entire class of
mammals, if not throughout the entire phylum of vertebrates, is indicated
as probable from the results of the comparative study now in progress,
which I hope to make the subject of a future publication. The prospects
seem to point to certain peculiar variations in the character of these cells
in herbivora and carnivora, and to striking and highly suggestive varia-
tions among herbivora themselves.

While these results do not prove that pancreatic cells do not transform
into islet cells, or vice versa, they furnish very strong reasons for holding
that under normal conditions the islets are physiologically independent
of the rest of the pancreas — a conclusion in accord with the observations
of De Witt and Flint as to the framework and architecture of these
structures, and with those of Opie, Pearce, and Helly as to the early
differentiation in' the embryo of the specific cells which are their histo-
genetic source.

It is but rational to conclude from the chemical evidence that the
substances produced by the two types of cells of the islets are not to be
classified with zymogen. If the cells of the pancreas have the power of

420 Cytology of the Areas of Langerhans

transforming themselves into the cells of the islets which I consider
improbable, that transformation must be regarded as a physiological as
well as a morphological one. In the course of an examination of many
hundreds of islets in the pancreas of the guinea pig I have been able to
find but one example of what might be interpreted as a vestige of a
lumen ; and this singular structure seemed to be formed by cells of the A
type.

There is one remaining aspect of the problem to be touched upon
before closing. This is the possibility that the A and the (3 types of cell
are in reality two different phases of the same cell — a notion by no means
improbable even in the face of the chemical evidence to the contrary.
Professor Bensley, who has examined my preparations with considerable
care, has pointed out cells which seem (from the anatomical side) to be
intermediate between the two types, especially in certain preparations
which, at his suggestion, were treated with Ehrlich's haematoxylin before
they were submitted to the neutral gentian bath. But even granting the
truth of this observation, the force of the general argument remains. If
the A and (3 cells are really phases of one and the same cellular structure,
their different chemical characters suggest that they are engaged in the
manufacture of two different secretions. If we conceive that the A cell
changes into the (3 cell, or vice versa, we must conclude that the change
implies the taking on of a different physiological activity. Whether or not
these two different secretions have to do with the pancreas itself or,
through the pancreas with functions lying, in their special or general
effects upon the chemistry of the body, near to or remote from, the
pancreas, is a matter to be determined by further research.

I have to thank Professor Bensley, who was kind enough to direct my
researches, for his lively interest in the work, for his invaluable sugges-
tions as to technique, for his assistance in the interpretation of difficult
nodi that arose as the work developed, and for having made preliminary
reports of the work to the Association of American Anatomists. My
thanks are also due to Mr. Leonard H. Wilder for the fidelity of the
drawings which accompany this paper.

LITERATURE.

1. BENSLEY, R. R. — The CEsophageal Glands of Urodela. Biological Bulletin,

Wood's Holl, Vol. II, pp. 87-104, 1906.

2. DALE, H. H. — On the Islets of Langerhans in the Pancreas. Phil. Tr.;

Lond., CXCVII, Bd., pp. 25-46, 1905.

3. DEWITT, L. M. — Morphology and Physiology of Areas of Langerhans in

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33

422 Cytology of the Areas of Langerhans

EXPLANATION OF FIGURES ON PLATE I.

FIG. 1. Section of an Islet of Langerhans of the Guinea pig, fixed in 70
per cent alcohol, stained with Bensley's Neutral Gentian, showing the A cells
of the islet filled with intensely stained granules, /3 cells orange. Zeiss apo.
2 mm., oc. 8.

FIG. 2. Islet of Langerhans of the Guinea pig; aqueous chrome-sublimate
fixation; neutral gentian stain; showing (3 cells filled with minute violet gran-
ules; A cells orange, the staining reaction being reversed. Pancreatic acini,
Pa. z. with zymogen granules are seen at the edges of the section.

THE CYTOLOGY OF THE AREAS OF LANTGERHANS

M. A. LANE

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