Sues Sst eerie Rieiegeriegees ee eee cooat plete corre set guns = bbl B44 bay { Cte i thea i HH viphetine beehebinenas ait veeaa) Pata i aN g3 7 WP Sah and aaiacce eA AT oe a oes ah J) lpn eli eT THE AMERICAN JOURNAL OF ANATOMY EDITORIAL CHARLES R. BARDEEN University of Wisconsin, HENRY H. DONALDSON, The Wistar Institute. THOMAS DWIGHT, Harvard University. SIMON H. GAGE, Cornell University. G. CARL HUBER, University of Michigan, BOARD GEORGE S. HUNTINGTON, Yolumbia University. FRANKLIN P. MALL, Johns Hopkins University. J. PLAYFAIR McMURRICH, University of Toronto. CHARLES 8. MINOT, Harvard University. GEORGE A. PIERSOL, University of Pennsylvania. HEN ?Y McE. KNOWER, SErEcrETARyY, Jouwns Hopkins University. VOLUME IX PUBLISHED QUARTERLY BY THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY 36TH STREET AND WOODLAND AVENUE PHILADELPHIA, PA. CONTENTS OF VOL. IX. PAGE I. Franxuin P. Matyi. On Several Anatomical Char- acters of the Human Brain, said to Vary According to Race and Sex, with Especial Reference to the Werehivotetne Wronitall Lobee= te njnie no ee as wees oe 1 With 38 Figures. II. Frepertc T. Lewis. On the Cervical Veins and Lymphatics in Four Human Embryos. With an Interpretation of Anomalies of the Subelavian and “ denlar Views an une Atle Tansemann. Ueber die Gehirne von Mommsen, Bunsen und Menzel, Stuttgart, 1907. *Stieda. Zeit. f. Morph. u. Anthropol., XI, 1907. Anatomical Characters of the Human Brain. 93 who spoke fifty languages gave a negative result, for nothing peculiar was found in it. However, Hansemann states that we should expect to find a morphological basis to account for geniuses of the first rank, for they possess qualities peculiar to themselves. In fact the config- urations of the brains of Helmholtz and Menzel showed some peculi- arities which may support this theory. The one ray of hope in the study of the peculiarities of the config- uration of the gyri and sulci comes from the comparison of brains of members of the same family which often show many similarities. This important discovery was made by Spitzka,** who observed that there were hereditary resemblances in the brains of three brothers. This was fully confirmed by Karplus*? in studying the brains of 21 groups of relations in each of which he found a marked similarity of the gyri and sulci. The configuration of the right side has a ten- dency to repeat itself on the right side, and the left on the left, but peculiarities on the right side are not found on the left in near rel- atives. There is an hereditary tendency in the fissuration of the brain as there is in the other features. Nevertheless, even if we should find that the brains of two eminent men of the same family were much alike we have by no means shown that the genius has an anatomical basis. Furthermore, it seems to have been established that anatomical variations often show different percentage in different communities. Schwalbe and Pfitzer** have shown, for instance, that the absence of the psoas minor is as follows. | MEN | WoMEN | = = —— = No. of | | No. of No. of times | Per Cent. No. of times Per Cent. Cases. absent. | | Cases. absent. St. Petersburg. .... | 800 27 “405- | 45: || 600 326 54.3 SEFASSOUIG... 6s 6 ss 386 | 219 | 56.7 175 99 | 56.6 OSU OMpe the relsec.0) 10) ah: 400 | 223 55.8 || 208 145 | 69.7 Brivlaridis. 2... os WssetO, hy 125 59.5 130 93‘) “aL5 *Spitzka. American Anthropologist, VI, 1904. “Karplus. Obersteiner’s Arbeiten aus d. Neurol. Inst., XII. Wien, 1905. “Schwalbe and Pfitzer, Morph. Arbeiten, Bd. 3. 24 Franklin P. Mall. In each group the percentage had reached a constant value, that is with an increase of the number of cases the percentage in a given locality did not change. The same condition may exist in brain configuration, and Merkel** states that the brains from cadavers used for dissection in Géttingen, and which come from Brunswick, of which Gauss was a native, were often very rich in gyri and sulci. On the other hand, in Mecklenburg, where Merkel also had had a large experience, brains of the Gauss type were never seen in the dissecting room, but instead a very simple type prevailed. It certainly would be important if it could be shown that the com- plexity of the gyri and sulci of the brain varied with the intelligence of the individual, that of genius being the most complex, but the facts do not bear this out, and such statements are only misleading. I may be permitted to add that brains rich in gyri and sulci, of the Gauss type, are by no means rare in the American negro.*° While there seems to be no evidence to show that the configuration of the brain of genius is different from that of other brains, there is some evidence in favor of the statement that there are slight differ- ences due to sex. It is often said that the brains of women are of a simple type, but if their weight is not considered it is questionable whether a collection of brains could be assorted according to sex with any degree of certainty. Furthermore, even the more pronounced differences of eurygyrencephaly and stenogyrencephaly are not easily recognizable because they are not easily measured. Of course, when gyri of the simple type are twice as broad as those of the complex type, as pictured on Plate 54 in Retzius’ Menschenhirn, it is not difficult, but there are many intermediate stages and the observer can only express an opinion, for there is nothing that can be weighed or measured. Waldeyer states that to determine whether a brain “Merkel. Top. Anat., I, Braunschweig, 1885-1890. *Spitzka, Amer. Phil. Soc. Vol. 21, has arranged a number of figures in plates showing the evolution of the complexity of the gyri. For example, in his Fig. 8 the gorilla with a simple brain is below, the brain of a Bush- woman is in the middle and that of Gauss, the most complex, is above. In another plate, Fig. 10, the brain of Gambetta holds the lower position, Altmann the middle and Skobeleff the upper. Comparing Figs. 8 and 10 it appears that Gambetta’s brain resembles the gorilla’s more than it does that of Gauss. Anatomical Characters of the Human Brain. 25 came from a man or woman is much like identifying the sex of the individual from which a given skull came. I am not so optimistic and would rather take my‘ chances with the skull. In the article by Schwalbe and Pfitzer mentioned above many anatomical variations are tabulated and there do not seem to be more variations in the male than in the female, but the percentage of vari- ations is by no means always alike in the two sexes. If there is a percentage difference according to sex in a special variation it tends to remain constant in various sets of statistics and does not become the same as the records are increased in number. Moreover, “bei den weiblichen Fallen werden in der Regel die Werthe viel rascher constant als bei den mannlichen.” In other words, a smaller number of records are required in the female than in the male to obtain the true percentage of variations. How much this indicates is by no means clear, but this conclusion should be that there is not a simpler type; but less variations in the female, which appears to be the opinion of Retzius regarding the female brain. We have tested this difference by grouping the illustrations of brains in the great Atlas of Retzius under simple and more complex types, without knowing whether the picture of a brain in question was from a man or from a woman and obtained the result given on page 19. In the first line in the table my estimates are found with the percentages below them. In the second line another estimation by ‘Dr. Sabin is given, and in the third line one by Dr. Mellus. In general the opinion expressed in these estimations does not bear out the notion that the configuration of the brains of women is of a simpler type than in those of men. This, however, is only our opinion regarding the complexity of the gyri and sulci of pictures of brains. But Retzius has tabulated in an excellent way a number of concrete data of 100 brains which can easily be tested in other specimens. These include a number of variations, such as the central sulcus communicating with the fissure of Sylvius, regarding which there can be little difference of opinion. There are in all 73 such records, 19 being of the norm and 56 of variations. Each of these records can be entered a second time by subtracting its frequency in percentage from 100. Thus, if the central 26 Franklin P. Mall. suleus communicates with the fissure of Sylvius in 3 per cent of the eases it is called a variation in 8 per cent of the cases, while in the remaining 97 per cent it is normal. In this way I obtained a column of 73 records, representing the norm as well as the variations for each hemisphere both of the male and the female. The average of these figures is as follows. MEN. | WomMeEN. Right Side. Left Side. | Right Side. | Left Side. = ~— | | | | | Norm. | Variations. Norm. | Variations.) Norm. | Variations.| Norm. | Variations. | | | | | = 2 | (= | | 78% | 2207, | 75% | 25% | 81% | 19% | 81% | 19% I | | This table indicates that the brain of woman is not nearer the norm but varies less than does that of man. Could all the variations found be grouped together in single brains, leaving the rest a8 per- fectly normal, then 76 brains of men and 81 of women out of our 100 would be exactly normal in the arrangement of the gyri and sulci. Retzius has done us a great service in pointing out the way by which this problem can be attacked by the statistical methods. A few remarks regarding his conclusion may be made, but before they can be criticised properly it will be necessary to tabulate many other brains, as he has done, of both men and women. In the first column of figures in Retzius’ table regarding the fissure of Sylvius both the norm and the variation is given, but the missing figures can easily be obtained by subtracting the given percentage from 100. In ease the average of a given record is more than 50 in both male and female, it is called normal, while when it is less it is called a variation. Thus the central suleus anastomoses with the sulcus precentralis superior in 18 per cent of the cases and therefore these do not anastomose in 72 per cent. It may be remarked that the number of brains of men studied by Retzius is somewhat small, while that of women is decidedly too small, for in the latter each single record equals 8 per cent when reduced to the scale of 100. The data given by Retzius regarding differences in the gyri and sulci due to sex may be criticized from two standpoints. Those in Anatomical Characters of the Human Brain. oT. which there is a marked difference between the brains of men and women may be tested by other records. For instance, according to Retzius the anterior branch of the fissure of Sylvius is divided and forms an operculum frontale intermedium in 82 per cent of the brains of men and in 100 per cent in those of women. At this point woman’s brain forms a perfect norm, being richer in all cases in gyri and sulci. However, only four specimens of brains of women without an inter- mediary operculum would have made the results for the two sexes exactly alike. No doubt a larger number of records would have shown, even in Stockholm, that the operculum frontale intermedium is not always present in the female brain. I notice that Karplus, in the article mentioned above, figures four brains of women without the operculum frontale intermedium, and states expressly that it is miss- ing in those four specimens which were found in a relatively small number of brains. His record will bring the chief difference, given by Retzius, pretty close to the male average of 82 per cent. The second criticism can only be made by collecting many more statistics along the lines laid down by Retzius in his great monograph. At any rate what has been written by Karplus is to the point: “Auf die von den Autoren angegebenen einzelnen Geschlechtsmerk- male der Gehirne, die ja von vielen bestritten werden, will ich hier nicht niher eingehen. Auch hier muss zunichst viel mehr Material ge- sammelt werden, bisher bin ich nicht davon tberzeugt, dass sich aus dem Furchenbild eine Inferioritit des weiblichen Gehirns ableiten hesse.”’ The question of the type of the female brain, a subject which has been discussed so much, is therefore still far from being solved in a satisfactory manner. Furthermore, it is by no means established that there are male and female types of the brain due to the form and arrangement of the gyri and sulci, as has been so frequently asserted. Each claim for specific differences fails when carefully tested, and the general claim that the brain of woman type is fcetal or of simian type is largely an opinion without any scientific foundation. Until anatomists can point out specifie differences which can be weighed or measured, or until they can assort a mixed collection of brains, their assertions - tH Se | fon bo - = 19 . . . . . -onownt Ow ws ase Ae es (OA N ORO . . 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SAREE SE ddd ae eS . aaana pa ed idee dee “I0TOD 108% POT fet, 9616 6826 GOLG 6G2E EGLG GGLS IGLG SFLG 9FLG EFLS CELE TELG GGLG 61LG L91 €0LG GST L69G LOT 116896 OLT E89G OLT GLOGS 99T L99G OLT 999G LOie a S99G €91 | 099% PLI | 649¢ HG 6 ~=LP9G GOT 6896 921 GEIG SLI | 669% ZLT | + GES moet) ‘Apoq | jo | ‘ON q4sue'T “OO Hid He es oS Ss SS eS eS cS) CNmMOAOH MOH OOrrdtdt Ot Ons Ssosoanesrnrwrn nr wnww wes es a) "N "OONneoOoOo ANN aN N N N re ND OMNNNANANANANN * © _ ot “SOD Cr N © 1g OONMMOODMrOOHMI9OD MWA SHH Ome O19 100 1900 0 190190 HHO HO ld NH Has | OFT O8T OLT 691 OST GOT O9T GST G61 OLT 0&1 OST gg LéT C61 GST OGG O9T GO’ O9T OLT G6l OLT S61 OST OLT O9T OLT OGE SOE GTE SIE STE GOS GOES 916 11096 066 GCE OGG OLS OGT 91616 OPE OGG OOF OPE OFE GoE GLE OOF ccé 08h G9E GLE GT€ O0€ OGE OLE OZE CIE GSE 6G COE ELE 21086 OO€ GCE G0G OSG OGT 906 OSE OSG OOF OFE crs cee CSE OF 098 OCP GLE OLE cos O0€ OcGT OOGT O0GT OSTT O0GT OS6 OLTT O&éT OL6 OFT OVP OSGI OGET OLET OOST OOFT OS9T OOST OSFT O0GT OSGI . . . SSSSSH SSS SSH SSeS SSeS “=~ a. a = . Smsssissisississas . BME OOo EE EE SEER RSE GEEE IEE ME ma Seed faalgeaMaaMan 89I ELT st €91 GLI quejuy queyuy 691 LOT LOT SST LOT OsT OLT I9T OST ool o9T OST LOT OLIG 8996 6961 6E$1 SEST 32 Franklin P. Mall. regarding male and female types are of no scientific value. It may turn out, however, that variations in the gyri and sulci will not be of the same percentage in both men and women and that the constant value in the latter will be found more readily, as is the case with other anatomical variations (Schwalbe). In this study of several anatomical characters said to vary according to race and sex, the evidence advanced has been tested and found want- ing. It is found, however, that portions of the brain vary greatly in different brains and that a very large number of records must be obtained before the norm will be found. For the present the crude- ness of our method will not permit us to determine anatomical characters due to race, sex or genius and which if they exist are com- pletely masked by the large number of marked individual variations. The study has been still further complicated by the personal equation of the investigator. Arguments for difference due to race, sex and genius will henceforward need to be based upon new data, really scientifically treated and not on the older statements. Notre To THE PrRrecEDING TABLE. The data given in the preceding table have been aranged in a great variety of ways, but only three of these bear upon the subject under discussion. They are given in Figs. 1 to 3. The individiual records are appended to enable those who are interested in the subject to make further comparisons with those given by Bean and by Spitzka, as well as for further use to those who may collect new data. The genu and splenium were outlined by Bean’s method, given on page 8. FOOTNOTE TO THE TABLE. 1Pia on left side. *Pia off on left side. *Boy. *Break not even on left side. ‘Pia off on left side. °Ventricle on right side greatly dilated. “Break unsatis- factory. S‘Sulci on both sides very irregular. *Pia off on right side. *The pos- terior left is decidedly larger than the posterior right. “Left operculum is very large and right parietal convolutions are very atrophic. “Curious inter- lacing of fiber bundles below central fissure on the left side. “Boy. *™Central fissure seems to be double on both sides. “Break unsatisfactory. “Large cavity in right brain; break also unsatisfactory; break on left side is not accurate. “Breaks unsatisfactory. “Insane murderer. ON THE CERVICAL VEINS AND LYMPHATICS IN FOUR HUMAN EMBRYOS, WiruH AN INTERPRETATION OF ANOMALIES OF THE SUBCLAVIAN AND JUGULAR VEINS IN THE ADULT. BY FREDERIC T. LEWIS. From the Department of Anatomy, Harvard Medical School. In order to explain an anomaly of the subclavian vein occurring in a man 68 years old, reconstructions of the cervical veins and lymphatics in four human embryos were made, with the following results. In an embryo having a maximum measurement of 10 mm., the right arm is drained by a primitive ulnar vein which unites with a thoraco-epigastric vein to form the dorsal subclavian vein. These vessels are shown in Fig. 1. The primitive ulnar vein is seen to receive a branch near the elbow, and distally it has many smaller tributaries which have been omitted from the drawing. There are no veins larger than capillaries along the radial border of the arm. The thoraco-epigastric vein, which is found in the lateral body wall, is represented in fishes and in all the higher classes of verte- brates. Unfortunately it has been given a great variety of names. In the rabbit it is called the external mammary vein (Krause,' Lewis”) and this name has been applied to it in man (Poirier*?). In man it has been called, in part at least, the long, lateral, or inferior thoracic vein, but the term thoraco-epigastric is preferable to any of these. The primitive ulnar and thoraco-epigastric veins unite to form a subclavian vein which passes dorsal to the brachial plexus to enter the "Krause, W. Die Anatomie des Kaninchens. Leipzig, 1868. *Lewis, F. T. The development of the lymphatic system in rabbits. Amer. Journ. of Anat., 1905, Vol. 5, pp. 95-111. °Poirier, P. Traité d’anatomie humaine. Paris, 1896. Vol. 2, p. 911. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 1. 34 Frederic T. Lewis. anterior cardinal vein. Small branches of the anterior cardinal vein are found ventral to the brachial plexus. In a slightly older embryo (Fig. 2) these ventral branches are continuous with the thoraco- epigastric vein so that the brachial plexus is enclosed in a venous ring. This arrangement of the subclavian veins has been described in the rabbit and man by Hochstetter,* and the corresponding stage in the rabbit has been figured.° In addition to the subclavian vein, the anterior cardinal has another important branch. This begins as a median vessel in the lingual region (Fig. 1). It turns sharply to the right, receives tributaries from the lateral superficial tissues and descends in front of the vagus nerve. In the lower part of its course it is close to the dorsal portion of the pericardial cavity. It passes on the lateral side of the vagus nerve to enter the anterior cardinal vein. In the older embryo this linguo-facial branch (V. ling-fac.) is easily recognized. The median vessel in the sublingual region is present, but it has not been drawn, since in this embryo it is drained by the linguo-facial branch of the left cardinal vein. As seen in the reconstruction, the linguo-facial vein has acquired a new outlet, which is anterior to the place where the hypoglossal nerve crosses the cardinal vein. The hypoglossal nerve, which before was lateral to the cardinal vein, is now sur- rounded by it,® and the venous loop through which it passes is shown near the top of Fig. 2. That part of the linguo-facial vein which descended in front of the vagus nerve has apparently disap- peared, and the vein is no longer in relation with the pericardial cavity. It is perhaps worth while to call attention to the scattered data regarding this linguo-facial vein. It was discovered by Grosser in embryo bats and described as follows :* “The veins of the branchial arches consist in young embryos of Rhinolophus of a median longitudinal vein near the ventral surface *Hochstetter, F. Ueber die Entwicklung der Extremititsvenen bei den Amnioten. Morph. Jahrb., 1891, Vol. 17, pp. 26-27 and 32-33. ‘Lewis, F. T. Amer. Journ. of Anat., 1905, Vol. 5, p. 98. °This accords with Tandler’s observation that the hypoglossal nerve is lateral to the internal jugular vein in human embryos of 8 and 9 mm. and medial at 12.5 mm. Anat. Anz., 1907, Vol. 31, pp. 478-480. "Grosser, O. Zur Anatomie und Entwickelungsgeschichte des Gefasssystems der Chiropteren. Anat. Hefte, 1901, Heft 55, p. 181. Cervical Veins and Lymphatics in Human Embryos. 35 of the mandibular and hyoid arches, which at its caudal end divides into two symmetrical parts. Each of these turns abruptly to one side, receives small tributaries from the lateral parts of the branchial arches, and empties, lateral to the aortic arches, into the anterior cardinal vein. Later the vessel cannot be demonstrated. A part of it may perhaps be incorporated in the external jugular vein.” Vling-fac. 2 q A \\ ii , Liifiaitaiiay Wa aK a\\ AN IM tit isi | V. th-ep. RiGee liek, Ys Fie. 1. Reconstruction, as seen from the ventral side, of the right arm and adjacent part of the body of a human embryo of 16 mm. (Harvard Embryo- logical Collection 1,000), to show the veins and lymphatic vessels. The latter, in this and the three following figures, have been heavily shaded. » 20 diam. Fie. 2. Similar reconstruction from a human embryo of 11.5 mm. (H. E. C. 189). « 20 diam. V. card. ant., Vena cardinalis anterior; V. card. com., V. eardinalis communis (duct of Cuvier) ; V. card. post., V. cardinalis posterior ; V. ling-fac., V. linguo-facialis; V. scl. d., V. subclavia dorsalis; V. scl. v., VY. subclavia ventralis; V. th-ep., V. thoraco-epigastrica; V. ul. p., V. ulnaris prima. This description is illustrated by an excellent reconstruction. Two years later these veins were recorded in pig embryos of 6, 12, 14 and 20 mm.’ Because of their course across the throat they were called “transverse veins.” It was stated that the median vessel, instead of bifurcating, sometimes passes wholly to the right and sometimes to the left. The early appearance of corresponding veins in the rabbit ‘Lewis, F. T. The gross anatomy of a 12 mm. pig. Amer. Journ. of Anat., 1903, Vol. 2, p. 221. 36 Frederic T. Lewis. was reported subsequently.® ‘They are present in an embryo of 914 days (3 mm.). Usually the linguo-facial vein arises from the anterior cardinal near its outlet, but sometimes it connects with the common cardinal vein (duct of Cuvier) and in one exceptional case—the left side only of an embryo of 12 days (5 mm.)—it emptied into the posterior cardinal vein. In a 7 mm. rabbit the median lingual por- tion of the vein has been seen to bifurcate symmetrically, agreeing with Grosser’s description for the bat. In rabbits from 9.5 to 29 mm. the main trunk of this vein has been shown in a series of six reconstructions.‘° Its terminal branches are the anterior and pos- terior facial veins, the latter receiving a posterior auricular branch; thus it corresponds with the external jugular vein of the adult rabbit as described by Krause, and it has been so labeled. This vein, how- ever, seems to be homologous with the common facial vein in man, the external jugular vein of human anatomy arising independently as will be seen presently. In a recent publication Grosser has shown that the lnguo- facial vein is homologous with the inferior jugular vein of fishes, amphibia, and reptiles,—a paired ventral vessel draining the floor of the branchial region.** In the same paper he states that this vein is well developed in cat and guinea-pig embryos and in a human embryo of 6.5 mm. Grosser’s comparative studies demonstrate the funda- mental importance of this vein. The name inferior jugular has, however, not been adopted in this paper since it is an unfortunate designation for a ventral vessel in fishes, which has nothing to do with the anterior (7. e., ventral) jugular vein of man, but gives rise to veins which empty anterior or superior to the other jugulars. The term linguo-facial is justified by the embryonic distribution of the vessel,—in part to the lingual region (hyoid and mandibular arches) and in part to the superficial tissues of the mandibular region. Although there is a complex rearrangement and new formation of branches, a single large vein drains this territory from early embry- *Lewis, EF. T. The intraembryonic vessels of rabbits from 81% to 18 days. Proc. Amer. Assoc. of Anat., 1908, pp. 12-13. ~Lewis, F. T. The development of the lymphatic system in rabbits. Amer. Journ. of Anat., 1905, Vol. 5, pp. 95-111. “Grosser, O. Die Elemente des Kopfvenensystems der Wirbeltiere. Verh. d. anat. Gesellschaft, 1907, pp. 179-192. F. R. Lillie refers to this vein in the chick as the external jugular. Development of the Chick, Chicago, 1908. Cervical Veins and Lymphatics in Human Embryos, 37 onic stages to the adult. Thus in man, according to Sebileau and Demoulin,'? ‘“Faraboeuf has well shown that the facial vein, lingual vein, and superior thyreoid vein unite to form a common canal very happily termed the thyreo-linguo-facial venous trunk. This arrange- ment is in fact very frequent.” Except for the superior thyreoid vein, which has not yet developed, this description may be applied to the embryonic vessels shown in Fig. 2. a—V card.ant. V.card.com. < ’ \\ AW Vicard. post. Fic. 3. From a human embryo of 16.0 mm. (H. BE. C., 13822). >< 20 diam. V. ceph., Vena cephalica. For other abbreviations see Fig. 1. The study of the veins in the embryos just described led to the following observations upon the lymphatic vessels. In the 10 mm. embryo (Fig. 1), the jugular sac is represented by a single lymph space in close relation with the anterior cardinal vein. It appears to be lined with endothelium, which in some sections has shrunken away from the surrounding mesenchyma. It contains a few blood corpus- “Sebileau, P., and Demoulin, A. Comment il faut comprendre le systéme des veines jugulaires antérieures. Bull. de la soc. anat. de Paris, 1892, année 67, pp. 120-132. 38 Frederic T. Lewis. cles, and appears to communicate with the vein by a slender oblique passage which is completely filled by a single file of blood corpuscles. This lymphatic space is larger and more irregular in outline than the neighboring small tributaries of the vein. No j = eZ, V. ling-fac. V.jug. int. > Vjug-ant. «— A, Y | V-th-ep. vA Vb: Fic. 4. From a human embryo of 22.8 mm. (H. E. C., 871). x 20 diam. The ribs, clavicle, scapula, and humerus have been stippled, and the sub- clavius muscle has been drawn. In addition to the veins shown in Fig. 1, the following are included: V. an. dexi., Vena anonyma dextra; V. an. sin., V. anonyma sinistra; V. br., V. brachialis; V. ceph., V. cephalica; V. jug. ant., V. jug. ext., V. jug. int., V. jugularis anterior, externa, interna; V. mam. int., VY. mammaria interna. lymphatics could be found in a 9.2 mm. embryo, so that the jugular lymphatics probably arise in human embryos of about 10 mm. This accords with the observation that they first appear in rabbits of 9.5-10.0 mm., but does not agree with Ingalls’ opinion that in a 4.9 mm. human embryo certain vessels represent “the first anlage, or Cervical Veins and Lymphatics in Human Embryos. 39 earliest forerunners, perhaps, of the lymphatic system in man.”’}% The vessels in question are clearly veins. In the 11.5 mm. embryo (Fig. 2) there are four or perhaps five lymphatic spaces, apparently separate from each other. They con- tain some blood corpuscles and in two cases they seem to connect with the vein, but the apertures are very small. At 16 mm. (Fig. 3) the lymphatics have increased and extend along a considerable por- tion of the anterior cardinal vein. There is a separate space in relation with the linguo-facial vein. A lymphatic vessel extends from the jugular sac dorsal to the brachial plexus, but the dorsal subclavian vein which it accompanied in the earlier stage has disappeared. Similarly in the rabbit the dorsal subclavian vein is accompanied by a lymphatic vessel and later both vein and lymphatic disappear. No outlet from the jugular sac into the cardinal vein was found in the transverse sections studied, but, as Professor Sabin has recently demonstrated, frontal sections are more favorable for detecting the valvular orifice. In an embryo of 22.8 mm. (Fig. 4) there is a very large jugular sac which has grown around certain nerves. The upper small aperture transmits a branch of the third cervical nerve, and the lower one is for branches of the third and fourth. In a rabbit of 14.5 mm. the jugular sae showed similar openings for the third and fourth cervical nerves. Returning to the veins in Fig. 3, it will be seen that the branch of the primitive ulnar vein near the elbow now joins the radial extremity of the ulnar vein, and from their junction a vessel can be followed along the radial border of the limb toward the shoulder. It is very probable that at this stage there is a capillary union between this cephalic vein and the lateral branch of the anterior cardinal which is shown in the figure. An obvious connection between them is seen in Fig. 4. Here the lateral branch of the anterior cardinal may be identified as the external jugular vein and it can be traced upward behind the ear as the posterior auricular vein. It does not yet connect with the linguo-facial vein. Fig. 4 shows the brachial vein (derived from the primitive ulnar) joining the larger thoraco-epigastric vein to make the (ventral) sub- clavian vein. Along the latter, several branches anastomose to make *Ingalls, N. W. A contribution to the embryology of the liver and vascular system in man. Anat. Record, 1908, Vol. 2, p. 348. 40 Frederic T. Lewis. a small vena comitans for the subclavian artery. Higher up both the subclavian vein and the vena comitans are separated from the artery by the sealenus anterior muscle, and both are dorsal to the sub- Vijug.int. Vijug.exrt. Vjug.an. Vceru.sup. V/ OY / Vitrcolli, yx V.th-dor. Fic. 5. Dissection of veins in a man 68 years old. Two-thirds natural size. V. br., Venae brachiales ; V. ceph., V. cephalica ; V. cerv. sup., V. cervicalis super- ficialis; V. cir. hum. post., V. circumflexa humeri posterior; V. cir. scap., V. circumflexa scapulae; V. jug. ant., V. jug. ext., V. jug. int., V. jugularis anterior, externa, interna; V. mam. int., V. mammaria interna; V. prof. br., VY. profunda brachii (with a branch, the V. circumflexa humeri anterior) ; V. th., V. thymica; V. th-ac., V. thoraco-acromialis; V. th-dor., V. thoraco- dorsalis; V. th-ep., V. thoraco-epigastrica; V. thy..ima, V. thy. inf., V. thyreoidea ima, inferior; V. tr. colli, V. transversa colli (from vertebral border of scapula) ; V. tr. scap., V. transversa scapulae (from scapular notch) ; VY. vert., VY. vertebralis. clavius muscle, which has been drawn in the figure. Ventral to the subclavius muscle there is a small branch distributed beneath the pectoral muscles; this branch probably is the principal factor in the Cervical Veins and Lymphaties in Human Embryos. 41 Fic. 6. The relation of the linguo-facial vein to the jugular veins in the adult. (From dissections. ) A. The primary relation. The linguo-facial vein is a branch of the internal jugular; its submental, anterior facial, lingual, and posterior facial branches are shown in the drawing. The linguo-facial vein has only small anastomoses with the external jugular vein, and none with the anterior jugular, the latter vessel being scarcely represented in this case. The similarity to the embryonie relations shown in Fig. 4 is apparent. B. ‘The linguo-facial vein has been tapped by the external jugular so that its branches appear to belong to the latter. C. The linguo-facial vein is drained chiefly by the anterior jugular, to which its branches appear to belong. D. The linguo-facial vein is sub-divided, so that its posterior facial branch empties into the external jugular, its anterior facial branch empties into the anterior jugular, and the lingual branch remains as a tributary of the internal jugular. 49 Frederic T. Lewis. anomaly to be described. At the outlet of the subclavian vein there are several branches, and among them the anterior jugular vein can be identified. The anomaly which led to the examination of the embryos is pic- tured in Fig. 5. If the small vein ventral to the subeclavius muscle in Fig. 4 is considered to have enlarged and anastomosed both with the cephalic vein and the subclavian vein, the conditions found in the anomaly will be strikingly reproduced. Both the clavicle and the subclavius muscle will then be surrounded by a venous ring. In the anomaly the “‘jugulo-cephalic” vein, which is above the clavicle, is as large as the subclavian vein. The ‘‘accessory subclavian vein,” which is between the clavicle and the subclavius muscle, is somewhat larger. The subclavian vein occupies its normal position between the sub- clavius muscle above, the scalenus anterior behind, and the first rib below. Finally it may be noted that the formation of a venous ring around the scalenus anterior muscle, occasionally recorded in the adult, is suggested in the 22.8 mm. embryo. Where the subclavian vein empties into the jugular, these vessels are molded about the muscle, and since a vertebral branch joins the jugular at this point, nearly two-thirds of the circumference of the scalenus anterior are in rela- tion with the veins. The completion of this ring by venous out- growths would account for the anomaly. The conclusion may be drawn that although the jugulo-cephalic vein in man is a persistence of an important and normal embryonic vein, the accessory subclavian vein, whether situated behind the sealenus or above the subclavian, is an abnormal vessel. Of wider interest is the conclusion that the linguo-facial vein is a morphological constant. In mammals it appears at an early stage, and although it often becomes resolved into a group of branches, it is present in the adult. Some of its transformations in man are shown in Fig. 6, on the preceding page. It may have large anastomoses with the external jugular vein or the anterior jugular vein or with both, but these can be readily described and understood on the basis of a primary linguo-facial vein. THE LYMPHATIC SYSTEM IN HUMAN EMBRYOS, WITH A CONSIDERATION OF THE MORPHOLOGY OF THE SYSTEM AS A WHOLE. BY FLORENCE R. SABIN. From the Anatomical Laboratory of the Johns Hopkins University. When we consider the history of our knowledge of the lymphatic system, it is clear that there have been two wholly different lines of thought with regards to our general conceptions. To establish its general morphology is the fundamental task for each of the systems of the body, and upon such a general conception is based all future elaboration of the system. I need only to refer to the neurone theory as establishing such a foundation for our knowledge of the nervous system. In connection with the lymphatic system, the idea that it arises from mesenchyme spaces dominates anatomical and zodlogical literature as is evidenced by examining most of the text books. This conception is based on the work of Budge, Sala, Gulland and many others. It allies the lymphatic system with tissue spaces and serous cavities. The other theory, which seems in a fair way to displace the earlier conception, is that the lymphatics are derived from the veins, that they are vascular rather than mesenchymal in origin. This theory, only recently crystallized, has had an interesting evo- lution; beginning with Langer and Ranvier, it has been formulated and developed by a group of American anatomists. In this paper I hope to add evidence for this theory and give a general picture of the primitive lymphatic system as a whole. The great usefulness of this theory, aside from the fact that we believe it to be true, is that it gives a key by which to work out the entire development of the lymphatie system down to its ultimate capillaries, and it will be readily conceded that the old theory of the relation of the lymphatics to the tissue spaces gave us no such point of attack. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 1. 44. Florence R. Sabin. The first theory, that the lymphatics arise from tissue spaces, received its strongest support from Budge." In his first paper Budge described injections of Berlin blue into the false amnion of three-day chick embryos. He found that the injection mass ran out into the area vasculosa in a series of irregular canals forming an abundant network immediately under the epiblast and hence dorsal to the vascular layer. This network of canals extended out to a marginal canal around the area vasculosa similar to the marginal vein. Budge interpreted this system of canals as a primitive -lymphatice system which in his injections arose in con- nection with the ccelom and its extra embryonal expansion, the false amnion. ‘This primative lymphatic system he said never had any connection with the veins, so that the interchange of fluid must have been through the walls. Dr. Mall? has studied Budge’s specimens and is convinced that they are injections showing simply the extent of the extra embryonal celom. I repeated Budge’s experiments, using India ink instead of Prus- sian blue, as it flows more readily, and found that I could duplicate Budge’s figures exactly.* The fluid ran out in blunt processes simulating canals, but readily distinguished from the lymphatic injections. The fluid runs exactly as it would, if forced between two glass plates held closely together, that is, blunt processes push out which form an advancing network, but this network soon fills into a solid mass. With a careful injection of true lymphatics on the other hand the individual vessels often remain absolutely distinct from the very point of the needle as is shown in Fig. 4, of the article in Volume I, of the American Journal of Anatomy, where the needle was introduced into two places, one just over the shoulder and the other over the crest of the ilium. The injections in the area vas- culosa of the chick are like the pictures obtained by injecting into a ‘Budge. Ueber ein Canalsystem im Mesoderm von Hiihnerembryonen. Arch. f. Anat. u. Phys., Anat. Abth., 1880, s. 320. Untersuchungen tiber die Entwickelung des Lymphsystems beim Hiihner- embryo. Arch. f. Anat. u. Phys., Anat. Abth., 1887, s. 59. *Buck’s Handbook of Medical Sciences. The Celom. *Sabin. The Development of the Lymphatic System. American Journal of Anatomy, Vol. I, 1901-1902. The Lymphatic System in Human Embryos. 45 mass of embryonic connective tissue which has no lymphatics. The fluid runs out in the lines of least resistance, simulating performed canals but easily distinguished from true lymphatic capillaries, both in form and from the fact that as the injection proceeds the network fills into a solid mass. Serial sections of the area vasculosa showed no preformed channels, but rather that the space between the germ layers is bridged by delicate fibrils, the processes of mesenchyme cells. It seems certain then that Budge’s primitive lymphatic system is simply a study of the extent of the early ceelom and morphologi- eally has no relation to the lymphatic system. In the understanding of the lymphatic system this point is of great importance, as will be shown later. None of the serous cavities, hollowed out of the mesenchyme, that is, the pleural and peritoneal cavities, the joints, the various burse, and the chambers for the vitreous and aqueous humors in the eyes, though they contain serous fluid ever form a part of the true lymphatic system. In Budge’s second paper, which is unfortunately just a fragment of his work published from the notes after his death, are pictured beautiful figures of true lymphatic injections made at a much later stage, namely in embryo chicks, 18 days old. These, the true lymphatics, Budge thought belonged to a second, the permanent system, distin- guished from the first by the presence of the thoracic duct which emptied into the veins. Budge thought that the thoracic duct arose from spaces derived from the celom. He also discovered the pos- terior lymph hearts in chick embryos between 10 and 20 days old. The theory of the origin of the lymphatic system from tissue spaces was further illustrated by Gulland.* He found spaces hollowed out in the mesenchyme along the course of the blood vessels of the limbs and thought that these flowed to- gether to form ducts. The next exponent of the theory that the lymphatics arise from the tissue spaces in Sala.® Sala has studied the origin and the development of the lymphatic *Gulland. Journal of Pathology and Bacteriology, Vol. II, 1894, p. 466. "Sala. Ricerche Lab. di Anat. Norm. d. r. Univ. di Roma, Vol. VII, 1899-1900. 46 Florence R. Sabin. system in chick embryos. Basing his work on Budge’s, he worked out with care the origin of the posterior lymph hearts which Budge had discovered. He found that the posterior lymph hearts begin at the middle of the seventh day in connection with the lateral branches of the first five coccygeal veins. He says that corresponding to these veins there are excavations in the mesenchyme which soon enter into communication with the lateral branches, and in fact one would say that these fissures are simply dilatations of the veins themselves. These two statements of course exclude one another, for the spaces can not be both fissures in the mesenchyme and dilatations of the veins. (‘‘Esaminando in serie le sezioni caudali di un emb. di g. 6 + ore 18, si scorge che nel mesenchima che sta lateralmente ai miotomi ed in corrispondenza dei rami laterali delle prime cinque vene coccygel, si vanno scavando dei piccoli spazi o fessure che ben presto entrano in comunicazione cogli stessi rami laterali venosi: si direbbe anzi che esse non sono che semplici dilatazioni, ramificazioni delle stesse vene.”’ ) Then he describes these fissures as becoming more abundant and confluent. By opening up communications with each other they form a sac or lymph heart in the mesenchmye. This sac he says is lined with flattened mesenchyme cells, which, if it were so, would, according to our standpoint, exclude it from being a vein. He found muscle in the wall of the hearts on the ninth day and was able to inject the heart directly by the second half of the tenth day. Sala’s description of the origin of the posterior lymph hearts in the chick is so clear and graphic that it is perfectly evident to those who are familiar with the method of origin of the lymph sacs in mammals, that the two processes are the same, that the sacs arise from the veins in both cases. The fact that Sala had the old conception of the lymphatic system as coming from the tissue spaces too firmly fixed in mind to really accept the evidence of his own material does not need to confuse the picture. The lymphatic ducts he thought began as fissures in the mesenchyme along the hypogastric veins on the ninth day. By the eleventh day these spaces communicated and formed a plexus of lymphatic ducts which connected with the lymph hearts and the thoracic duct. The Lymphatic System in Human Embryos. 47 The thoracic duct, which he found extended only from the beginning of the celiac artery to the outlet of the superior vena cava, began on the eighth day in the following manner. First.a series of mesen- chyme spaces around which occur clumps of mesenchyme cells which develop into a solid cord. ‘These solid cords become excavated and form the thoracic duct. There is nothing to correspond with this in connection with the lymphatic system in mammals. To trace the development of the idea that the lymphatic system is derived from the venous system it is necessary to begin with the work of Langer,® published in 1868. In this important paper, Langer makes clear a number of funda- mental points. He distinguished the lymphatics in the tadpole’s tail from the arteries and the veins by injecting them. He found the two longitudinal lymphatic vessels of the tail, and the branches forming a plexus leading from them. He distinguished the lym- phatic vessels clearly from the surrounding connective tissue, and determined that the lymphatics were closed tubes. He was studying a border zone of developing lymphatics and saw that the lymphatics here were really terminal blind ends. He noted the endothelial sprouts from the sides and ends of the vessels and interpreted these sprouts to mean that the lymphatic vessels grow by the same method as do blood capillaries. Thus he says: “Ich zweifle nicht, dass Lymph und Blutcapillaren nach dem einen und demselben Bildungsmodus sich vermehren, die Elemente sind dieselben.” This in reality is his great contribution and upon this idea as a foundation rests the new conception of the lymphatic system as derived from the veins. Another of his obser- vations must not be omitted, namely that in the course of a lymphatic capillary, a portion of the vessel may be greatly narrowed, that is to say, even completely collapsed. “Ich traf aber auch Rohrchen, welche sich ziemlich rash verengten und in der Mitte ihres Verlaufes einen diinnen, anscheinend ganz soliden Faden darstellten.” The meaning of this phenomenon and its relation to the general theory will be made clear later. *Langer. Ueber das Lymphgefiisssystem des Frosches. Sitzb. d. k. Akad. d. Wissensch., LVIII Bd., I Abth., 1868. 48 Florence R. Sabin. Between the years 1895 and 1897, Ranvier published a series of articles on the development of the lymphatic system.’ He also studied the development of the lymphatic system in the frog and added an extensive study of the growth of the lymphatics in pig embryos from 9 to 18 em. long. He observed endothelial sprouts in growing lymphatics and interpreted them as Langer had done 27 years before to mean that the growth of the lymphatic capillaries is by the process of sprouting. Some of the very large lymphatic vessels which he found in the mesentery he interpreted to mean degeneration or retrogression of the system. Ranvier suggested the theory that the lymphatic system comes from the veins, on the basis that the growth is from centre to periphery rather than from the connective tissue spaces to the veins—but he did not prove his theory, for he did not find lymphatics in embryos below 9 em. in length, at which time the lymphatic capillaries have already covered the surface of the body. W. J. MacCallum was the next one to call attention to this method of growth by sprouting and he has given graphic descriptions of the process. He studied developing lymphatics in the skin of embryo pigs, 5 to 15 em. long, watching the injection under the microscope in order to determine the relation of the lymphatic capillaries to the connective tissue cells and spaces.§ In studying the growth of the lymphatic capillaries in the skin of the embryo pig, I found that the early lymphatics started from certain centres and gradually spread over the surface of the body.® The first of these areas is in the neck, from which vessels grow over the head, shoulder and back. The second is over the crest of the ilium for the vessels over the back and hip, while subsequent centres form the axilla and inguinal region for vessels to the ventral aspects of the body wall and limbs. By studying the figures in Volume ITI, "Ranvier. Comptes Rendus de l’Acad. d. Sciences, 1894 to 1896, and Archives d’Anatomie microscopique. Paris, 1897. SMacCallum. Die Beziehung der Lymphgefiisse zum Bindegewebe. Arch. f. Anat. u. Phys., Anat. Abth., 1902. *Sabin. American Journal of Anatomy, Vol. I, 1901-1902, Vol. III, 1904, and Vol. IV, 1905. The Lymphatic System in Human Embryos. 49 of the American Journal of Anatomy, which show complete injec- tions of the skin for each stage, it will be seen that the lymphatics in- vade non-lymphatie areas, even in the last figure of the series where all of the systems have anastomosed over the body there is a marked non-lymphatic area over the top of the head as well as over the feet. In pigs longer than 5.5 em., it is difficult to obtain such extensive injections because valves begin to develop and tend to make the lymph flow from periphery to centre. During this early period of the spread of the lymphatics over the body there are no valves whatever, which accounts for the wide extent of the injection shown for a pig 5.5 em. long. To trace these vessels back to their source was fundamental to an understanding of the lymphatic system, and I began with the group in the neck as it was the primary group. ‘The vessels in the neck converge to a sac which is readily demonstrated by injection as is shown in Fig. 1, Vol. IV, American Journal of Anatomy. This sac, which lies against the internal jugular vein, is the beginning or anlage of the lymphatic system. In embryo pigs from 14.5 to 16 em. long there are symmetrical jugular sacs opening into the vein. Saxer made mention of these sacs as a part of the lymphatic system, but did not realize their fundamental significance.’ These sacs are either empty or contain a few blood corpuscles. F. T. Lewis worked on the stages before this lymphatic sac is formed and carried our knowledge a step farther by showing that they are preceded by a plexus of veins opening into the jugular vein.1t This plexus of veins gradually becomes cut off from the main vein and by the coalescence of the small veins a sac is formed which is entirely free from the jugular vein for a time. Subsequently the symmetrical sacs rejoin the veins. The endothelial lining of these saes is thus derived from the endothelium of the veins. In studying the lymphatics Dr. Lewis - used the method of graphic reconstruction. The fact that the jugu- lar sacs are transformed venous capillaries, I was able to entirely confirm by the method of injection in pig embryos.1?_ In pig embryos *Anat. Hefte, Vol. VI, 1896. “Lewis. The Development of the Lymphatic System in Rabbits. : Amer. Jour. Anat., Vol. V, 1906. “Sabin. Anat. Record, Vol. II, 1908. 50 Florence R. Sabin. 13 and 14 mm. long there is an abundant plexus of capillaries anterior to the junction of the primitive ulnar vein with the internal jugular vein, readily injected from the veins. In embryos slightly older this plexus of capillaries is being transformed into a sac, and these sacs are less readily injected from the veins. For example in an embryo 15 mm. long, the sac was injected on the side from the vein and not in the other. About this time then the primary connections with the vein become severed. In my specimens the sacs are filled with blood. When the secondary opening into the veins is formed the sacs become empty and this is true in pig embryos 16 mm. long. In connection with human embryos I shall show how to determine the presence of these secondary openings or valves. This method of formation of the jugular sacs was also confirmed by Huntington and McClure in studying cat embryos.’* They have followed all the details of the transformation of the simple veins to the abundant venous plexus and the sac formation by Born’s method of reconstruction. Thus the origin of the jugular sac has been worked out in the pig, the rabbit and the cat by the methods of injection and of reconstruction both in two and in three dimensions. The formation of this jugular sac will also be illustrated in the human embryos in this paper. Besides the jugular sacs two other paired sacs and two unpaired have been described. Lewis described symmetrical subclavian sacs in the rabbit, which in human embryos are, however, an extension of the jugular sacs; the other paired sac is the posterior or sciatic one, noted in the pig and more fully marked out in this paper for human embryos. The unpaired sacs are the cisterna chyli and the mesenteric or better retroperitoneal sac. This sae was discovered by Lewis its origin and development have been worked out by Baetjer; its significance is brought out in Heuer’s work in connection with the lymphatics of the intestine. Mr. Baetjer** has shown conclusively that the retroperitoneal sac begins as a series of small veins which bud off from the renal veins. In his figures are shown the small veins in the root of the mesen- 8Huntington and McClure. The Anatomical Record, Vol. II, 1908. “Baetjer. Amer. Jour. Anat., Vol. VIII. The Lymphatic System in Human Embryos. 51 tery of a pig embryo 17 to 19 mm. long. It is readily noted that these small veins are injected from the main veins as the drawings show the injected ink of the specimens. As the embryo develops, these small veins enlarge and coalesce to form a sac, which shows a few connections with the veins, as proved by injection until the embryos are 23 mm. long. The sac is completely formed at 30 mm., when it is eut off from the veins entirely and clearly connected with the cisterna chyli. Baetjer’s series of nine drawings show every stage in process of the transformation of the veins into the sac and its subsequent connection with the lymphatic system. Thus to sum up, it will be seen that the lymphatic system begins as a series of sacs of which eight haye been described; three paired, the jugular sacs, the subclavian and the posterior lymph sacs; and two unpaired, the retroperitoneal and the cisterna chyli. In the human embryo there are only six, for the subclavian sacs are extensions of the jugular sacs. All of the sacs are shown in Fig. 12, in a human embryo 30 mm. long. The method of origin of two of them, namely the jugular sacs and the retroperitoneal sacs, has been worked out with care showing that they are clearly derived from the veins. The jugular sacs form a secondary connection with the jugular veins, the other sacs forming in regions where there is great shifting of veins do not form secondary communications with their own veins but join the other lymph sacs to make a primitive system. The question now arises whether these sacs can be considered as analogous with the amphibian lymph hearts. None of the mamma- lian sacs studied develop any muscle in their walls; throughout their history they have a lining simply of endothelium, but they all are in regions from which ducts radiate out to drain wide areas, so that as the system begins to function the lymphatic stream converges to these sacs and in this sense they represent the lymph hearts. In the chick the posterior lymph sacs are true lymph hearts, for they develop a muscular wall, and from Sala’s description it is easy to see that these hearts really arise by exactly the same process as the mamma- lian sacs. The fate of the lymph sacs has some bearing on the subject.1> This has been followed for the jugular sac in the pig and ®Sabin. Amer. Jour. Anat., Vol. IV, 1905. 52 Florence R. Sabin. for all the sacs in the human. They all become completely trans- formed into a group of lymph nodes except the cisterna chyli, which is partially though to a varying degree transformed. The lymph sacs make the great primary groups of nodes for each region through which lymph must pass before entering the veins. Thus for example in the intestines the preaortic nodes are the primary group and they come from the retroperitoneal sac, while the nodes of the mesentery are secondary, tertiary, ete. Thus we may define primary lymph nodes as those that are derived from the lymph sacs, and they are also primary in the sense of being the first to develop for a given region. It therefore seems to me that it is fair to conclude that the lymph sacs of the mammals, which represent the lymph nodes, take the place of the lymph hearts of the amphibia. They do not of course represent the same function, for they never have any muscle, so they never pulsate, and from the beginning they must cause a slowing of the lymph flow rather than a hastening of it and this slowing must become much more marked as they are transformed into lymph nodes. Thus they seem to me analogous to amphibian lymph hearts. From the preceding analysis of the literature, it is clear that there is a general agreement among recent workers that the mamma- lian lymph sacs precede the lymph vessels, and hence form a primary lymphatic system and that these sacs are derived from the veins. This position has been very greatly strengthened by the work of Favaro’® and Allen,’ on the lymphatic system in fishes, and by Knower’s and Hoyer,*® in the amphibia. Favaro discovered that in fishes the lymphatics come from the veins, and that here the relation of the lymphatics to the veins is much more primitive than in mammals. Lymph hearts and vein hearts may be present, moreover one and the same vessel may carry either blood or lymph either at the same time or at different times. Thus tHavaro. Atti R. ist Veneto di se. lett. ed arti, 1905-06, T, 65, Parte seconda. Appendice alla Dispensa 10. Octobre 1906. S. 279. Venezia 1906. “Allen. Proceedings of the Washington Academy of Sciences, Vol. IX, 1907. SKnower. Anat. Record, Vol. II, 1908. “TToyer. Bulletin de l’Acad. d. Sciences d. Cracovie, 1908. The Lymphatic System in Human Embryos. 53 he speaks of venee lymphaticee and vasa lymphatice. The system varies much in the different forms; in Urodeles he finds that the lymph hearts begin as a swelling of one of the primitive lateral lon- gitudinal veins. This abstract is taken from Schwalbe’s Jahres- berichte. } Knower and Hoyer have shown independently that the funda- mental points maintained for mammals are true also for the amphi- bia. They have found that the anterior lymph hearts are the first structures of the lymphatic system to appear in the embryo and have described their origin from the veins. They have found that the first lymph vessels are derived from the lymph hearts, this being stated by Hoyer on page 463 of his article as follows: ‘“‘Beriicksichtigen wir weiterhin die Art und Weise, in welcher sich die Lymphgefasse entwickeln, namentlich das Auftreten der vorderen Lymphherzen an der vorderen Vertebralvene sowie der Lymphgefasse, welche aus dem Lymphherzen hervorgehen, so kann man sich dem Gedanken nicht verschlieszen, dass das Lymphgefasssystem eben an diesen Stellen symmetrisch auf‘beiden K6rperseiten seinen Anfang nimmt und sich von dort aus iiber den ganzen Korper verbreitet. Als wichtige, diese Ansicht stiitzende Tatsachen hebe ich aus der vor- hegenden Arbeit hervor: Die weite Kommunikation des sich entwick- elnden Lymphherzens mit der Vene, die anfanglich mit einer kegel- formigen Zelle endigende freie Spitze des spindelférmigen Lymph- herzens, welche sich spater zu einem Zellstrange verlaingert und sich schlieszlich zu einem Lymphgefisse umbildet, ferner die rege Zell- vermehrung im Gebiete des sich entwickelnden Lymphherzens und schlieszlich die Entwicklung der zwei auf den Kanten der Myomeren einander parallel verlanfenden Lymphgefiisse.”’ Both of them state that they will give further evidence of the central origin of the lymph vessels and their growth toward the periphery in their final papers. We come now to the relation of the peripheral lymphatics to the saes and to the origin of the thoracic duct. Here we have a diversity of opinion and certain unsettled points which for the sake of the development of the subject it is fundamental to have perfectly clear. In the first place, Sala, who, in connection with the origin of the posterior lymph hearts, really describes them as coming from the 54 Florence R. Sabin. veins, though he confuses the picture by considering them as coming from tissue spaces at the same time, describes the thoracie duct as coming from solid cords of mesenchyme, and the peripheral vessels as derived from spaces in the mesenchyme. We find nothing to correspond to the solid cords of mesenchyme as an anlage of the thoracic duct in mammals, and believe that the lymph vessels grow out from the primitive sacs. That is, we believe that the conditions found by H. Hoyer for amphibia, that the vessels come from the hearts, is true also in mammals. This being the disputed point however, it will be necessary to review the literature in this connec- tion with care. In 1901 I showed that the jugular lymph sacs are the primary lymphatics in mammals, that they are derived from the veins, that from these sacs, and others, vessels grow out to invade the body and that therefore there are non-lymphatic areas and one can study the invasion of these areas by lymphatic vessels. In the study of the skin this general law was found to hold, that there are areas which at first cannot be injected either directly or through the sacs. This I believe to be because there are no lymphatics to inject. That gradually lymphatics invade these areas and at first a primary subcutaneous plexus can be injected, later a secondary more super- ficial plexus, and finally terminal capillaries in the papille. The same law holds for the lymphatics of the intestine as shown by Dr. G. Heuer in the same number of this journal. In the intestine the lymphatics first form a plexus in the submucosa; secondarily a mucosal capillary plexus forms and from this mucosal plexus the lacteals grow out. In connection with the intestine the fundamental point that the lymphatics grow out from the sacs is also shown. In all the early work the injections of the intestine were made through the thoracic duct, but later it proved that by far the best place to inject is the retroperitoneal sac. This sac gives the key for working out the development of the lymphatics of the viscera. For years I have been trying to get injections of the lymphatics of the lungs and diaphragm and have never succeeded until I intro- duced the needle directly into the retroperitoneal sac. In connec- tion with the intestine, injections into the retroperitoneal sac at The Lymphatic System in Human Embryos. 55 first show no vessels in the mesentery, next vessels inject from the sac into the mesentery and these vessels gradually extend to the bowel wall which they reach in embryos between 4 and 4.5 cm. long. Thus injections of the retroperitoneal sac make it possible to trace the development of the lymphatics to the viscera, and this is an im- portant point in the proof of the general theory. In 1904 F. T. Lewis published an important contribution to our knowledge of the lymphatic system. He studied perfect serial sections of rabbit embryos, worked out the early history of the jugu- lar saes, and discovered the retroperitoneal sac as has been mentioned. In studying the peripheral lymphatics, Lewis pictured a series of small isolated vessels extending along the external mammary and umbilical veins. These isolated vessels are distinguished in sections by being slightly larger in caliber than the neighboring vein. In sections they are clearly isolated. I have had the privilege of examin- ing Dr. Lewis’ specimens and can confirm his observations entirely. In one or two places there was evidently great difficulty in determin- ing whether some of these vessels were isolated or were connected with the vein. Moreover, I can find some of these isolated vessels in pig and human embryos. These numerous lymphatic anlagen of Lewis are now the crucial point in connection with the lymphatic system. They exist undoubtedly in perfect sections, they are always lined by a perfectly formed endothelium and never show any tran- sitions toward tissue spaces. The question is simply, Are they lym- phatie vessels which have grown from the sacs and are only apparently isolated or do they arise in situ? I believe them to be true lym- phaties derived from the sacs and will give my reasons shortly. In connection with Lewis’ observations it is important to make clear the work of Huntington and McClure.?? They have strength- ened the theory that the lymphatics come from the veins the more because they began with a vigorous attack upon the theory. In 1906 they described elaborate models of the developing lym- phatics in cat embryos which showed lymphatic vessels along the veins previous to the formation of the lvmph sacs. These early lymphatics *Wuntington and McClure. Anat. Record, Vol. I, 1906-07, and Vol. II, 1908. 56 Florence R. Sabin. which they termed subintimal, proved to be only tissue spaces and they withdrew this work in 1907. At this time they presented the development of the jugular lymph sacs in the cat, agreeing entirely with the work of Lewis and myself; but in connection with the rest of the system they at that time agreed entirely with Sala, believing that the peripheral vessels were dilated tissue spaces. In the Anatomischer Anzeiger of 1908, McClure gives up entirely the theory of the origin of the lymphatics from tissue spaces and comes to agree with Lewis that sections show multiple anlagen. As I have already said, the multiple anlagen of Lewis are undoubtedly in sections and to interpret them is the crucial point. They cannot be interpreted through sections alone; and merely repeating the observation of them in sections does not add to our knowledge of their interpretation. They must be subjected to some kind of an experiment. The inadequacy of simple observa- tion to interpret them was clear to Lewis for he refrained from mak- ing an interpretation. The experience of Huntington and McClure serves to emphasize strongly the inadequacy of sections alone, and the large part of that personal equation plays in interpretation, for from practically the same type of material they have taken succes- sively three different standpoints. In this laboratory under the direction of Professor Mall a group of people have been subjecting these numerous anlagen of Lewis to some sort of experiment. Dr. Eliot R. Clark?! has been studying the blood vessels and lymphatics in the living tadpole’s tail. His speci- mens prove Langer’s suggestion that lymphatics grow by the sprouting of their endothelial lining cells by making it possible to watch them grow. His observations and descriptions of these lymphatic eapilla- ries, sending out long sprouts that now move forward, now bend out of their course to pick up some stray blood corpuscle and now retreat, make one realize how little sections show us. Certain of his observa- tions are exceedingly fundamental, first in the non-lymphatie zone in the living specimen there are no isolated anlagen. This you can never say with certainty in sections because as will be shortly proven lymphatics can be demonstrated by injection where they cannot be ™Clark, Anat. Record, Vol. III, 1909. Or bo | The Lymphatic System in Human Embryos. seen in sections. But in these specimens of the entire living tail, endothelium can be distinguished from mesenchyme, and the lym- phatics grow out from their own endothelium and do not add any peripheral anlagen. A second point which Dr. Clark observed, but did not publish, is the sudden collapsing of a part of a lymphatic vessel. Once or twice while a red blood cell was pushing its way into the vessel, its central end collapsed suddenly to an endothelial thread, while the peripheral end remained dilated. These collapsed lyn- phatics right in the middle of a vessel were noted and figured by Langer; they have been noted many times in blood capillaries, for example, see Fig. 49 of Stricker’s Handbuch der Histologie, but they have been interpreted as evidences of growth simply, while it may be that these collapsed vessels are a part of the functional activity of the lymphatic capillaries. The reverse of this process of collapsing of the vessels, namely the sudden opening up of tiny vessels during an injection, I have often observed and used as an argument in favor of continuous lymphatics rather than isolated anlagen. (See Symposium on the Lymphatic System. Anat. Record, Vol. IT, 1908.) It can be readily seen that in cross section these en- tirely collapsed vessels might be wholly lost and only the dilated por- tions shown, and thus the suggestion is that Lewis’ anlagen represent a transitory phase of the functional activity of the lymphatic capil- laries. The question of the multiple anlagen has resolved itself wholly into a question of method, with the study of the living lymphatics and injected lymphatics on the one hand and the method of serial sections on the other. Ludwig’s famous phrase, “die Methode ist alles,” was never more apt than in this connection, for it sums up the whole situation. Having long worked with injections we are con- vinced that uninjected serial sections are wholly inadequate to show all the blood capillaries or lymph capillaries, moreover, we are con- vinced that Dr. Lewis has carried the observations as far as they ean be carried with sections and that sections will always show these apparently isolated anlagen. ‘To put the contention that serial sections are inadequate to the test, Dr. Clark has made the following 58 Florence R. Sabin. ' experiment. He made a careful camera lucida drawing of the lym- phaties in the living tadpole’s tail, then killed the tadpole and cut it in serial sections and tried to reconstruct the lymphatics. The failure in reconstructing these amphibian lymphatics confirms similar attempts of my own on mammalian lymphatics and makes me feel sure that uninjected capillaries cannot be completely reconstructed. The same point in connection with the blood vascular system, namely that the blood capillaries cannot be reconstructed from unin- jected specimens no matter how perfect, will be conceded, but has been brought out much more strikingly by the work of Dr. Evans, soon to be published from this laboratory, for he has shown that a blood capillary plexus can be demonstrated by injection where it was not known to exist before. Thus the question of the relation of the peripheral vessels to the sacs is becoming more and more clear. There is a primary lymphatie system which consists of sacs that are formed directly from the veins. These primary lymphatic sacs are transformed from a series of isolated sacs into a system by means of the thoracic duet and the right lymphatic duct. These two structures form a part of the primary system. The secondary system consists of the peripheral vessels which, it is beconiing more and more sure, are an outgrowth from the sacs. Thus we can say that the primary system, as far as it is made up of sacs, comes from transformed veins, and that the secondary system, characterized by being formed of lymphatic ducts and capillaries, develops by endothelial sprouting from the sacs. It remains now to be determined whether the thoracic duct develops after the manner of the primary sacs as transformed branches of the azygos veins or whether it develops as the other lymphatic duets of the body do, from endothelial sprouts from the sacs. No theorizing can decide between these two ideas. We must wait some decisive method of getting at the facts. The presumption seems to us to lie on the side that the thoracic duct develops in the same manner as all the other ducts, since wherever the isolated anlages of ducts can be tested, as, for example, in the living tadpole’s tail, they prove not to exist. Moreover, Dr. McClure, who is at present the advocate of the idea that the thoracic duct arises as a series of The Lymphatic System in Human Embryos. 59 independent spindle spaces along the aygos vein, shows himself the weakness of his own position. He says, referring to serial sections,?* “These outgrowths, in the writer’s estimation, constitute the veno- lymphatic anlages of the thoracic and right lymphatic ducts.” That is to say, the entire argument rests on the interpretation of appear- ances in sections, and it is becoming more clear each year that inter- pretation of sections is not proof. Tur Lympuatic System In Human Embryos. Based upon these studies of the lymphatic system in the pig, rabbit and cat embryos, I have studied through the Mall collection of human embryos. I do not believe that the subject could have been worked out with human embryos alone, for the real advances in the study of vascular problems have always come from the method of injection. The method cannot be well applied to human material on account of its searcity, but the points determined in other mamma- lian embryos can be verified in serial sections of human embryos. Moreover, the Mall collection is sufficiently ample to illustrate all the essential points of the origin of the lymphatic system. In several points I think it adds new evidence to that already gained from other mammals; for example, in connection with the history of the thoracic duct, in gaining a conception of all of the primitive sacs as forming a primitive system, in tracing the posterior sacs which had thus far been seen only in pig embryos among mammals, and in follow- ing the transformation of all the sacs into lymph nodes. It is a very great pleasure to thank Professor Mall for the privilege of studying his valuable collection and for many helpful suggestions during the progress of the work. In the Mall collection no trace of a lymphatic system can be made out in embryos of the first four weeks, from 2 to 8 mm. long. In these there are certain spaces which might be confused with a lym- phatie system, first certain areas where the meshes of the connective tissue are especially large, as, for example, around the developing celom, and secondly spaces which follow the course of the nerves. ™=MecClure. Anat. Anz., XXXII Bd., 1908, p. 536. 60 Florence R. Sabin. These spaces along the nerves, which may be termed perineural spaces, are especially important to note both on account of their physiological significance and because they have been confused with lymphatics. They may be injected from the space around the spinal cord and they are especially large around the growing tips of the nerves. Their constancy, their presence in perfectly prepared speci- mens, and especially their size at the growing tips of the nerves, leads one to think that they are physiologically of great importance to the nerves, but they never form a part of the lymphatic system. No injections of these spaces ever run over into the lymphatic system. An injection into the developing arachnoid spaces around the spinal cord will often pass into the veins, entering them around the fourth ventricle, but I have never succeeded in injecting any lymphatic vessels from the arachnoid nor in tracing any lymphatic vessels to the arachnoid, so that I believe the older anatomists, for example Breschet, were right in believing that the lymphatic system does not drain the great arachnoid lymph space which rather retains its primitive relation to the veins while other parts of the body become drained by a new system of capillaries, namely the lymphatics, derived from the veins. In studying Professor Mall’s collection it seems that there are two stages to be made out in the development of the system as a whole. This has been illustrated in the table on the next page. The first includes a study of the origin of all of the primitive sacs and their fusion into a primitive lymphatic system through two factors, namely the formation of the valves of the jugular sacs which make the per- manent openings into the veins, and secondly the connection of the various sacs by means of the cisterna chyli and thoracic duct. This period includes embryos up to 30 mm. in length, of which there are seventeen in the series. ‘This first stage may be divided into two periods, one of which there are fourteen specimens, measuring up to 20 mm., which have the jugular sacs alone; the other shown in three specimens, which mark the time of origin of the other sacs and of the thoracic duct. The second stage involves the transformation of the sacs into the primary lymph nodes and the spread of the peripheral lymphatics. The Lymphatic System in Human Embryos. 61 The first point is well illustrated in the Mall collection; the second, namely the spread of the peripheral lymphatics, needs ample material for injection. It has been worked out only for the lymphatics of the intestine and skin in the pig. To return to the first stage, namely that of the origin of the sacs, one can outline the course of development as follows. The jugular lymph sacs begin in an embryo 8 mm. long, the valves are first seen at 10.5 mm. The sac reaches its maximum development at 30 mm. when it attains a size of 5x 3.6 mm. The beginning of the process of the bridging of the sac, which is the process by which the sac is ultimately turned into a chain of lymph nodes comes early, namely in an embryo 14 mm. long. The process of the transformation of the jugular, sac into nodes is about complete in an embryo of 80 mm. At 20 mm. there begin to be signs of the formation of the other sacs in the presence of a plexus of veins in the region of the mesenteric sac and the posterior lymph sacs, and at 23 mm. there is a definite retroperitoneal sac and a cisterna chy. By 24 mm. all three of the sacs are well formed, namely the mesenteric, the cisterna chyli and the posterior lymph sacs. All the sacs together with the thoracic duct are illustrated in Fig. 12, for an embryo of 30 mm., which marks the stage of the completion of the primitive system. The posterior lymph sae which is second in size to the jugular apparently reaches its maximum in an embryo 80 mm. where it measures 2.8x2x38.5mm. Ihave no higher stages but judge from its appear- ance that it will soon be entirely cut into lymph nodes. The retro- peritoneal sac, so large in the pig embryos, is always small in the human, and the cisterna chyli is the smallest of all. The thoracic duct is complete in an embryo 30 mm. long. These facts are summed up in the accompanying table. In the table, where one measurement is given, it represents the length of the sae or its antero-posterior diameter; where two measurements are given the first is the length, the second the width or lateral diameter. I shall now describe in detail the lymphatics in each of the 22 embryos listed in the table. The earliest specimen in the Mall collection to show any traces of the beginning lymphatic system is an embryo (No. 397) 8 mm. long. In this embryo, as shown in 62 Florence R. Sabin. : F ie i Ih ah 6B | BS a a8 ce Direction g 5 Ey I e say Hs Jugular Lymph Sac. | Other Lymph Saes. poy ieee ae | a | ss ES | bis | aed» Size in mm. | 8 397 | Trans-| 10 | .3x.19 | Prelymphatic | | verse plexus of veins. | 9 | 163 | Trans-| 20 | ..36x .14 Same. | verse 10.5 | 109 | Trans-| 20 | .7x .28 |Symmetrical| | verse sacs,empty, with valves. 11 353 |Coronal| 10 122 Sac full of blood (Ant. post.)|Extensive plex-) | us of veins along jugular vein. Valve formed but apparently not open. Ex- tension of jug-) ular sac along primitive ulnar | vein. 12.5 | 317 |Coronal; 20 1.5 Definite longsac out of preceding plexus of veins. | | Valve. | 14 144 |Sagittal 40 1.5. |Sac empty, be- ginning of bridg-| | ing. 15. 350 |Coronal| 10 Very abortive sac. 15 423 | Trans-| 50 a) Very small sac. | verse © | V7 106 | Trans- , 50 Very abortive verse © sac. | ibe 424 Region damag’d. 17, 296 |Coronal 20 1.5 Sacs large,valve undoubtedly open. Small ex- | tension along | primitive ulnar vein. Length of | Embryo in mm. _ [=r] 20 20 The Lymphatic System in Human Embryos. 24 30 46 50 50 50 80 No. of Embryo in Mali Collection. 74 22 128 382 Other Lymph Sacs. Saes wider, cut |Small groups of vessels along by developing|renal anastomosis. Large median anastomosis of sciatic veins. Groups of veins along sciatic vein, anlage of sacs. Median A a Direction Z g | o g st Jugular Lymph Sac. Section. ee | ‘Size in mm. Trans-| 50 | 1.8 Symmetrical verse | jugular sacs. No extension along primitive ulnar | vein. Trans- | 50 | 1.6 verse | | nerve. First vessels from the | | sac to the skin. Coronal, 50 .75 |Abortive. | Sagittal; 50 2x1_ |Sacs and valves Trans- 20 Large. verse ‘Coronal 50 5x3.6 |Maximum size, begin’g lymph- nodes along | jugular sac and | subclavian vein. Sagittal, 10 |3.75x1.5\/Sac with few | lymph nodes. Sagittal | 100 4x1.5 |Sac turning into lymph nodes. Trans-| 50 | 3x1.5 |Many follicles. verse | | Sagittal 5|0and 4x1.75 |Fine bridges all | | 100 — thin. | Trans-, 100 1.75x1 |Chainsof lymph | verse | | nodes. Posterior Lymph Sac. | a Present 4.6x1 with begin- ni’g lymph- hnode. Present 25x 21D Sse de 7s Sae with nodes meas- uring 2.8 x Pg SENG Ny posterior lymph anastomosis of renal veins. Cisterna | Retroperi- | Chyli. jtoneal Sac. Present | Present | Present | Present | Present, | Present thoracic duct com- plete. | Not Present, | found. |no nodes. Not Present, found [no nodes.) 12 Large | Lox | Dam|aged | Present, |Mass of | surround-| lymph- ed by | nodes. nodes 64 Florence R. Sabin. diagrammatic form in Fig. 1, there is a group of vessels lying external to the internal jugular vein near its junction with the primitive ulnar vein. The figure can be interpreted by reference to Fig. 4. These vessels are completely filled with blood and yet I cannot find any openings from them into the veins, or into each other, and thus interpret them after a study of corresponding stages in other mammalian embryos as a plexus of veins which have separated from the jugular vein preparatory to the formation of the anterior or jugular lymph sac. These vessels are small, the largest measuring Q » aD ‘Deon, prea (| Fie. 1 Fic. 1. Reconstruction of the plexus of small veins lateral to the V. jugularis interna in a human embryo, 8 mm. long, (crown rump). Mall collection, No. 397. % about 50. The plexus of veins is shaded. D. C., ductus Cuvier; V. j. i., vena jugularis interna; V. u. p., vena ulnaris primitiva. Fic. 2. Reconstruction of the small veins lateral to the right V. jugularis interna in a human embryo 9 mm. long. Mall collection, No. 1638. > about 50. Of the veins, six are shaded, indicating that they are full of blood, while the two with heavy outlines are nearly empty. Lettering same as Fig. 10. approximately .3 mm. in the antero-posterior diameter, by .19 mm. laterally. The next specimen in the series is an embryo (No. 163) measuring 9 mm. This specimen shows a similar condition but with certain differences. In the first place the plexus of isolated vessels occupies a slightly different place, as seen in Fig. 2. They lie farther ventral- ward, extending over the body wall external to the heart. Most of these vessels are well filled with blood, while two are nearly empty. This embryo differs also in having an asymmetrical development, the The Lymphatic System in Human Embryos. 65 vessels representing the forerunners of the lymphatics being much larger on the right side than on the left. The three largest sacs of this series measure .27 x .19 mm., .86x.14 mm. and .27 x.14 mm. ° respectively. The next specimen is an embryo (No. 109) measuring 10.5 mm. In this embryo there are symmetrical jugular sacs as seen in Fig. 3, just external to the internal jugular vein. The relation of the sac to the venous system as a whole is shown in Fig. 4, which is a reconstruction from serial sections. This embryo has been figured Fic. 8. Transverse section through the neck of a human embryo, 10.5 mm. long, showing the symmetrical jugular lymph sacs. Mall collection, No. 109. A., artery; N. X., n. vagus; m. s., n. sympatheticus; Oe., oesophagus; P., pericardium; S. 1]. j., saccus lymphaticus jugularis; T. trachea. The VY. jugularis are filled with blood and lie just medial to the sacs. by Bardeen and Lewis, American Jour. of Anat., Vol. I, 1901-1902, and by Dr. Mall, American Jour. of Anat., Vol. IV, 1905; the outline and some of the details of the figure are taken from their reconstructions. As will be seen in Fig. 4, the sac lies external to the jugular vein and anterior to the primitive ulnar. In this embryo the question of a valve is an interesting one. In studying through Dr. Mall’s collection it has proved that the finding of the valves depends wholly on the plane of the section. There is only one plane which is at all adequate for determining the << fs "Uy izes Fic. 4. Reconstruction of the right jugular lymph sacs, shown in solid black, in a human 10.5 mm. long. Mall collection, No. 109. » about 14. G. N., gasserian ganglion; S$. v., sinus venosus; V. c., V. cephalica: V. ¢. i, vena cava inferior; V. h., vena hepatica; V. j. i., vena jugularis interna; V. p.,. vena porta; V. p. ¢., vena cardinalis posterior; V. s. ¢., vena subcardinalis ; V. u. (p.), Vena ulnaris (primitiva) ; V. u., vena umbilicalis; W. b, Wolffian body. The Lymphatic System in Human Embryos. 67 valves, namely the coronal. This will be readily seen in Fig. 10, which shows that the valve is made by a long projection of the lymphatic duct into the angle of the internal jugular vein with the cephalic vein. Imaginary cross sections through this figure will show that the place of the valve would be represented by a small duct in the angle between two veins and this is exactly what is seen in Fig. 5 for this embryo. A study of Fig. 10 will also show that there could be nothing distinctive of the actual opening of the lymphatic to the vein in cross sections, for they would consist simply of a double layer of endothelium between the veins. In like manner sagittal sections are still more difficult than transverse ones for locat- ing the valves and indeed only in an occasional, fortunate section can it be accurately done. DaFeters Fic. 5. ‘Transverse section through the jugular sacs of a human embryo, 10.6 mm. long. Mall collection, No. 109, to show the left valve. ~ 28. A., artery; N., nerve; Oe., esophagus; P., pericardium; S. 1, j., vena jugularis; V. j. i., vena jugularis interna. To return to the embryo 10.5 mm. long, I think that the valve is present, for, as is seen in Fig. 5, there is a small duct in the angle between two veins and the duct connects with the sac as traced in serial sections. Secondly, the lateral vein in this section is the cephalic, see Fig. 4, and is therefore in the exact position of the undoubted valves seen later in Figs. 10 and 13. Whether this valve is open or not it is impossible to say. The sacs are both empty, and in the earlier stages where there are no valves they are often, though not always, full of blood. They measure .7 x .28 mm., showing a con- siderable increase over the two preceding specimens. This embryo 68 Florence R. Sabin. shows one further point of interest, namely a possible beginning of the thoracic duct in the shape of a duct running over toward the aorta as shown in Fig. 6. The entire question of a thoracic duct will be discussed later, on page 77. The next embryo of the series (No. 353) is 11 mm. in length. This embryo is cut in coronal sections which proves to be the best plain not only for seeing the valves but for understanding all of the cervical lymphatics. This embryo is represented in a series of three figures, 7, 8 and 9, two of them sections and the third a diagram Fig. 6. Transverse section through the left jugular sac to show the possible beginning of a vessel growing down to form the upper part of the thoracic duct. x 40. A., aorta; N. X., N. vagus; Oe., cesophagus; S. 1. j., saccus lymphaticus jugularis; V. j. i.. vena jugularis interna. from the same series. In Figs. 7 and 8 will be seen the extension of the lymphatic plexus along the external border of the jugular vein. These two figures show a number of important things, first in connection with the veins, they show the relations of the primitive ulnar and the cephalic to the jugular vein; for the lymphatics they show the relation of the lymphatics to the cephalic vein and in general to the arm bud. These relations are all brought together in the diagram of Fig. 9. Here it will be seen that this plexus which appears isolated in Fig. 7 is really continuous. The plexus is actually much more complex than is shown in Fig. 9. Measuring The Lymphatic System in Human Embryos. 69 from the valve, which is in the angle between the cephalic and internal jugular veins, it extends 1.2 mm. along the jugular vein. By a comparison with the reconstruction of the preceding stage, Fig. 4, I think that the sections shown in Fig. 7 and 8 suggest that the sac is extending along the jugular vein by means of a plexus of veins. The next point of interest is the location of the valve. In Figs. 7 and 8, it will be seen that the beginning cephalic vein is easily recognized by its position opposite the upper curve of the arm bud. The lymphatic sac runs deep into the angle between the cephalic and the internal jugular veins, Fig. 7, but in no section is there any break in the endothelium of the sac, which leads one to think that the valve may not yet be open and that this fact may account for the complete filling of the lymphatic sacs with blood. The in- ternal jugular vein is only partially filled with blood. The blood of the vein itself was omitted in the drawing. In this embryo there is an extension of the jugular sac along the primitive ulnar and lateral thoracic veins. This extension forms the subclavian sac which gives rise to the lymphatics of the arm, Fig. 9. This is especially interesting in connection with F. T. Lewis’s observations on the subclavian sac in rabbits where it begins as an isolated sac. I was able to confirm Lewis’ observations on his specimens of rabbit embryos, but feel sure that in human embryos the sac in the arm bud is an extension of the jugular sac. The sac along the ulnar veins measures .8 mm. beginning from the valve. This makes 2 mm. the total extent of the lymphatics in this embryo. By putting together Figs. 7 and 8, relating them by the position of the cephalic veins, it will be noted that following along the ex- ternal border of the internal jugular vein there are a series of branches which we might call segmental, some of them, as for example above the lymphatics in Fig. 7 or between the lymphatics and the primitive ulnar vein in Tig. 8, are obviously small veins, others like the cephalic and primitive ulnar are large veins, while still others are being transformed into lymphatics. This suggests the process of transformation of the various branches of the internal jugular vein from the original simple segmental type into the adult system. In these transformations some of the branches become 70 Florence R. Sabin. enlarged, others reduced and dropped out, while still others are changed into lymphatic sacs. The next embryo of the series (No. 317), measuring 1214 em., has not been illustrated because it is exactly like the preceding except that the lymphatics have much less blood, and the plexus along the - xe ¥ er ae 4) Ue. TE: 2 re Fic. 7. Coronal section through the arm bud of a human embryo, 11 mm. long. Mall collection, No. 353, to show the plexus of veins or lymphatic sacs along the internal vein. This section is to be related to Fig. 8 by means of the composite section, Fig. 9. \ about 36. S. 1. j., saccus lymphaticus jugularis; V. c., vena cephalica; V. j. i., vena jugularis interna. Fic. 8. Coronal section through the arm bud of the same embryo as Fig. 7, to show the relation of the lymphatic sac to the primitive ulnar vein. The larger lymphatic sac is the upper part of the extension along the prim- itive ulnar vein, shown in Fig. 18. about 36. S. 1. j., saccus lymphaticus - jugularis; V. ¢., vena cephalica; V. j. i., vena jugularis interna; V. t. l.. vena thoracicus lateralis; V. u. (p.), vena ulnaris (primitiva). jugular vein has been definitely transformed into a single sac. The extent of the lymphatics along the jugular vein is practically the same. The valve is definite, showing the same type as seen in Fig. The Lymphatic System in Human Embryos. rial Fic. 9. A composite diagram made by superimposing the sections showing the jugular sacs, as shown in Figs. 16 and 17, of human embryo, 11 mm. long, Mall collection. » about 36. D. C., ductus Cuvier; V., position of valve; V. j. i, vena jugularis interna; V. t. 1., vena thoracicus lateralis; V. u. (p.) vena ulnaris (primitiva). 72 Florence R. Sabin. 10, except that an opening cannot be made out. It is impossible to say whether the opening is not present or whether the shrinkage of preservative is sufficient to conceal it. The sixth embryo of the series (No. 144), 14 mm. long, shows only one new point in the formation of the jugular sac, namely the beginning of the process of bridging of the sac which is illustrated for a later stage in Fig. 14. This cutting of the sacs by slender con- nective tissue bridges, which has already been described in the devel- opment of the jugular lymph sacs in the pig, is, I believe, the beginning of the transformation of the sac into a lymph node.” This will be considered later. Two of the embryos of the series, one (No. 350) measuring 15 mm. and the other (No. 106) measuring 17 mm., have very abortive sacs near the junction of the primitive ulnar with the jugular vein. In both instances the preservative is not good enough to show the en- dothelium, so there is no way of telling these small sacs, which measure less than half a millimeter in their longest diameter, from mesenchyme spaces except by their position in comparison with other embryos. They certainly are an evidence that there are marked irregularities in the development of the lymphatic system. Another embryo of 15 mm. (No. 423) has also only a small sac, this one measuring .9 mm. In the collection there are some embryos in which the preservative is too poor to admit determining the lymphatics at all, but out of the series of 22 which have been studied there are four cases of abortive jugular sacs, or 18 per cent. These embryos measure 15, 17 and 20 mm. The next specimen (No. 424), measuring 17 mm., is valuable, for it has a double vascular injection. An extravasation along the jugular region interferes with a study of the jugular lymph sacs, but the vascular injection of the posterior part of the embryo gives conclusive evidence that the other sacs, namely the retroperitoneal, the posterior and the receptaculum chyli have not begun. Embryo (No. 296) measuring 17 mm. is the earliest specimen in which I have found a valve undoubtedly open. This is shown in 4Sabin. Amer. Jour. Anat., Vol. IV, 1905. The Lymphatic System in Human Embryos, 73 Fig. 10. It is in exactly the position found in the embryo 11 mm. long (Fig. 7) and in No. 317 which is 12.5 mm., which are cut in coronal sections, but in the two earlier embryos I could not make out the break in the endothelium. ‘The extent of the lymph sac in the section is 1.5 mm. and there is a slight extension along the prim- itive ulnar vein. The next embryo of the series (No. 74) measures 16 mm. In Dr. Mall’s catalogue it is placed after those measuring 17 mm., for Bee, EP By OWN pa ee, Pate as, ope ae “4 “Wath se gee oe dy. “ntay oe 0: Ae Sef ORAS Vege oh aytalenbe ‘ So eee Fic. 10. Coronal section through the arm bud of a human embryo, 17 mm. long, Mall collection, No. 296, to show the open valve of the jugular lymph sac in relation to the veins. » about 26. S. 1. j., saccus lymphaticus jugu- laris; V. c., vena cephalica; V. j. i, vena jugularis interna; V. u. (p.). vena ulnaris (primitiva). it is undoubtedly further developed. This embryo, in which the sec- tions are 50 microns thick, is a very satisfactory one for determining the sacs, for the veins are unusually distended with blood and the lymph sacs are filled with a serum which takes a definite stain. The sacs extend a distance of 1.8 mm. along the internal jugular vein. There is no sac on the primitive ulnar vein and there are 74. Florence R. Sabin. no traces of the other sacs in the posterior part of the body. The veins are especially large in the posterior part of the body. The sections are too thick to show the valves well. The sacs appear as in Fig. 5 except that they are larger. The next embryo of the series (No. 22) measures 20 mm. and shows several interesting points. The series is cut transversely and the sacs also appear much as they are shown in Fig. 3 for an embryo 10.5 long except that they are much wider. The sacs measure 1.6 x 7. The new point of interest is, that in this series the third nerve cuts through the sac; in a later stage, in an embryo measuring 30 mm., Fig. 12, three nerves cut through the sac, namely the third, fourth and fifth. For the first time, in this stage, there are vessels extending from the sac toward the skin.. It will be remembered that this is the point in which the recent American workers on the lymphatic system differ. A further point of interest in this series is a group of small ves- sels along the renal anastomosing vein. These vessels, I think, are forerunners of the mesenteric sacs. The indications of lymphatics for the posterior part of the body appear at this stage. In another embryo (No. 128) measuring 20 mm. the jugular sacs are again abortive, measuring only .75 mm. ‘The specimen is, however, very interesting in connection with the lymphatics for the posterior part of the body. In the neck, as we have seen, the early lymphatics are the two jugular sacs, with either an extension or a supplementary sac along the primitive ulnar vein, in the arm bud. In the posterior part of the body three sacs have been found, two of them median, the mesenteric sac and the cisterna chyli; and one paired, namely the posterior lymph sac. In this embryo, in the place of the future cisterna chyli, there is an extensive median vein connecting the two sciatic veins. Just ventral to this, compare with Fig. 12, is the renal anastomosis running through the great mass of the sympathetic system in the hilum of the two adrenal bodies. Around these two large median anastomosing veins there is as yet no evidence of the future median lymphatic sac. However, to the side of the two sciatic veins, just posterior to the median anastomosis, is an abundant plexus of veins on the one side and a possible beginning pos- The Lymphatic System in Human Embryos. 75 terior lymph sac on the other, making a definite indication of the pos- terior lymphatic sacs. This stage is, I believe, just preliminary to the formation of the three abdominal sacs. In the next specimen these sacs become definite. Thus in the first fourteen specimens of the series, measuring from 8 to 20 mm., simply the jugular sacs are present. From now on, that is in embryos above 20 mm., we shall have to follow not only the jugular sacs but the mesenteric sac, the cisterna chyli and the posterior: lymph sacs as well. , The first embryo above 20 mm. in the series, is one (No. 382) measuring 23 mm. ‘This series is cut in sagittal sections. It shows the jugular sac beautifully, which has now reached a size of 2x1 mm., and lies opposite the third, fourth and fifth cervical vertebree. The series, however, is much more important in connection with the other lymphatic sacs. I cannot find the posterior lymph sac, but both the mesenteric sac and the cisterna chyli are present. For these two median sacs the sagittal plane proves to be by far the best. In Fig. 11, which was made by graphic reconstruction, is shown the retro- peritoneal sac in its relation to the renal vein and the suprarenal body. It is designed to relate the mesenteric sac and cisterna chyli to the surrounding structures. The point at which the vena cava turns ventralward, opposite the second lumbar vertebra, marks the position both of the renal veins and also the suprarenal branch which is a large vein running anteriorly along the ventral surface of the supra- renal body. The retroperitoneal sac extends along the renal and suprarenal veins, the latter being hidden in the diagram by the vena cava. In following the suprarenal veins the lymphatic vessels ap- proach the superior mesenteric artery, along which they subsequently grow out into the mesentery, as has been shown by Heuer.?” The line of mesentery is shown in the diagram. The diagram shows the mass of sympathetic ganglia closely related to the suprarenal body. In this early stage the lymphatic ducts are not likely to be confused with the sympathetic ganglia, but later when nodes begin to develop care must be exercised to distinguish them. The diagram also shows “Heuer. Amer. Jour. Anat., Vol. IX, No. 1. Fic. 11. A composite diagram made by superimposing the sections showing the relations of the mesenteric sac and cisterna chyli to the veins, in a human embryo measuring 23 mm., Mall collection, No. 382. X about 8. A. m. s., A. mesenterica su- perior; C. ¢., cisterna chyli; G. s., gangli sympathetica; S. 1. m., sacecus lymphaticus: mesentericus; S., suprarenal body; V. a., v. azygos; vy. c. i., vena cava inferior. The Lymphatic System in Human Embryos. Ll interesting relations of the cisterna chyli. It arises opposite the second, third and fourth lumbar vertebre, closely adjacent to the inferior vena cava where it anastomoses with the azygos veins. In studying through Dr. Mall’s collection I have become convinced that the cisterna chyli forms one of the primitive sacs and that the thoracic duct may grow forward, 7. e., anteriorly from it. Baetjer has shown ‘that the mesenteric sac soon becomes connected with the cisterna chyli. In this series I cannot find any evidence of a thoracic duct. The cisterna chyli differs from the other sacs simply in not being completely transformed into lymph nodes, though its lower border develops into a large group of them, as will be shown in the last series. The next embryo of the series (No. 6), measuring 24 mm., has a large jugular sac. The series is incomplete so that I cannot get the length of the sac, but its width is the same as the preceding, namely 1 mm. The valves are present and the sac shows much bridging. There is a well defined subclavian sac. This series is also more in- teresting in connection with the other sacs. It shows three things, the retroperitoneal sac, the cisterna chyli, the beginning thoracic duct and the anlage of the posterior lymph sacs. A point of especial interest in this series is in connection with the cisterna chyli. This sac is present as a few vessels dorsal to the aorta; and from the sac duets extend anteriorly immediately adjacent to the azygos veins. On the left side, this duct extends into the thoracic cavity almost to the neck. I cannot trace it to the jugular sac nor is the series perfect enough to enable one to say whether it is present in every section or not, but there is sufficient evidence to indicate that the thoracic duct may be an outgrowth of the cisterna chyli. The thoracic duct has proved to be the most difficult part of the lymphatic system to work out for this reason, we have not yet found a way to inject it in early stages and uninjected sections are not adequate. The evidence of sections is as follows, the jugular sac and the cisterna chyli, which the duct subsequently connects, develop before the duct. The question is, does the duct develop from multiple anlagen from the azygos veins for which there is no proof except that lymphatic vessels can be seen in sections adjacent to The Lymphatic System in Human Embryos. 79 these veins, or does the duct grow from the two sacs, the cisterna chyli and the jugular one. For this second view the evidence is also weak, it consists in this, that other lymph ducts wherever we can study them grow from the sacs; and secondly in pig embryos and in human embryos one can trace a duct forward from the cisterna chyli and caudalward from the jugular sac, and in later stages these two ducts have joined. The weakness of this evidence lies in the fact that in the earlier stages the picture is always liable to be con- fused by Lewis’ multiple anlagen. In both pig and human embryos the stages to be studied for the thoracic duct lie between 20 and 30 mm. In an embryo pig the complete thoracic duct can be injected at 27 mm. It should be quite clearly noted that whichever method of formation of the thoracic duct proves ultimately to be correct, that is whether it arises from the azygos veins in situ or from an out- growth of the lymphatic sacs, the most fundamental point remains the same, that its endothelium is vascular. However, it should be stated here that wherever growing lym- phatice capillaries have been absolutely tested, they grow by the sprouting of their own endothelium rather than by additions of new anlagen trom the veins. This has already been noted for the living lymphatics; it is also shown by the work of Dr. H. M. Evans”* on the growth of new lymphatic capillaries into a sarcoma of the intestine. His injections show that the new lymphatic capillaries are derived from the mucosal plexus and that these new vessels are analogous with the central lacteals of the villi. In the tumor, how- ever, they are developing beyond the normal limits of the terminal lacteals into a spreading plexus. This plexus shows all gradations as seen in Evans’ figure to the normal lacteal. __ From the next specimen (No. 86), measuring 30 mm., a graphic reconstruction has been made of the entire primitive lymphatic sys- tem, Fig. 12. It does not show the extent of the peripheral lym- phaties, but does show the relations of the primitive system. At this stage, as will be seen in the reconstruction, the lymphatic system 2Hvans. On the occurrence of newly-formed lymphatic vessels in malignant growths. Johns Hopkins Hospital Bulletin, 1908. 80 Florence R. Sabin. consists of the large jugular sac, measuring 5 x 3.6 mm., with its large extension along the ulnar vein to the arm bud. Emptying. into the jugular sac on one side is the thoracic duct which connects with a small cisterna chyli. Ventral to the cisterna chyli is the sec- embryo of 30 mm., Mall collection, No. 86. > about 11. The level of the section is shown on the reconstruction of Fig. 21. The section shows the complete lymph sac on the right side and is cut to show the valve on the left. S. 1. j., saccus lymphaticus jugularis; V. i., V. innominata; V. j. i. V. jugularis interna; V. 1. s., vasa lymphatica superficialis. ond median sac, the retroperitoneal, which is adjacent to the renal veins. At this stage a connection between the cisterna chyli and the mesenteric sac, which has been so well shown by Baetjer for the pig of the same size, could not be made out. The posterior sac has now become a long narrow sac, along the course of the primi- The Lymphatic System in Human Embryos. 81 tive sciatic veins and inferior vena cava. It measures 4.6 x .5 x .96 (dorso-ventral), and runs almost to the cisterna chyli, with which a connection cannot be made out. The plane of the section, coronal, is not especially advantageous for determining whether the connection has been made or not. py Morsthy Peters. | Fic. 14. Coronal section through the jugular lymph sac of the same embryo, at the level shown in Fig. 21, to show the simple bridging of the sac which is the anlage of the first lymph node. » about 19. S. 1. j., saccus lymphaticus jugularis. The jugular sacs show a number of points of interest. First their increase in size, this being the stage of the maximum size. The valve is very beautifully shown, as is seen in Fig. 13. The level of this section is shown on the diagram. A second point of interest is the extensive bridging of the sac. This occurs especially near the dorsal border, as is shown in Fig. 14. At this stage the bridges of connective tissue, which cut the sac, show more connective 82 Florence R. Sabin. tissue cells than the surrounding mesenchyme. This thickening of the mesenchyme around a plexus of lymph ducts makes the anlage of a lymph node. A third point of interest is the spreading of the ducts from the jugular sac to the skin. I want to call especial attention to the great size of these ducts, one especially which leaves the lateral sur- face of the sac. These ducts are the first lymphatics to reach the skin; as has been said, they first reach the skin in a human embryo of about 20 mm., and by this stage they have grown over the head and down over the shoulders. These peripheral vessels have not been reconstructed. Fig. 12 shows that the sac has now been cut through by three of the cervical nerves, the third, fourth and fifth. This is interesting in connection with the shifting of the structures in the neck and in the placing of the sacs. - Just at the edge of the subclavian sac is a second small beginning lymph node. This lymph node is like the jugular one, consisting of bridges of thickened connective tissue between a rich plexus of lymphatic capillaries. The beginning of the deep lymphatics for the arm is also shown. I could not trace - them farther in the sections. The thoracic duct shows beautifully in the sections. It begins at the cisterna chyli as a double duct, but the right one soon crosses obliquely in the plane of the coronal section to the left side and joins its fellow. The duct lies adjacent to the azygos veins and has many irregularities. At this stage, the duct reaches the jugular sac, an advance from embryo No. 6, of 24 mm., in which it. only extended into the thoracic cavity. In the angle of the bifurcation of the trachea in this embryo is a clump of lymphatic vessels which possibly connect with the thoracic duct, though the connection could not be made out in the sections. These vessels extend a short distance along the bronchi and are the first visceral lymphatics I have found in the series. The retroperitoneal sac is shown in Fig. 15, which corresponds with the line on Fig. 12. The section shows the relation of the sac to the renal vein and brings out the especially large masses of the sympathetic ganglia in this region. The-Lymphatic System in Human Embryos. 83 The posterior lymph sac shows on one side in Fig. 15, but much better in Fig. 16. The posterior lymph sac is a double sac extending Fic. 15. Coronal section through the retroperitoneal sac of the human embryo, at a level indicated on Fig. 21. » about 39. A., aorta; K., kidney; S. l. m., saccus lymphaticus mesenterica; S. 1. p., saccus lymphatica posterior ; V. ¢. i, vena cava inferior; V. r., vena renalis. along the primitive sciatic veins. The reconstruction is made of.the left side, but shows where the left primitive sciatic vein joins the 84 Florence R. Sabin. right to form the inferior vena cava, and shows that the sacs now extend forward almost to the cisterna chyli. The cisterna chyli being median and the posterior sacs being lateral, the plane of the section made it impossible to trace whether the connection has been made or not. The two sacs, however, run to the same level and probably do connect. The posterior sac measures 4.6 x.2 (lateral x .9 (dorso-ventral). In the angle where the femoral vein branches * | Ss o ™ FOR ‘ave bh. 29") Vb at, a ¢ AS PAL, ¥:/: oF MLE i ite BF © hot ey ‘ a; pee DH a eanerhy ereR ake: 5 WE et Pe + along the primitive sciatic veins, of the same embryo, at a level shown in Fig. 21. x about 49. S. 1. p., saccus lymphaticus posterior; V. ¢., vena eandalis; V. s., vena sciatica primitiva. off from the primitive sciatic is a lymph node and it will be seen that deep lymphatic vessels follow both the sciatic and femoral veins. There is also a group of superficial lymphatics covering the skin of the hip in the groove between the body wall and the leg. These superficial lymphatics could be traced to a connection with the sac on the opposite side, but not on the side reconstructed. This gap is probably due to the accidental plane of the section ; it comes where The Lymphatic System in Human Embryos. 85 the vessels turn directly outward and are so cut in cross section. These gaps to be found in serial sections have already been discussed, they occur in thin sections, but much more often in thick ones like these, this embryo being cut at 50 microns, where the slender lym- phatics must often be missed. The extent of these superficial lym- phatics has not been shown in the reconstruction, they are readily made out in the skin over the back and hip. There is no difficulty in telling them, they are so sharply lined by endothelium, are empty and about three times the size of the blood capillary. This specimen then shows all the primitive sacs and their relations to the thoracic duct. It marks also the beginning of the peripheral lymphatic system, both visceral, to the lungs, and superficial to the skin. The next specimens consist of a group of four embryos of about the same stage, No. 95 measuring 46 mm. and three others (No. 96, No. 84 and No. 224) all measuring 50 mm. They all prove to be ‘especially interesting in connection with the development of the posterior lymph sac. In connection with the jugular sac the measure- ments are given in the table. These sacs show certain differences. In No. 95 the transformation into lymph nodes is not extensive and is chiefly at the upper end. No. 224, on the other hand, shows a fine bridging throughout the sac. The other two specimens show an important stage in the evolution of lymph nodes. By referring back to Fig. 14 it will be seen that when the nodes first begin in an embryo, 39 mm. long, they consist simply of a thickened connective tissue between a plexus of ducts. But at this stage, 50 mm., there appear round clumps of lymphocytes in the connective tissue bridges. These clumps of lymphocytes are the primary lymph follicles and they occur around the blood vessels of the connective tissue bridges. These primary follicles are well illustrated in Fig. 17, in the femoral lymph node, or in Fig. 18. The evolution of the lymph node depends on the balance between the two elements; the lymph ducts which multiply until they are sinuses and the vascular part with its at- tendant lymphocytes which make the follicles and cords. It will be seen in the figures of these embryos, that in early stages the lymphatic element by far predominates. In embryo No. 84, the size of the lymph ducts coming from the 86 Florence R. Sabin. jugular sac is particularly striking. In one section, one of these ducts measures 2.75 x .5 mm. When it is considered that these vessels are really capillaries, being lined by a single layer of endo- thelium, one sees that they are really enormous in size, almost as Mp a Fic. 17. Sugittal section of a human embryo measuring 50 mm., Mall collection, No. 96, showing the posterior lymph sac within the pelvis and its extension along the femoral vein. » about 8. F., femur; Lg., lympho- glandula (femoralis) ; O. s.,.os sacrum; S. 1. p., saccus lymphaticus posterior with lymph node in the border; V. s., vena sciatica; V. 1. v., vertebra lum- balis v. big as the inferior vena cava itself. In general, the lymphatic vessels are considerably larger than the blood capillaries. The cisterna chyli could not be found in No. 95, but there is a small lymph node near its usual location and there is a thoracic duct. The second embryo (No. 96) was damaged at the area; the The Lymphatic System in Human Embryos, 87 other two show the cisterna chyli well with large connections with the mesenteric sac. This is especially true in No, 84, when the series is transverse, the sections looking like Fig. 9 of Mr. Baetjer’s series. Both the cisterna chyli and the retroperitoneal sac are easily located from Fig. 11. They are bridged from the very beginning. These four series, however, are much more interesting in connec- tion with the posterior lymph sacs. As we have seen, these sacs begin in an embryo about 24 mm. long as sacs along the primitive sciatic veins. In an embryo of 30 mm. they are long, narrow sacs. In Fig. 17 it will be seen that in an embryo 50 mm. long they have become large sacs lying in the side of the pelvis opposite the first three sacral vertebre. The entire dorsal wall of the sac is occupied by a lymph node. In one of the other series it is plain that the sac is opposite the bifurcation of the vein into the sciatic and femoral veins, and that there is a large lymph node in the angle of these two veins. From the sacs extend vessels, both along the femoral vein, as shown in the figure and along the sciatic; both of these groups of vessels have developing lymph nodes. These are secondary nodes in contrast with the primary nodes which come from the sacs.. The primary groups of nodes are the jugular, subclavian, retroperitoneal and posterior. The early secondary nodes are near the sacs, a point also in support of the outgrowth of lymphatics from centre to periphery. The last embryo of the series (No. 172), measuring 80 mm., is especially valuable in connection with the fate of the jugular lymph sacs, the development of lymph nodes and the spread of the peripheral lymphatics. The jugular sac is fast becoming transformed into a large group of lymph nodes. In a few sections there are remnants of the sac measuring 1.75 x .5 or even 1.75 x 1 mm., but most of the sac has disappeared. There are also secondary lymph nodes along the other veins of the neck, for example along the external jugular vein next the parotid gland, and along the facial vein at the angle of the jaw. In connection with the arm there is an extensive group of nodes over the shoulder, In the axilla there are four groups—one posterior to the vessels and nerves, one along the subclavian vein, and two groups anterior to the pectoralis minor muscle. 88 Florence R. Sabin. Along the trachea is a group of nodes, of which the mass at the bifurcation is especially large. Nodes are also seen along the bronchi within the hilum of the lung, and large lymph vessels extend into the pleura while smaller ones are to be seen in the septa of the lung itself. No nodes are to be made out within the lung. The thoracic duct is easy to follow as a plexus of vessels along the aorta. Along the vertebral column there are three chains of lymph nodes—one on either side of the bodies of the vertebre not far from the mid-line and closely associated with the thoracic duct. The other two sets are farther to the side, against the body of the vertebre near the base of the transverse processes. These drain the body walls. So abundant are these vertebral lymph nodes that scarcely a section lacks them, the sections being 100 microns thick. In passing into the abdominal cavity the cisterna chyli is readily located. Along its lateral borders is a complete chain of nodes, and at the lower end is a large clump of similar nodes. The retroperitoneal sac has been transformed into a group of nodes except at the upper end, just below the superior mesenteric artery where the sac still persists. Fig. 18 is taken just below the more open part of the sac and shows the bridging and some extension of the sac to the right. The retroperitoneal sac then becomes the group of nodes ventral to the aorta. It will be remembered that at the beginning, the sac extended along the veins of the adrenal bodies. At this stage there is an extensive mass of lymphatic tissue contin- uous with the mesenteric sac, extending along the hilum of the suprarenal bodies. ‘The same mass of lymphatic tissue lies at the base of the mesentery at the portal of the liver. In no section, however, are there any nodes within the hilum of the liver. The most extraordinary development has taken place in the mesentery. A group of nodes follows the pancreas and there is a small node at the hilum of the spleen. A similar node lies against the stomach. In the center of the mesentery is an exceedingly large node, measuring 2 mm. on a side, see Fig. 18. This large central lymphatic mass in the mesentery is connected with the mesenteric sac by a chain of nodes running along the superior mesenteric artery. From this central mass vessels run out in the mesentery toward the intestine. The Lymphatic System in Human Embryos. 89 The only structures with which the developing lymph nodes could be confused are the sympathetic ganglia. On this account care must be exercised, especially around the retroperitoneal sac, where both structures are very abundant. The sections of these stages are thick (50 to 100 microns). and the low powers of the py, v “canndlpsss Fie. 18. Transverse section through the abdominal cavity of a human embryo, 80 mm. long, Mall collection, No. 172. It shows the kidneys, a little of the liver, and many loops of the intestine. about 8. A. m. s., arteria mesenterica superior; C. ¢., cisterna chyli at its lower border; Lg. m., lymphoglandulae mesentericae; S. 1. m., saccus lymphaticus mesenterica. microscope are inadequate to distinguish them, especially when the connective tissue around the ganglia is broken. With care and serial sections the lymph nodes can be absolutely determined. The significance of this retroperitoneal sac is brought out in the injected specimens in Dr. Heuer’s paper. It will be noted that in 90 Florence R. Sabin. _— EP aie ly \ Co ae Wen FON he B97 ON PS OLS x ee. y At a \\ W\ \ \ i ah Th if 1 1 ° — SPP gS) ? (at Fie. 19. Transverse section through the pelvis of a human embryo, 80 mm. long. Mall collection, No. 172, to show the posterior lymph sacs. xX about 9. B., bladder; Lg., lymphoglandula; R., rectum; S. 1. post., saccus lymphaticus posterior. the injected pig embryos the sac seems much larger than in the sections of human embryos. Its importance is that it is the anlage of the visceral lymphatics; it is transformed into the preaortic nodes of which the most anterior group is around the celiac axis. In Fig. 18 is shown the lower part of the cisterna chyli; here the sac is being transformed into lymph nodes while farther an- Lymphatics in Small Intestine of the Pig. 91 terior the sac itself persists. In tracing the series caudalward, the central mass of lymph nodes corresponding with the cisterna chyli, can be traced to the pelvis, where the mass turns a little to the side and joins the posterior lymph sacs. The posterior lymph sacs are really enormous in size, measuring 2.8 x 2 x 8.5 mm. (dorso-ventral). These measurements include the glandular masses in the edge of the sac. The sacs are well shown in Fig. 19, which illustrates that the posterior sacs are being transformed into lymph nodes. In some sections of the pelvis these masses of lymphatic tissue seem to take up almost two thirds of the area of the cross section. From the posterior sac two sets of vessels extend, one along the sciatic vein and the other along the femoral. There is one lymph node along the sciatic vessels and a chain of nodes along the femoral. In Fig. 19 is a tiny lymph node, labeled Lg., which illustrates well the simplest form of a lymph node, a central mass of lymphocytes with a plexus of lymph ducts around. This plexus of ducts is so close that it may already be termed a sinus, so the node consists of a single follicle with its peripheral sinus. It is the structural unit of the lymph node. : From the description of this specimen it will be seen that the foundations of the lymphatic system as it is found in the adult have been laid down in an embryo of 80 mm. The primitive system is complete, and the sacs are forming the primary nodes. ‘The peripheral vessels have extended to the skin and to the viscera, and secondary nodes are forming along these vessels. I think that we have the key for working out the peripheral spread of the lymphatics and carrying them to their capillary bed. Injections of the retroperitoneal sac give us the material for tracing this development. THE DEVELOPMENT OF THE LYMPHATICS IN THE SMALL INTESTINE OF THE PIG. BY GEORGE HEUER. From the Anatomical Laboratory of the Johns Hopkins University. WitTH 17 Ficures. The recent American work on the lymphatic system has given us a new conception of the morphology of the system as a whole. The primitive lymphatic system consists of a number of sacs which are derived from the veins and which become united into a system for the most part by the thoracic duct. A further essential of this primitive system is that all of the sacs give up their connection with the veins, and only the two in the neck rejoin the vein to form the permanent opening. Thus far three sets of paired sacs and two unpaired ones have been described: the jugular sacs, the subclavian which in human embryos are an extension of the jugular, and the sciatic are the paired, the retroperitoneal sac and the cisterna chyli are the unpaired. It is the retroperitoneal sac which especially concerns us in this paper, for it is the source of the lym- phatics of the intestine. This primitive system is complete and can be injected in pig embryos 2.7 em. long. The idea that the sacs form a primitive lymphatic system is not the most fundamental conception of this new theory, but rather that these sacs arise from the veins and in turn give rise to the lym- phatic vessels, so that we may say that the lymphatic system as a whole is derived from the blood vascular system, that lymphatics are modified veins, and that the growth of lymphatics is always from center to periphery. There is now a general agreement in regard to the origin of the primitive sacs, but in regard to the second THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 1. 94 George Heuer. exceedingly important point, namely, that the lymphatic vessels grow from the sacs, there are still differences. It was to test this point that the present study was undertaken at the suggestion of Dr. Sabin. I believe that this work, showing that the vessels of the intestine grow from the retroperitoneal sac, strengthens her position that the lymphatic system is derived from the sacs. Cer- tainly the theory is a fruitful one, for injections of the retro- peritoneal sac give the key for tracing the growth of the lymphatic vessels to the viscera. ; The literature and general relations of the problem are given by Dr. Sabin in the article on the development of the lymphatics in human embryos in this same journal and therefore only the work on the retroperitoneal sac will be mentioned here. This sac was discovered by F. 'T. Lewist who described it as a part of the lymphatic system in 1906. It has been thoroughly worked out by Mr W. Betjer,? in this laboratory. Mr. Betjer has shown that in pig embryos 17-19 mm. long there are small branches of the large renal anastomosing vein in the root of the mesentery. These small veins ‘are ventral to the renal vein and run in an antero-posterior direction. In embryos 19 and 20 mm. long these veins increase markedly in size and number, and by the time the embryo is 21 mm. long show sac-like dilatations which are still readily injected from the renal vein. From this. time on the sac formation goes on rapidly; in embryos 22-23 mm. long, these small sacs have been completely transformed into a large median sac entirely cut off from the veins and likewise independent of the cisterna chyli. By the time the pig is 2.7 em. long, this sac is abundantly connected with the cisterna chyli, which forms. from the veins dorsal to the aorta, and an injection into the thoracic duct will flow through the cisterna chyli into the retroperitoneal SAC.) Methods and Material. This paper begins with the stage at which Mr. Beetjer left off, namely, where the retroperitoneal sac ‘Lewis, Amer. Jour. of Anat., Vol. V., 1906. *Betjer, Amer. Jour. of Anat., Vol. VIII. Lymphatics in Small Intestine of the Pig. 95 is connected with the thoracic duct through the cisterna chyli. Such an injection is shown in Fig. 1 from an embryo 3 cm. long. The earliest stage in which the thoracic duct and sac have been injected is 2.7 em., where the appearances do not differ from the specimen shown in Fig. 1, except that the sac is a little smaller. Fig. 1—An embryo pig 3 cm. long, to show an injection of the lymphatics made by puncturing the thoracic duct. x 2.5, R. o., reproductive organ; Lr. s., retroperitoneal sac; s., stomach; s. b., suprarenal body; t. d., thoracic duct around the aorta; W. b., Wolftian body. The method of injection is as follows: the embryo is opened by cutting away the thoracic wall and turning the left lung far to the right or removing it entirely. For injections of the retroperitoneal sac it is better to remove the liver. The aorta and azygos vein should be thoroughly exposed, and the hypodermic needle plunged down behind the aorta, inserting the needle just at the arch where 96 George Heuer. it is easy to distinguish the vein and thus avoid it. In Fig. 2 is shown a section of an embryo 20 mm. long, taken at the level of the apex of the lung, to show the thoracic duct in its relation to the aorta and to the azygos veins. It shows why it is necessary to insert the needle behind the aorta. It is best to use the finest possible needles; my injections are made with No. 28. For an injection mass either saturated aqueous Prussian blue or India ink are used; the Prussian blue gives beautiful total specimens which Fic. 2.—Transverse section of an embryo pig, 2 cm. long at the level of the apex of the lung to show the relation of the thoracic duct to the azygos veins and to the aorta. x 50. A., aorta; a. v., azygos vein; E., esophagus; p. ¢., pleural cavity; t. d., thoracic duct; v. c., vertebral cartilage. can be kept either in formalin or hardened in bichloride acetic and kept in alcohol; the India ink runs farther, and the total specimens can be cleared by the Schultze method. The ink is more useful for determining complete injections. For studying the development of the lymphatics to the intestine it was found best at first to inject by means of the thoracic duct in embryos from 3.0 to 12.5 em. Subsequently after the exact position of the retroperitoneal sac was determined, it proved that it was far better to introduce the needle directly into the sac. This Lymphatics in Small Intestine of the Pig. 97 is done as follows: The embryo is thoroughly opened and the two Wolffian bodies spread apart as far as possible and the entire in- testine turned to the right. This exposes the sac, as can be seen in Fig. 8. By looking closely one can sce three or four small arteries Fic. 3.—Embryo pig 3.5 cm. long to show an injection of the retroperitoneal sae made through the thoracic duct. x 2.5. r. s., retroperitoneal sac; r. 0., reproductive organ; s, stomach; W. b., Wolffian body. to the Wolffian body running perpendicular to the rectum; the needle must enter just dorsal to these arteries. The retroperitoneal sac is the best point for injecting the viscera. To determine the zones in the intestine, the sac has been filled until it ruptured. Good injections are only to be obtained in embryos in which the heart is 98 George Heuer. still beating. For any stage it is easier to obtain complete injections. through the sac than through the thoracic duct, for in the later case the injection mass must fill the sac before it runs out into the vessels,. and the size of the sac decreases the pressure. Figs. 1 to 4 and 7 show that the thoracic duct is fairly sym- metrical below the heart, that there are two vessels, one on either side connected by many cross channels making a plexus around the aorta. Dorsal to the heart, the right duct crosses over and joins the left. Dorsal to the kidneys, the two ducts unite in a median cisterna chyli. In embryos above 12 cm. it was found impossible to obtain in- jections of the intestinal wall through the thoracic duct. This is. due to the developing lymph nodes, which at first do not check the flow of the injection mass, but later retard it very much. It has been shown that in human embryos the retroperitoneal sac is changed into the group of preaortic lymph nodes extending from the celiac axis to the bifurcation of the aorta, and this change is being made- in an embryo 8 em. long. A secondary, larger group of lymph nodes forms in the center of the mesentery, along the superior mesenteric: artery, and this group is also being formed in the same embryo. The early lymph nodes, however, consist of a great plexus of wide- lymphatic ducts with very few follicles and hence injection through them is easy. By the time the pig is 12 cm., however, the injection mass passes to the nodes, and increased pressure results in an extravasation at the node. For these stages, therefore, it was found necessary to inject into the wall of the intestine itself. To get good injections by this method it was found best to pierce both muscle coats, and thus have the needle just enter the sub-mucosa and inject slowly under low pressure. In embryos from 12 to 13: em. this was difficult, in the older stages, from 16 cm. up, it could be done readily. The areas injected by these two methods, in the younger embryos. through the thoracic duct or retroperitoneal sac, in the older pigs directly into the wall of the intestine, were of course very different. By injecting into the sac, a general lymphatic injection was obtained, the lymphatics reaching in embryos 5 or 6 em. long all the thoracic: Lymphatics in Small Intestine of the Pig. 99 and abdominal viscera. By injecting into the wall of the intestine only the lymphatics of the intestine and the mesenteric lymph nodes were filled with the injection mass. The lymphatics were studied both macroscopically and micro- scopically. In the younger stages, 2.7 to 12 cm., in which a general lymphatic injection was made, a single embryo could be used for both purposes. The best view of the lymphatics is obtained while injecting; there is absolutely no difficulty in distinguishing the lymphatics nor in telling any extravasations whatever. After noting the extent of the injection, small pieces were removed for microscopic sections, while the rest of the embryo was placed in a large amount of strong alcohol, 95 to 96 per cent, until shriveled, then cleared in caustic potash, 1 to 2 per cent, and mounted in glycerine, ac- cording to the Schultze method. Specimens cleared in this way show the course of the lymphatics beautifully and with a dissecting microscope they can be followed to all the organs. The walls of the lymphatic vessels are so delicate, however, that the specimens are not permanent. For the study of the lymphatics of the intestine, cleared pieces of the intestine were mounted in glycerine in hanging drop slides and studied with the microscope in conjunction with serial sections. The portion of the intestine which was most readily identified and which was in all cases particularly examined is a loop of the duodenum which coils around the root of the mesentery. This loop is seen in Figs. 1, 3 and 4. It is the loop most apt to be injected if the needle is introduced into the thoracic duct, probably for mechanical reasons, for when the needle enters the retroperitoneal sac itself, the mesentery becomes uniformly injected. General Description. The lymphatics of the intestine arise from the retroperitoneal sac. This sac is shown in Fig. 1 in an embryo’ 3 em. long. The specimen was made by injecting into the thoracic duct. It will be seen that the sac is triangular in shape and lies opposite the hilum of the Wolffian bodies. In an embryo 2.7 em. long the sac measures about 2 mm. in length, at this stage 3 cm., it is about 2.7 mm, long. That it is connected with the cisterna chyli is proved by the injection and is shown in Beetjer’s Fig. 9 for an embryo of the same stage. 100 George Heuer. Fic. 4.—Embryo pig 4.5 cm. long to show an injection of the retroperitoneal sac and mesentery made through the thoracic duct. x 2.5. K., kidney; r. o., reproductive organ; r. s., retroperitoneal sac; sp., spleen; s., stomach; W. b., Wolffian body. Lymphatics in Small Intestine of the Pig. 101 In Fig. 3 is shown a lymphatic injection of a pig 3.5 cm. long. At this stage the sac is readily injected either through the thoracic duct or directly. It is diamond shape with a slight indentation op- posite the suprarenal bodies. ‘This indentation indicates a division of the sac into two portions, an anterior portion which, as is seen in Fig. 4, sends vessels to the stomach, spleen and duodenum while the ducts for the intestine come from the posterior portion. At this stage, namely, at 3.5 cm., there are a few vessels extending on to the suprarenal bodies, as well as numerous blunt processes to the Wolffian body. It is especially to be noted that the sac is a solid mass, in the injected specimen, that is, it has a single cavity in contrast to the later stage of Fig. 7, where the sac is broken up into a mass of vessels making the anlage of a lymph node or a group of nodes. In Fig. 4 is shown an injection of an embryo 4.5 cm. long. This stage is particularly interesting to us, for it shows the lym- phatics extending into the mesentery. The thoracic duct is plain, and there is a vessel running to the heart, as well as a back flow from. the point of injection to the jugular lymph sac. Below the diaphragm, the stomach has been pulled up and the spleen turned over to the left to show the lymphatics passing to its dorsal border. The mesenteric sac is now considerably larger, measuring 5 by 4 mm. From its anterior border three groups of vessels are seen, one to the spleen, a large group which reaches the stomach wall, and the third group which pases on to the duodenum. In the mesentery of the coil of the lower end of the duodenum, the vessels form a beautiful plexus and have reached the mesenteric border of the gut. The sac itself, which still retains its character as a large sac, is connected with the thoracic duct in three places, one, the principal group of several ducts opposite the hilum of the kidney which is the primary connection seen in sections at 3 em., as shown in Betjer’s Fig. 9; secondly, a small duct which connects the anterior end of the sac with the thoracic duct; this duct runs just anterior to the suprarenal body; and thirdly, an anastomosis between the posterior end of the retroperitoneal sac and the pos- terior lymph sac. These relations are all shown in Fig. 5, 102 George Heuer. which is taken from a pig of the same litter as Fig. 4, and is drawn with the Wolffian body and kidney removed in order to show the cisterna chyli which les dorsal to the aorta. In watching an injection from the thoracic duct, the fluid usually runs down the duct to a point just anterior to the suprarenal body, here the stream divides and part runs into the anterior end of the mesenteric sac, while the rest runs on into the cisterna chyli. From here the central part of the retroperitoneal sac fills up. As soon as this central part is filled, the fluid runs from it into the anterior part of the sac. An incomplete injection might lead one to think that the Fic. 5.—The cisterna chyli and posterior lymph sac from a pig 4.5 cm. long, from the same litter as Fig. 4. The retroperitoneal sac has been almost entirely removed to show the lymphatics dorsal to it. The three points of anatomosis are marked in the drawing as 1, 2 and 3. The Wolffian body and kidney have been removed from the left side. x 2.5. ¢. c., cisterna chyli; p. l. s., posterior lymph sac; t. d., thoracic duct. anterior end of the sac was a separate sac, while as a matter of fact there is one continuous sac, corresponding with the position of the entire group of preaortic nodes of the adult, a chain of nodes, which extend from the level of the celiac axis to the bifurcation of the aorta. It does not correspond in position with the so-called mesenteric lymph nodes which are within the folds of the mesentery. Therefore the sac is termed here the retroperitoneal lymph sac, rather than retaining the name of mesenteric sac used by Dr. F. T. Lewis and Mr. Betjer. The anatomosis of the posterior lymph sac with the retroperitoneal sac 1s shown in Fig. 5. This posterior sac receives three sets of Lymphatics in Small Intestine of the Pig. 103 vessels, a sciatic group, a femoral and an umbilical. It is clear then that in the embryo, lymph from the legs has the direct course through the posterior sac to the cisterna chyli, or the indirect path through the posterior sac and retroperitoneal sac. This, in connection with the fact that the abdominal viscera, the diaphragm and the lymphatics of the lungs are most readily injected from the retro- peritoneal sac, is of importance in emphasizing the significance of the retroperitoneal sac and the preaortic group of nodes into which it develops. In an embryo 5.5 em. long a single puncture into the retroperitoneal sac injected the lymphatics of the abdominal viscera, the skin of both hips and legs, the diaphragm, lungs, esophagus and lymphatics of the skin of the head. Such very ex- tensive anastomosis of all the lymphatic vessels of the embryo is of significance as a basis for variations in the adult. To sum up the lymphatics at this stage, namely at 4.5 em., the pri- mary system is complete, that is the primary lymph sacs are formed and connected into a system by the thoracic duct. Above the diaphragm the vessels have reached the heart, and the esophagus, probably the lungs also. Below the diaphragm vessels from the anterior part of the sac have reached the spleen, the stomach, the intestinal wall, the kidneys, suprarenal bodies and Wolffian bodies. There is an anastomosis with the posterior lymph sac. From the posterior lymph sac vessels follow the sciatic, femoral and umbilical veins. For the small intestine the lymphatics extend along the superior mesenteric artery. In human embryos it has been shown that the mesenteric sac spreads along the suprarenal veins to the root of the superior mesenteric artery. In the study of the growth of lym- phatic capillaries, it proves that these delicate walled vessels grow along some thicker walled vessel; the earliest lymphatics grow along the veins, but in the case of some of the viscera other vessels or ducts may be followed, as for example the mesenteric arteries or the bronchi in the lungs. The border zone of the injected lymphatic capillaries is marked by the rounded blunt ends which are characteristic of injections of terminal lymphatics. In studying the border zones of growing lymphatics it has been shown that the growing tips are either smooth and rounded or have long slender endothelial sprouts 104 George Heuer. which Dr. Clark has shown are collapsed vessels capable of rapidly expanding according to their state of functional activity. Hence we may say that the border zone consists of terminal vessels either collapsed or expanded. An injection mass may either open up these ends or may rupture them. A considerable increase of pressure will always rupture the terminal vessels of a border zone. The best evidence that we have at present of these border zones of growing lymphatics is in Dr. Clark’s work in the living tadpole’s tail where one can see a row of growing tips beyond which there are no lymphatic vessels whatever, only blood capillaries. The next best evidence Fic. 6.—Injected lymphatics in the stomach wall of an embryo pig 6 cm. long. The large mass is at the lesser curvature. comes in making numerous injections of succeeding stages and noting that as the embryos increase in size the zone of injected lymphatics spreads. . Injections at 5.5 and 6 cm. show an increase in size of the retro- peritoneal sac; for example, at 5.5 cm. the sac measures 7 mm. in length, and there is a great increase in the number of ducts from it and an extension of their zone. At 6 cm. lymphatic vessels have extended far over the surface of the stomach and intestine (Fig. 6). The retroperitoneal sac is being cut up into a mass of vessels so that it has not the solid appearance in injections as at 3.5 and 4.5 emi. The breaking up of the sac into a plexus of Lymphatics in Small Intestine of the Pig. 105 vessels is marked at 8 cm., as is shown in Fig, 7. During the stages from 4 to 10 em. long, a number of changes take place, in the first place the plexus in the mesentery becomes exceed- ingly complex. For example, at 8.5 cm., an injection directly into the retroperitoneal sac will fill the entire mesentery with an abundant plexus of ducts; moreover, at the root of the mesentary, along its entire length, the injection mass fills in so as to appear solid to the naked eye. ‘This long line of exceedingly abundant injection cor- responds with the long line of single mesenteric nodes which are characteristic of the pig. Thus both the primary, that is retroperi- toneal, and the secondary or mesenteric lymph nodes are forming at this stage. To return to the wall of the intestine, it is shown in Fig. 4 that lymphatic vessels have reached the stomach and intestine in an embryo 4.5 cm. long. Injections made directly into the sac show that the vessels reach the intestine in embryos about 4 cm. long. The injections shown in Figs. 8, 11 and 12 were all made into the thoracic duct before it was found that puncturing the sac gives better results; and some of them are incomplete, but they serve to illustrate the progression of the lymphatics. In Fig. 4, it is seen that the lymphatics form a plexus in the mesentery and from this plexus a series of lymph vessels grows into the intestine along the arteries. These vessels enter the submucosa and form there a primary plexus. This primary submucosal plexus, at a stage when there is no secondary plexus, is shown for the stomach for a pig 6 em. long. The drawing is not made so that it can be oriented readily, but the heavy mass is at the lesser curvature, and the plexus shown is in the submucosa. ¥ In Fig. 8, is shown a section of the duodenum of a pig 8.7 cm. long. By this time there is not only a submucosal plexus but a secondary mucosal plexus as well. As has been said, the lymphatics which grow into the intestine at the mesenteric border penetrate the longi- tudinal and circular muscle coats, and enter the embryonic submucosa. In following through a large number of series, no deviation from this course has been observed. The point at which the lymph ducts penetrate the intestine, however, ts subject to variation within certain 106 George Heuer. limits, and the lymphatic ducts follow one of two courses. In the one type, the lymphatic trunk penetrates the wall of the intestine immediately on reaching it, that is that portion along the mesenteric A Fic. 7.—Injected lymphatics in a pig embryo 8 cm. long to show the thoracic duct and that the retroperitoneal sac is being transformed into a plexus of ducts the anlage of a group of lymph nodes. x 1.3. attachment included between the two folds of the peritoneum, as shown in Fig. 8. In the other type the lymph vessel may run in the serosa a variable distance, usually not more than one quarter or Lymphatics in Small Intestine of the Pig. 107 one half around the intestine, before penetrating the muscle coats, as shown in Fig. 8. In the latter case, by the branching of this vessel, a subperitoneal plexus may develop, a consideration of which will be taken up later. The variations in the course of the lymphatic vessels above de- scribed may be seen in any part of the intestine and in all stages of embryos where lymphatics have entered the intestine as well as es eS Seer Fic. 8.—Injected lymphatics in the duodenum of a pig embryo 8.7 cm. long to show the entrance of the lymphatics into the submucosa, the submucosal plexus, and the beginning of the mucosal vessels. C. m., circular muscle; 1. m., longitudinal muscle; m. |. n., mesenteric lymph node. in the new-born and adult pig. The same relation is seen in an older embryo, 16 em. long, in Fig. 14. In injecting the lymphaties of the intestine of the new-born or adult pig, the variations in the course of the large collecting trunks through the intestinal wall to reach the mesentery is frequently observed. This variation in the course of the lymph vessels through the bowel wall is dependent upon the distribution of the blood vessels. It was shown in tracing the growth of the lymphatics through the 108 George Heuer. root of the mesentery that these vessels followed the artery in their development. It is equally true that the lymphatic ducts follow the arteries through the wall of the intestine. This can be seen in serial sections and in whole specimens which have been cleared by the method of Schultze. In serial sections of injected material, one can readily find a section showing an artery passing through the wall of the intestine; and in such a section often one and sometimes two lymphatic vessels are seen closely accompanying the artery through the bowel wall. The arteries in their course to the intestine show the variations which we have described for the lymphatics; that is, the artery either penetrates the wall of the intestine immediately on reaching it, or passes some distance under the serosa before doing so. Since the lymphatic vessels follow the arteries to the intestine, the variation in this course is accounted for by the distribution of the blood vessels. To return to Fig. 8, from an embryo 8.7 em., beside showing the beginning of the mesenteric lymph nodes and the method of the en- trance of the lymphatics into the wall of the gut, the section also shows that the submucosal plexus has spread entirely around the. wall of the intestine. On entering the submucosa, the lymphatic trunk divides into two branches, one of which extends around either side of the wall of the intestine. In its course, each branch lies entirely in the sub- mucosa. In Fig. 9 is shown an important point in connection with these branches of the intestinal wall. It is from a pig 9 em. long, and shows that these primary branches form a series of more or less complete lymphatic loops lying in the submucosa, near the circular muscle coat. This is, probably, an incomplete injection at this stage, but it serves to illustrate the point, for in making the injections the primary vessels of an area the vessels that develop first, fill first, and those that develop later inject later. Thus a vas- cular unit can be shown by a partial injection as seen in Fig. 17 for the new born pig. This can be observed even in the adult. The growth and the arrangement of these vessels indicate a seg- mental development, and each with its future branches may be thought Lymphatics in Small Intestine of the Pig. 109 of asa unit of structure. These primary lymph ducts can be followed in the ascending series of embryos and appear as the large collecting trunks, such as are seen for example in Fig. 14. The formation of simple lymphatic loops in the submucosa as the primary event in the development of the lymphatics of the intestine has been demonstrated by repeated injections in embryos from 6 to 10 cm, long. It has been found constant that lymphatic vessels first appear in the submucosa and that the vessels branch to form loop-like structures. By means of serial sections, these loops are found to be continuous with the lymphatics of the mesentery. ¥ic. 9.—Loop of small intestine of an embryo pig 9 cm. long to show the primary lymphatic loops in the submucosa. By the branching of these primary loops and the fusion of these branches with those of adjacent loops, a primary lymphatic plexus is formed in the submucosa. This is shown in Fig. 10, from a pig 10 em. long. In cleared whole preparations and in serial sections, branches from the primary lymph ducts are seen extending out into the submucosa toward one another. In different preparations it is found that some of the branches from neighboring trunks have fused, while others have approached one another but have not fused. Cleared specimens from embryos 8.5 to 11 em. long show this plexus formation very well. It seems clear, therefore, that a primary lymphatic plexus is formed in the submucosa by the branching of the primary loops and 110 George Heuer. the fusion of these branches. One can readily demonstrate out- growing branches in injected material, and from the appearance of the peripheral ends of these branches and by the section one feels reasonably sure that the injection has been complete. Moreover, if one takes a series of embryos from 8.5 to 11 em. long, injected from the same place along the thoracic duct and under about the same pressure, he finds in each succeeding larger embryo a more complete plexus formation. Fic. 10.—Loop of small intestine of an embryo 10 cm. long to show the development of the submucosal plexus from the primary loops shown in Fig. 9. The plexus thus formed in the submucosa never becomes complex and close-meshed. In younger embryos it appears most complex, for here the primary lymph ducts are close together. With the in- crease in the size of the embryo and the elongation of the intestine, it becomes a wider meshed and less complex appearing structure and is recognized as the plexus of large vessels in embryos 16 em. long, as in Fig. 14, and in new-born and adult pigs. From the primary submucosal plexus above described, there is developed a second lymphatic plexus in the mucous membrane of the intestine, namely, a plexus in the mucosa at the base of the villi. From a study of the material at hand,it is believed that this plexus, Lymphatics in Small Intestine of the Pig. 111 like the preceding, is the result of the peripheral spread of the lym- phatics through the intestine, a plexus being formed by branching and the fusion of neighboring branches. The beginning formation of this second plexus is seen in stages in which the primary plexus is but incompletely formed and, therefore, we must think of the two plexuses as developing more or less simultaneously. In embryos 8.5 to 9.0 em. long, in which primary lymph ducts and an incom- plete submucosal plexus are found, branches are seen coming off from the primary plexus, these branches extending toward the villi. These branches mark the beginning development of the mucosal Fic. 11.—Section of the duodenum of a pig 8.7 cm. long to show the sub- mucosal plexus, the mucosal plexas and the beginning of the lacteals. 1., lac- teals; m. p., mucosal plexus; s. p., submucosal plexus. SS plexus. They have been found in embryos 8.7 cm. long, as seen in Fig. 8, and still better in Fig. 11. Fig. 11 is from a section 100 microns thick and not all the lymph vessels lie in the same plane, as shown in the drawing. It will be seen that the primary plexus is repre- sented by an almost complete loop lying nearest to the circular muscle coat. From it branches have been given off extending inward toward the villi. In the older embryos and in the new-born and adult pigs, these branches remain as the connecting vessels between the mucosal and submucosal plexuses. In two places in the section, these branches have been themselves branched to form lymph-vessels running along at the base of the villi. This is an important step 112 George Heuer. in the development of the plexus in the mucosa, for it shows the way in which this plexus is formed. In embryos 9, 10, and 11 cm. long, the branching of the vessels has increased and numerous branches have united to form a close-meshed plexus at the bases of the villi. The vessels of this mucosal plexus are of smaller size than those of the preceding plexus and remain ‘so through the ascending series. The plexus remains a close-meshed one throughout, which was seen not to be the case in the submucosal plexus. The central lymph vessels of the villi of the intestine develop from and almost simultaneously with the secondary lymphatic plexus of Fic. 12.—Composite section made from four adjacent sections to show the central villus, from the small intestines of a pig embryo 9 cm. long. the mucosa. From a study of injections, they, like the plexuses of which we have spoken, seem to be the result of the peripheral extension of the lymphatics throughout the wall of the intestine, the villi being the peripheral limit of this extension. Repeated injections in the younger embryos have shown no lymph vessels in the villi. In an embryo 8.7 em. long, as we have seen, a beginning plexus in the mucosa has been formed and in following through this series a few short branches have been found which have extended into the bases of the villi. In embryo 9 em. long, the central lymph vessels of the villi have been injected, and they appear as straight Lymphatics in Small Intestine of the Pig. 113 ducts with bulbous rounded ends extending from the mucosal plexus of the center of the villi. Fig. 12, combining four consecutive sec- tions of a series from an embryo 9 em. long, shows such a vessel. Fic. 13.—Section of the mesentery and intestine of an embryo pig 9.5 cm. long. This is an incomplete injection. It shows the size of the mesenteric - vessels, their course along the artery and a tertiary lymph node. D. 1. n., developing lymph node. We see here the branches of the primary plexus near the circular muscle coat, those of the secondary plexus near the base of the villi, while connecting the two plexuses are vessels from which the second- ary plexus is derived. From the secondary plexus the central lymph 114 George Heuer. duct is seen extending into the villus. The artery and vein are shown in the drawing. There are, then, in the mucous membrame of an embryo 9 em. long all the essential structures which go to make up the lymphatic plexuses as found in the mucosa and submucosa of the adult, and it is by the growth of these parts that the latter are derived. Fic. 14.—Cleared specimen of a loop of the small intestine from an embryo pig 16 cm. long to show the mesenteric vessels with their valves, the large meshed submucosal plexus, the deeper, closer-meshed mucosal plexus, and the central lacteals. In Fig. 13 is shown an incomplete injection of the duodenum to show the very large ducts at the root of the mesentery, the fact that the vessels follow the artery in the mesentery, and thirdly, that there are beginning tertiary lymph nodes along the ducts in the mesentery. In Figs. 14 and 15 are shown injected lymphatics in Lymphatics in Small Intestine of the Pig. 115 5 Fie. 15.—Thick section from the same specimen as Fig. 14, to show the exact position of the two plexuses. the intestine of a pig 16 cm. long. Here the characteristics of the adult system are all laid down. In the gross specimen of Fig. 14, the characteristic submucosal plexus is well seen. The vessels are no longer capillaries, but are collecting trunks, as shown by their size and the obvious valves. The well known “‘collarettes’” indicate the position of these valves. 116 George Heuer. The plexus of these vessels is wider meshed, and shows clearly the primitive units, in contrast with the finer meshed mucosal plexus of capillaries. This mucosal plexus gives rise to the central lacteals, as is seen in both Figs. 14 and 15. The development of the third lymphatic plexus of the intestine of the pig, namely that confined to the muscularis and serosa, has been the most difficult to study. It apparently develops later than the plexuses in the mucous membrane and an injection of what could be definitely termed a plexus has not been obtained in embryos under 10 em. in length. In embryos up to 9 em. in length no injected lym- Fic. 16.—Injection of the serosal plexus in a loop of intestine from a pig 20 cm. long. phaties have been found between the muscle coats; and it appears that the lymphatics in these younger stages are developed only in the mucous membrane. In following the lymphatics through the wall of the intestine, it was found, as previously stated that the lymph ducts may pass in the serum some distance around the wall of the intestine before penetrating the muscle layers. Branches may be given off from the lymph ducts, while lying in this layer and by the intereommunica- tion of these branches the beginning of a subperitoneal plexus may be formed. Such vessels have been found as early as 10 em. In older embryos, as for example at 20 cm., it is easy to demonstrate lod Lymphaties in Small Intestine of the Pig. 117 a serosal plexus. In Fig. 16, from an embryo 20 cm. long, the serosal plexus was injected by introducing the needle directly into the wall of the intestine. Sections of this specimen proved that the lymphatics are in the serous covering of the bowel. The origin of this plexus is then similar to that of the plexuses in the mucous membrane; that is, it is by the growth and extension of lymphatic vessels through the serosa, these vessels being derived from the primary lymph ducts. The mode of origin is indicated in embryos younger than 10 em., in which short, blunt-ended lymph PL Mi si ety Fie. 17.—A lymphatic tree injected in the intestinal wall of a new-born pig to show that the injection of a single mesenteric trunk may isolate one of the units even after the plexus is complete. ducts can be seen extending a short distance into the serosa. While this seems to be the origin of the subperitoneal plexus, it is difficult to say that another process docs not enter into its formation, that is a growth outward of lymphatics from the primary plexus in the submucosa. In embryos 10.3 em. long, serial sections of the intestine show a few lymphatic vessels extending from the primary plexus through the circular muscle coat toward the serosa and from the 118 George Heuer. series we can feel fairly certain that these are not vessels coming into the intestine. In embryos 16 cm. long, such branches are more numerous and can be traced for some distance between the muscle layers before penetrating the longitudinal coat, to enter the serosa. Such vessels extend into the serosa and may help to form the plexus there. They form the connecting vessels between the plexuses in the mucous membrane and the serosa. | From the preceding study it may be seen that the evolution of the lymphatic system of the intestine is from the center to the periphery. The retroperitoneal sac is the origin of the lymph vessels of the intestine; by repeated injections it has been shown that this sac is the best place from which to inject the intestinal vessels, and that corresponding with the growth of the embryo an increasing zone of lymphatics can be injected. From the sac vessels grow out into the mesentery making a plexus which extends to the wall of the gut. The primary mesenteric lymphatics enter the mesentery with the artery. From the mesenteric plexus a series of vessels enters the wall of the intestine with the branches of the mesenteric artery. These lymphatic vessels penetrate to the submucosa and form there a series of loops extending around the wall of the gut. These loops represent lymphatic units, which soon become united into a complete but coarse-meshed plexus. From this submuosal plexus, the mucosal plexus of smaller vessels develops. The mucosal plexus is fine meshed. From the mucosal plexus, the lacteals grow into the villi. The serosal plexus develops late from the lymphatic trunks as they are entering the bowel wall. The retroperitoneal sac, which is the origin of the lymphatic vessels of the intestine, is the anlage of the retroperitoneal, preaortic chain of lymph nodes. The first evidences of the formation of the nodes from the sac occur as early as 3 cm., when the sac begins to be bridged by connective tissue bands. The preaortic nodes are all primary ones for the intestine; the mesenteric nodes develop along the course of the lymphatic ducts and form the secondary group. Thus, the primary nodes, are those that come from the primitive sacs, secondary and tertiary nodes, etc., develop along the course of the vessels. ON THE PRENATAL GROWTH OF THE HUMAN BODY AND THE RELATIVE GROWTH OF THE VARIOUS ORGANS AND PARTS. BY C. M. JACKSON. From the Anatomical Laboratory, University of Missouri, Columbia. WITH 4 FIGURES AND 6 TABLES. Although numerous observations on various phases of the growth of the human embryo and fetus are scattered throughout the anatom- ical literature, they have never been collected and presented so as to give a comprehensive view of the subject. It is the purpose of this paper to present, in addition to the data already available, the results of an extensive series of original observations. These obser- vations were made primarily in order to fill some of the existing gaps in our knowledge regarding this subject, particularly concerning the rate of growth during the earlier months. It is now possible to de- scribe (though imperfectly and still subject to correction by further data) the general course of prenatal growth in the human body, and in its various organs and parts. The material used for these observations includes 43 specimens from my collection of human embryos and fetuses. The specimens range all the way from 6 mm. up to the full-term fetus. Upon 32 of these specimens, the observations include the total volume, and the volume of the head, trunk, extremities, and of each of the principal organs of the body. For supplementary data concerning human em- bryos of the first month, the volumes of seven of the His-Ziegler models were measured. For obvious reasons, the volume rather than the weight was chosen for measurement in the case of the models. In the small embryos THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 1. 120 C. M. Jackson. also the volume may be determined, where it is difficult or impossible to ascertain the weight. Even where the organs are large enough to be dissected out and weighed, one does not like to sacrifice valu- able specimens for this purpose, if it can be avoided. On the other hand, it is comparatively easy, though somewhat tedious, to measure the volumes of embryos which have been cut into serial sections. The sections must first be drawn to a definite scale of enlargement. Then one may proceed in either of two ways. In the first embryo meas- ured (11 mm.) a rough model was constructed by Born’s wax-plate method, and the volumes of the body and of the various organs and parts were measured by water displacement.t An easier method, which is equally accurate, was used with other small embryos. In the enlarged drawings of the sections, the areas of the body and of the various organs were measured by means of a planimeter. The volumes desired were then easily calculated by multiplying the areas (corrected for magnification) by the thickness of the sections. From the third month onward, it was found possible to measure directly the volumes desired by means of water displacement in graduated glass cylinders of various sizes. In some fresh specimens the volume and specific gravity were determined by weighing successively in air and suspended in water. Certain sources of error must be recognized in the use of these methods. First is the effect of the reagents used. Most of the speci- mens used had been fixed and preserved in 5 per cent to 10 per cent formalin solution. It is well known that in general formalin causes a certain amount of swelling or expansion of tissues. In one fetus. of the 5th month in which this point was carefully observed, the swelling amounted to nearly 13 per cent of the total volume, after: three months in a 10 per cent formalin solution. Furthermore, it is stated that the swelling is not equal in all of the various organs, though the amount of difference and the conditions of occurrence are not yet fully known. Alcohol, on the other hand, causes shrinkage. *This embryo was studied by Bonnot and Seevers (6) under my direc- tion. I am also under obligations to J. A. Watkins, M. L. Clint, and R. Lhamon for assistance in making a part of the observations used in this. paper. Prenatal Growth of the Human Body. 121 For the specimens sectioned, it must also be remembered that the process of dehydration and embedding in paraffin causes a shrinkage of at least 20 per cent, or more than enough to counterbalance the swelling due to the formalin fixation. It is improbable, however, that the errors from these sources are large enough to affect materially the main conclusions concerning growth, especially concerning the relative growth of the various parts. In the following pages there will be considered briefly : first the pre- natal growth of the body as a whole, then the relative growth of its principal parts, and finally the relative growth of most of the indi- vidual organs. For the organs and parts, it has been found more convenient and useful to record the relative size, expressed in per- centage of the entire body. From these data, the absolute size of any part can easily be calculated, if desired (that of the whole body being given). 1. GrowTH OF THE Bopy as Aa WHOLE. In Table I, a list is given of the 48 specimens upon which my own observations were made. In the first column, the catalog numbers (in my collection) are indicated. In estimating the age of the speci- mens, Mall’s rule was used for the first four months and Hasse’s rule for the last five months. In the fifth month, a compromise was used between figures derived from Mall’s method and those from Hasse’s. In Table II, some observations upon the volumes of the His-Ziegler models are recorded. ‘The embryos corresponding to these models have been figured and described in detail by His (23), who gives no data concerning their weight or volume, however. While a considerable amount of data has accumulated concerning the growth of the fetus from the 4th to the 10th month, very few observations have been made upon the earlier, embryos. In fact up to this time no data have been published which allow any accurate conclusions concerning growth in the human embryo during the first three months. My own observations include, in addition to the seven His-Ziegler models, eighteen embryos within this period. Two of these embryos (6 mm., 7.3 mm.) are of the 1st month, six of the 2d month; and ten of the 3d month. Four of these embryos (6 mm., 122 C. M. Jackson. 11 mm., 17 mm., and 31 mm.) were measured from sections, the remainder by the direct method described (7.8 mm. by weighing). The data obtained from these specimens form the basis for the figures given for the first three months in Table III. The volume of the human ovum (the diameter being assumed to be .2 mm., as usually stated) is about .000004 ce., which, assuming the specific gravity to be 1.0, corresponds to a weight of .000004 g. The 7.3 mm. embryo (volume .026 cc.) was probably somewhat shrunken by the alcohol and embedding process so that the volume of .041 ec. obtained from the His model of a 7.5 mm. embryo is perhaps nearer the true size at the end of the first month, corresponding to a weight of about .04 g. Thus we obtain for the relative monthly growth rate? of the 1st month, 9999; for the 2d month, 74; for the 3d month, 11. Fehling (16), whose figures are often quoted, gave no estimate for the 1st month, but (without reliable data) estimated the relative monthly growth rate for the 2d, 3d, and 4th months at 3, 4, and 5 respect- ively, the greatest relative growth being in the 4th month. My ob- servations, however, prove beyond doubt the conclusion of Miihlmann (36), recently emphasized by Minot (34), that the relative growth in the human embryo is by far the greatest during the 1st month, declining rapidly at first, then more slowly hroughout succeeding months. Even the large number, 9999, representing an increase of nearly one million per cent, is in reality too small for the relative growth of the human embryo during the 1st month. For as a matter of fact, ‘not the entire ovum, but only a portion of it, actually goes to form the embryo. The remainder is concerned with the formation of the membranes, etc. Since it is not known what proportion of the ovum goes for each of these purposes, the problem may be approached in another way. Table IIIa shows the weight of embryo plus mem- branes and enclosed fluids, at the end of the 1st, 2d and 3d months, *The relative growth rate is the ratio of the gain during a given period * to the weight at the beginning of the period. and is the most accurate index of the rate of growth. It indicates the increase in a unit of weight during the given unit of time. Thus while the total amount of gain in absolute weight increases steadily for each prenatal month, the gain per gram of body weight (as shown by the relative growth rate) is constantly decreasing. Prenatal Growth of the Human Body. 123 according to observations by Waldeyer (44) and Daffner (10). This gives the enormous figure of 574,999 for the relative growth during the 1st month, corresponding to an increase of over 57 million per cent. This number is undoubtedly too high, however, since the fluids enclosed in the membranes and making up a considerable proportion of the total weight, can hardly be fairly considered as products of em- bryonic growth, in the ordinary sense of the term. The true relative growth for the 1st month therefore les somewhere between 1 million and 50 million per cent. From the foregoing, it appears that the relative growth of the human embryo is enormous in the 1st month, declining thereafter, at first very rapidly, then more and more slowly. The next question TABLE IIIa. GROWTH OF THE HumMAN Empryo PLUS MEMBRANES AND ENCLOSED FLUIDS. | Weight at Beginning Weight-at mids” |) “= Heletve Growl of Month. | of Month. ea (a. (b.) (23) Ist Month. (Ovum .000004 g.) | 2.3 g. (Waldeyer.) | 574999. BAe 2.32. | 25 g. (Daffner.) 9.9 ad ieee 25 g. 100 g. 3.9 which naturally arises concerns the growth within the 1st month. Some light is thrown upon this question by the observations on the volumes of the His models, recorded in Table II. First it may be noted that the yolk sac is relatively large in the early embryos. In the 2 mm. embryo, it makes up more than three- fourths of the total volume. In the 2.6 mm. embryo, the yolk sac remains at about the same absolute size, but owing to the increase in the size of the embryo proper, it here forms less than two-thirds of the total volume. In my 6 mm. specimen (No. 176) the volume of the yolk sac was .0056 ce., forming a little more than one-third of the total volume, the embryo proper measuring .0098 cc. According to Mall (29) the diameter of the yolk sac is approximately 1 mm. at the age of 1 week, increasing 1 mm. each week up to the 6th. It is therefore evident that although the growth of the yolk sac has 124 C. M. Jackson. been relatively very great during the first two weeks, its later growth is much less rapid. As to the embryo proper, the actual volume at the end of the 2d week (2.2 mm.) is seen to be .000781 cc. As the volume of the ovum at the beginning is about .000004 ce., this corresponds to an increase of 195 times in volume during the first half of the 1st month. During the second half of the 1st month, the embryo proper increases from .000781 to about .04 ee., or about 50 times. It is therefore evident that the growth of the human embryo is relatively more rapid during the first half of the first month than during the second half. The difference would appear still greater, if the growth of the yolk sac, membranes, etc., was taken into account.? From what we know of the development in lower animals, as Donaldson (11) has pointed out, there is probably no increase in volume during the early segmentation stages of the ovum; so that the increase must be all the more rapid when it actually begins. In addition to the observations in Tables I and II, a considerable amount of data concerning the prenatal growth of the whole body from the 4th to the 10th months has already been published. Ahl- feld (1), Fehling (16), Legou (25), Faucon (15), Michaelis (35) and others have recorded the weight of fetuses whose age was es- timated from menstrual histories. Curves of absolute growth for the prenatal period, based upon these data, are shown in Fig. 1, (curves 1, 2, 4, 5). No curves are shown for the data of Faucon and others which do not differ materially from those given. From all the data available, I have ventured to construct a normal curve (Fig. 1, curve 3), which is intended to represent the absolute pre- natal growth, according to our present knowledge. As will be seen, it does not differ greatly from that based upon the data of Fehling (who utilizes also observations from Hecker and Schroeder). Ahl- feld’s figures seem entirely too high for the average weight at the ’Daffner (10) gives the weight of a fresh “ovum” of fourteen days as 0.82 ¢g., which is more than 200,000 times the weight of the ovum at the beginning. During the second half of the third month, however (accept- ing Waldeyer’s observations of the 2.3 g. for the weight at the end of the first month), the embryo plus membranes, etc., increases only about three times in weight. Prenatal Growth of the Human Body. 125 corresponding ages, in spite of his statement that “Nur solche Kinder verwendet werden deren Mutter den Tag der Conception genau an- zugeben wussten.” Hennig (19) has published a curve of growth in fetal weight, but without the data upon which it is based. His curve shows a marked increase in the growth rate in the 6th and 8th months, followed by retardations in the 7th and 9th months. Donald- son (12) believes that a new phase of growth in the human fetus begins with the 6th month, where the curve of absolute growth begins to rise more rapidly. A study of the growth rate, however, as ex- pressed by the figures for the relative monthly growth rate in Table III (or corresponding figures in Fehling’s table) reveals no evidence of any marked change at this particular time. All of the data being considered, it seems most probable that the normal curve of fetal growth is fairly regular, though the uncertainty regarding the age of specimens and the degree of individual variation makes it very difficult to determine this curve accurately. The curve as drawn (curve 3, Fig. 1) is fairly regular, corresponding roughly to the formula y = x*, or A Weight (g) = (eae From this formula, the weight may be calculated approximately from the age, or vice versa, for any time beyond the first month. By some such growth formula the age should be determined more accu- rately than by the length (which theoretically should vary as the cube root of the volume, or weight).* The majority of previous investigators have concluded that for determining the age, the length is a more reliable criterion than the weight; probably because the skeleton, which determines the length, is thought to be less variable than the soft parts, which make up most of the weight. This is still an open question, however. *Roberts (40) has worked out a rule, assuming that the weight increases as the cube of the age; but this results in figures somewhat too high for the average weight at the various months. This is also the case with the formula: Weight (g) = 50 (months — 2)? recently proposed by Tut- tle (42). 3250 3000 2750 2500 2250 1250 1000 750 500 Age in Days Fic. 1. Curves of absolute prenatal growth. Curve No. 1, data from Ahl- feld; 2, from Fehling; 4, from Legou; 5, from Michelis. Curve No. 3 repre- sents the normal curve of growth (weight) constructed by the author, based on Table III, from all data available. The dots represent the total body vol- ume in cubic centimeters of the specimens studied, the age being estimated from their lengths. Prenatal Growth of the Human Body. 127 Although the growth rate at the end of the fetal period is far less than at the beginning, it is still very rapid as compared with the growth rate after birth. If the relative monthly growth rate of the last fetal month (.45) were maintained during the first year after birth, the weight of the body at the end of the first year would be over 250 kilograms! This marked diminution in the growth rate after birth indicates that the prenatal conditions are far more favor- able to growth than the postnatal. As may be seen by the dots in Fig. 1, the volume in ce. of the fetal specimens studied, the age being estimated from their length, cor- responds roughly to the curves of growth in weight, where the age was determined from menstrual history. We should expect the curve of growth in volume to differ slightly from that of weight, on account of slight changes in the specific gravity of the fetus. In the earlier months, the specific gravity of the embryo is very little over 1, though in later fetuses it reaches 1.04 or 1.05. 9. RELATIVE GRowTH OF THE PRINCIPAL Parts oF THE Bopy. Since growth is not uniform in the various parts of the body, these must be separately considered. No data have been published showing the relative size of the head, trunk and extremities in the various prenatal months, although it is well known that the head is at first relatively large and the extremities small. My own observations on the relative growth of the various parts in 32 specimens are included in Table IV. The relative size of the head was also observed in 5 of the His models (Table IT). The growth of the various parts in the specimens observed is illus- trated graphically by the curves of relative growth shown in Fig. 2.° The unbroken lines connect points corresponding to the observations on the 32 specimens in Table IV. The dotted line at the beginning of the “Head” curve indicates approximately the relations found in the His models representing embryos in the latter part of the 1st "It must be borne in mind that the curves of relative growth merely indicate whether the part is growing more or less rapidly than the average rate of the body as a whole, whose absolute growth curve is shown in Tost yaa 128 C. M. Jackson. month. The dotted lines on the right indicate, for convenience of comparison, the relative growth of the corresponding parts between birth and adult life, based chiefly upon observations by Meeh (31). The method followed in dividing the body was to separate the head from the trunk by a plane passing just below the mandible anteriorly, and just below the cranium posteriorly. The neck is therefore in- cluded in the trunk. The upper extremities were separated from the trunk by an approximately sagittal plane through the shoulder joint, and the lower extremities by an oblique plane through the hip joint, parallel to Poupart’s ligament. As it is impossible to pass these planes always in exactly the same way, the measurements on different specimens are not exactly comparable to each other, though the error is comparatively small. There is also a certain loss of blood, ete., especially in the case of the fresh specimens. TTead. As may be seen in Table II, the head in the His models of embryos in the latter half of the 1st month forms from 34 per cent to 39 per cent of the entire body volume. Table IV and the “Head” curve in Fig. 2 show that the head reaches its maximum relative size, about 45 per cent of the total body volume, during the 2d month. There- after it declines gradually in relative stze, forming only about 26 per cent or 27 per cent of the total body at birth. His (23) from a study of the profile areas in embryos of the 1st and 2d months, concluded that the head is at first relatively small, increasing from about 30 per cent in the latter part of the 1st month to 56 per cent of the total body at the end of the 2d month. He thought that the head and trunk during this period are in a sort of race for supremacy, first one being larger, then the other. Profile areas, however, lacking the third dimension, are not necessarily in the same proportion as the volumes. His calculates the profile areas of the head in his embryos 4 mm., 5 mm. and 7.5 mm. to be 32.3 per cent, 30.7 per cent and 30.6 per cent of the total body; but I find in his models of these same embryos the volume of the head to be respectively 34.9 per cent, 38.7 per cent and 36.6 per cent of the Prenatal Growth of the Human Body. 129 total volume. In no specimen examined by me does the head exceed the trunk in size, though it sometimes approaches it closely. The steady decrease in the relative size of the head from the 2d month onward is in part due to a corresponding decrease in the relative size of the brain (which will be described later). As the brain throughout bears a fairly constant ratio to the volume of the head (somewhat less than half), however, it would appear that the facial*portion of the head must also decrease in relative size at about the game rate as the brain. This does not agree with the conclusion ot Merkel (32), who found the facial portion of the head of about the same relative size in a series of fetuses of different ages, excepting the youngest (3d month), where the face was relatively larger. In the adult, according to Meeh’s (31) observations, the head forms from 6 per cent to 11 per cent of the total body volume; or, according to Harless (18), 6 per cent to 9 percent of the total body weight. Trunk. In Fig. 2, the curve of the relative growth of the trunk is not repre- sented in the 1st month. Since, however, the extremities are very small at this time, it is evident that the trunk must be relatively very large. When the head forms 35 per cent of the body, the trunk would necessarily form nearly 65 per cent. At the beginning of the 2d month, as is shown by the curve, the trunk has decreased in relative size, so that it forms about 50 per cent of the total body volume. From the 2d month onward, the trunk continues to de- crease (somewhat irregularly) in relative size. During the first half of the fetal period, the curve of relative growth of the trunk descends nearly parallel with that of the head. The curves diverge in the sccond half of the fetal period, however, that of the trunk remaining on the whole nearly horizontal, fluctuating between 40 per cent and 45 per cent. Meeh (31) separated the trunk from the lower extremities by a horizontal section at the level of the perineum, which apparently adds about 6 per cent to the relative size of the trunk. The average in 8 adults measured by him is about 54 per cent, which would cor- 130 C. M. Jackson. respond to approximately 48 per cent of the total volume by my method. Between birth and adult life the trunk therefore apparently increases slightly in relative size. a5[- ay as EEE LN ma ee) ee ee ee ames || Bah) sae WT | MBO apabete eres ee CP RB aA eee... 0 40 60 80 100 120 140 160 180 200 220 24 0 260 280 New born Days of Gestation Fic. 2. Curves showing relative prenatal growth of the various parts of the body, in specimens studied, the size being expressed in percentage of the total body volume (Table IV). The dotted lines at the right represent the postnatal growth of the corresponding parts (based on data from Meeh). The Extremities. The extremities are seen to be at first relatively very small, each forming between 2 per cent and 3 per cent of the total body volume during the 2d month. The upper extremities are usually slightly larger than the lower until early in the 3d month. Then both begin to increase in relative size, the lower more rapidly. The increase in the relative size of the extremities during the first half of the fetal period counterbalances the relative decrease of the head and trunk during that period, as shown by the curves in Fig. 2. In the second Prenatal Growth of the Human Body. 131 half of the fetal period, the extremities continue to increase in relative size, but more slowly; the increase of both counterbalancing the con- tinued decrease in the relative size of the head, the trunk remaining nearly unchanged. At birth, the upper extremities form about 10 per cent of the whole body, the lower about 20 per cent. In the adult (judging from the data of Meeh and Harless), the upper ex- tremities have increased but slightly, if at all; while the lower ex- tremities have increased to about 35 per cent, or nearly twice the relative size at birth. In general, it may be said that the period of maximum relative growth passes somewhat wave-like over, the body from the head toward the foot. The head, as we have seen, reaches its maximum relative size in the 2d month. In the trunk, the upper portion, including the thorax and the upper abdominal viscera, is relatively largest during the earlier half of the fetal life. The lower part of the ab- domen becomes more prominent toward the end of the fetal period, due chiefly to the rapid expansion of the intestines at this time. The pelvis and lower extremities do not reach their greatest relative size until early adult life, although the upper extremities have reached their maximum relative size at birth. It may also be noted that the organs lying dorsal to the body axis (brain and spinal cord) grow at first far more rapidly than the organs ventral to the body axis. The volume of the brain and spinal cord together at the beginning of the 2d month is nearly 3 times as great as the combined volume of the organs lying ventral to the body axis, At birth, they are about equal. In the adult, the ventral organs are 6 times as large as the brain and spinal cord. The significance of the rapid growth of the brain and spinal cord in determining the marked flexure of the body in the early embryo has already been pointed out by Merkel (32) and by Keibel (Normentafel zur Ent- wicklungsgeschichte des Schweines, 1897). 3. GrowTH oF THE INDIVIDUAL ORGANS. As the growth rate of the whole body is the resultant of the growth rate of the various parts, so the growth of the various parts depends 132 C. M. Jackson. in turn upon the growth of their component organs. ‘The relative size of the principal organs in the specimens examined is given in Table IV. In Table V the average relative size of the principal organs is given for the various lunar months. In this table, all the available data published in the literature have been utilized, measurements on about 800 embryonic, fetal and newborn specimens being used. Lunar Months Fie. 3. Curves showing relative prenatal growth (percentage of the total body weight) in the brain, spinal cord, liver, lungs and heart. Based upon Table V, from all data available, grouped by months. The dotted lines on the left indicate probable relations of the first month, as explained in the text; those on the right connect the data for still-born with the corresponding figures for live-born. Still-born = Live-born These include observations by Welcker and Brandt (45), Legou (25), Faucon (15), Arnovljevie (3), Brandt (8), Anderson (2), Boyd (7), Lomer (28), Meeh (31), Liman (26), Thoma (41), Oppen- heimer (38), Miihlmann (36), Collin and Lucien (9), and Beneke (4). cms 14,6 — 68 | 115 mM: 4) 520 = vat 194 fea Gs as 75 148 m..|°5.8 — 76 197 PEG. 2 9.0 79 123 Mew Ges 9.2 82 $26). EAR TSS 12 87 1302. ile ms |). 9: 1327 95 129 m,) 9x5 14.5 97 130 £. A MOR5 16. 105 143 far s(t = 110 162 m. {11.5 = 115 186 mae 20.3 130 £995. |. fe (1342 23. 132 Tt de lha.. 22.5 140 AS ER ria vs 2125 | 2140 F1S.10) ane hoes ee 146 195 m. |16. 25.5 152 154 fee deze he OG: 158 210 foe WEES, 00 Be De? GO 172 mi segs | 275 165 Bot ec 120: P eI" 170 | Abo. \20- 31: 174 | 192 i120» 33. 185 Dias ee, 2199" 34. 192 | 193". m...|26. 37/27. Ble 209. | 208. "|< mz ° (8155 46. 258 201 | m. |30. 46.5 | 260 234 m. |31. 49. 274 202 a (BD 50. 280 198 m. (36. 54. 302 | | Volume. Fixation. 01544 ce.| Formalin. .026 | Alcohol—Formalin. .0976 Alcohol. .3788 Formalin. 183 fs Bi) | Alcohol—Formalin. 2.29 | Fresh. 1.693 | Formalin. 5.6 | Alcohol. 6.0 Formalin. 4.1 sé 8.8 | £6 5.0 | ‘e 10.0 | @ 14.75 4 15eo | ee iL5),b5: “ 24.0 of 54 | Alcohol. 80 | ce 108 | <4 122 | oS 95 Formalin. 97 6c 243 os PASH | of 370 180 | Fresh. 214 ! ss 375 Formalin. 464 Alcohol. 383 Fresh. 460 Formalin. 605 s 690 775 re 791 Fresh. 941 Formalin. 1981 Fresh. 2310 Formalin. 2727 Fresh. 3470 Formalin. | 3830 ag ] | 156 C. M. Jackson. TABLE II. OBSERVATIONS ON VoLUME OF His-ZieGuEeR MOopELs. Number. 1(SR.) | 4 (M,) |-3 (BB.) | 5 (Lr.) 6 (@.) 7 (R) 8 (A.) Embryo length. | 2.2mm. | 2.6 mm.) 3.2mm.| 4.2mm.) 4mm. | 5 mm. |7.5 mm. Age His: 14 da. 20 da. 23 da. Mall’s | | Rule..... 15da. | 16da. | 18da. | 20da. | 20da. | 22da. | 28 da. Total volume: of | model........| 208 cc. | 236 ce. | *95ce..-|/;*218 cc. |; “86ce: 124 cc. | 328ce. Magnification | | of model.....| 40 diam. 40 diam. | 40 diam. | 40 diam. | 20 diam. | 20 diam. 20 diam. Corresponding | | (actual) vol- ume of em- | | Pryostes etl .00325ce .003687ce .001484cc .003406cc).01075 ec. .0155 ec.) .041 ce. Actual volume | | of embry ° | | | proper. . '.000781¢e wpe, .001484cc .003406cc|.01075 cc.. .0155 ce. .041 ce. Actual columel | of yolk sac..... .002469ee 002406ee | | Head = % of | | Total body ee ah eer ould | 87.9% | 338.9% | 34.9% | 38.7% | | 36. 6% *In Nos. 3 ai 5, the an of the volume of the eae are somewhat uncertain since in each a portion of the ventral body wall is deficient in the model. Cor- rection was made for this by adding 5 cc. to the volume observed for No. 3, and 9 ec. to that for No. 5. TABLE III. PRENATAL GRowTH oF Human Bopy By Monrtrus. | > “s 2A ; ae : Relative Monthly Lamar Month, | Weightat Beginning | yeh | Growth. (P=) it .000004 g. .04 g. 9999. ui .04 3: all) 74. III. 3.0 36 te ive 36. | 120. Dee V. 120. | 330. ile ris VI. 330. 600. 82 VII. 600. 1000. .67 VIII. 1000. 1500. 750) IDE 1500. 2200. AT xX | 2200. 3200. .45 Prenatal Growth of the Human Body. TABLE IV. RELATIVE SIZE OF VARIOUS FETAL ORGANS (IN PERCENTAGE OF TOTAL Bopy VOLUME) IN SPECIMENS OBSERVED. No. and Sex. 60 (—) 58 (f) Crown-rump length {eerie | el eecme Volume of body...... .122 ce. | .4735 ce. 15 Coie Ween eal Pea tas See | 44.42% | 45.76% Upper extremities ....| 2.91 2.13 Lower extremities ....) 2.43 2.26 “Dra Ta ea eam an Saar serra rar) a8) Ue 49.86 IB igre Sete ens Bee 20.26 2230 PMA COR. ..0< osc. 8% 4.85 3.43 Thyroid gland ....... | Thymus eaten aera: van PIGHRap aatoct a e «cect | 3.64 174, Right lung ..........| :18 .276 Left lung ...... Es Se .18 aoe BI VER Sage sb scien s : ll 4°85 6.91 BS Deemer sea neck eat oh .019 IPANGrEASH ena | .032 Right kidney ........| .044 heft. kidney... 5.5.0). | .042 Right suprarenal ..... .169 Left suprarenal ...... 172 Mtomachsetes oc enn. .56 JNGEStINES:..ss0s oe els. pias 02 MEX PANGS tssajsree + she .0852 a2 57 (f) | | 185 (f) 148 (m) SS = 3.lem. | 4.5cm, | 5.8em. 2.117 ce. | 8.8ec. | 15.5ce. 45.4% 2.64 Peak 49.76 18.81 1.53 10.56 -0357 | 4.67 5.68 | 46.47 | 22.72 1.25 NR RH. Bets . (0°) | 43.18% | 41.94% 5.87 7.61 | 44.58 18.58 UA 1.06 1.26 1.03 6.45 .516 . 048 31 ol .452 2.0 197 (f) 6.2em. 5. o.Gee 36.8 % 5.75 6.58 50.9 .03 74 23 71 OOo) te .29 .309 . 258 . 258 .58 Norse. To correct for shrinkage in the first three embryos (which were em- bedded in paraffin), 25 per cent was added to the observed body volumes given in Table I. OFINMESF. pee eS TABLE IV (Continued). RELATIVE SIZE OF VARIOUS FETAL ORGANS (IN PERCENTAGE OF ToTAL Bopy VOLUME) IN SPECIMENS OBSERVED. No. and Sex. | 123 (m) 128 (f) 181 (m) | 129 (m) | 130 (f) 143 (m) Crown-rump length | 6.8cm.) 7.5 cm. | 9 em. | 9.5 cm. | 10.5 cm.| 11. em. Volume of body....... 24. ce. | 54. ec. | 80. ce. | 108 ce. | 122 ce. | 95. ee. Pleads ty Sat fe cea 43.75% | 40.74% | 40. % | 39 81% | 39.35% | 40. % Upper extremities..... 4.17 6.11 | 6.88 6.02 | 5.33 6.11 Lower extremities....| 6.25 | 10.2 9.75 9.07 9.43 8.42 Trunk 145.83 |42.96 |43.88 | 45.09 | 45.9 45.47 1B) ari Ta ROR oes a | 15.0 h06 | 719") 1.08 59 Right Wines..3| 14 | 136 | 1.51 .988 .909 Left luve..:s2..| 11° | 1.08 1.16 .79 BGSSy oases MIVEr She. 5s Sb e474. 63S 4.35 2.0 4.84 Gpleel co ass vo 078.) W227) 59 201 108 | .24 Pancreas....... 074 | 060) 069 | .067 | 054) 12 Right kidney... .338 | .468 | .499 f gas {| -286| .37 Left kidney..... .368 | .484 | .478 |S ° ie 26 Al Right supra- | | renal...... 1B ieik6 4) cll? | .087 118 Left supra- | 231 Tomales 05 « 176) | 160, | 4128 |} be 087 | -.138 Stomach.......| 22 | .519 .213 : 273 || 9909 | Intestines...... S240 | 8.772. (3,08 a 308: |e Sex glands..... 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A ContrIBUTION TO THE MorRPHOLOGY OF THE MamMMALIAN SKULL. BY CHARLES SEARING MEAD. WITH 11 TrxT FIGURES AND 4 PLATES. CONTENTS. PAGE JN EO RKO ELON ies st Srsrevoetr che Sheets Cio Dic OSE OR DRIES EERO PERCE oR UTA Prater iecrnesione 167 PIO S Ko AS A AAW OL peaches, cates avons aves a alis: © Sic War alee al aon gee rele beteaar eee recov oneeenee 169 JET OWIUTAS BSP IES Seat ece KA oe aOR Ent ee RNR nE TIS Ei choo mente CRI ICTS Choscky peor oss 170 EVOAT OO) CCU UTA S ater chats: sis: eis\ ey spate at ceva hiss e ptoNeia ee rawei os Sie echol so eRe oreo eneeeets 175 ECC PT Oe OGL CA Rete cre nial. ei sVSIes cee sNiopances lous fel aver are yehatel SEetene meee al ocetone oiclonete ete eee es else 178 AUIGUCORY CAPSULES. 52a cha ctors cust ova chet wel orarat eek eke ev over SER aoe wactie Saceiens 180 Soung- Conducting rAppara wus) saleeiciss «seve ele oee elaronstecare) ottned ch nite eee revere 185 Nerve Foramina in the Region of the Ear-Capsules.................. 188 REAOFOLDILOLEMIPOLALISH secrecy Scie ae race ore oO Here ae oars ot ora tae erent 192 FUG O HGH OLAS re hcrchacra tarsi sieee isis) Senos oue ev erele ter Siete oor'ere aero Gir aait) avsiave arenarouene 199 CONCIUSTONS seer core c pers eete hems fete ater aca sles operas eres, eee eo PER e ene Shee arate - .206 SUIT O Sy orareterar tekereiel yeteuster cook wie. o anole fo 5 SvePe atieie Sie. clone a Sree ae saislertne as 208 INTRODUCTION. The study of the chondrocranium of Sus is of value not only in assisting us to understand the structure of the adult skull in this form, but also on account of its bearing on the general morphology of the mammalian cranium. Owing to the generalized dentition and the structure of the feet, Sus has been placed relatively low in the ungulate series. Hence, we would expect many primitive char- acters to be retained in its cartilaginous cranium, and, indeed, this is the fact, for a number of reptilian characters are present. This chondrocranium is also valuable for comparison with those of pri- mates and insectivores. THH AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. 168 Charles Searing Mead. The Sus embryo studied was 30 mm. long (head-rump measure- ment), and the length of its head 12 mm. The head was cut trans- versely into 795 sections, each 0.015 mm. thick. The cartilage in all the even-numbered sections was drawn with the aid of a projec- tion apparatus, the drawings being enlargements of 25 diameters. In making the reconstruction, Born’s wax plate method was used. For comparison I have had Ziegler’s wax models of the chondro- erania of man, Gallus, Lacerta and Rana. In addition, Professor Eugen Fischer, of the University of Freiburg, loaned me his re- constructions of the Semnopitheeus, Macacus, Tarsius and Talpa skulls, and the series of sections from which his Talpa reconstruction was made. Also a reconstruction of the primordial cranium of Lepus, prepared by Dr. Max Voit, has been of service. Of the models and reconstructions used for comparison, the one of Talpa resembled more closely that of Sus than did any of the others, and, hence, has been referred to most frequently in the com- parisons. The literature dealing with the development of the mammalian skull is very extensive. The publications of Parker, Spondh, Kolliker and Decker, on the chondrocrania of the mammals, are the principal works belonging to the old school, in which the skulls were prepared principally by the maceration method. The introduction of Born’s wax plate method of reconstruction has made possible not only a more exact study of the embryonic cranium itself, but has also enabled one to study the surrounding tissues as well. A more fundamental view of the skull is thus obtained. Among the more recent papers on the embryonic skull may be mentioned Gaupp’s “Die Entwickelung des Kopfskelettes” in Hertwig’s Handbuch (1905 b), valuable on account of its general survey; also his ex- tensive contribution on the development of the skull of Echidna (1908). The papers of Fischer on Talpa (1901 b) and the apes (1908) and a forthcoming paper of Voit on Lepus (1909) likewise contain valuable results. This investigation was undertaken at the suggestion of Professor Ernst Gaupp. The work was conducted in the laboratory of the Comparative Anatomical Institute of Freiburg in Baden. The Chondrocranium of an Embryo Pig. 169 I will here take the opportunity to express to Professor Gaupp my sincere appreciation not only for the use of his extensive series of sections, but also for his very valuable help and suggestions. Dr. Max Voit I wish to thank for the use of his Lepus reconstruction and for the assistance which he has rendered me. And for the loan of the reconstructions of the Semnopithecus, Macacus, Tarsius and Talpa skulls, and also for the series of sections from which the Talpa reconstruction was made I wish to express to Professor Eugen Fischer my sincere thanks. THE SKULL AS A WHOLE. The form of the primordial cranium of the pig represents well the generalized mammalian type of chondrocranium, No part is markedly underdeveloped and no part is greatly overdeveloped at the expense of the surrounding portions. As a whole, the chondrocranium at this stage of development, 7. ¢., in an embryo 30 mm. long, resembles a pear in shape, with the small end anterior, forming the nasal region, and the large end posterior. The large brain-box is widely open dorsally, a feature characteristic of the amniotes. Later this region of the skull is completed by the formation of the large roofing membrane bones, the frontals and parietals. Ventrally the general contour is completed by the man- dibular, hyoid and branchial arches. ‘The brain-case is large and extends forward over the posterior half of the nasal capsules, forcing the fenestrae cribrosee from their primitive vertical position into a horizontal one. Its side walls are formed by a broad continuous plate of cartilage on each side, thus standing in sharp contrast to the condition in similar embryos of man and the primates, in which this region is very rudimentary. The nasal capsules are of moderate length, not long as in Talpa, nor short as in man. Taken as a whole, the chondrocranium is complete except for some minor details. None of the cartilaginous bones (Hrsatzknochen, Gaupp) have as yet begun to ossify. Some of the membrane bones have not yet appeared and the others are only in the very early stages of their formation; con- sequently they have not been included in my reconstruction (Pls. I-IV). With the exception of the mandibular, hyoid and branchial 170 Charles Searing Mead. arches (the visceral skeleton), the different parts of the skull are not separated by sutures, the cartilage composing the neural cranium forming one continuous unit. Kolliker has divided the skull into the posterior part, the pars chordalis, through which the notochord runs, and the pars prechordalis, situated anterior to this. Each of these divisions Gegenbaur has again divided into two regions, which Gaupp has named, from behind forward, as follows: regio occipitalis, regio otica, regio orbitotemporalis and regio ethmoidalis. For de- scriptive purposes it will be convenient to follow these divisions, ex- cept that the basal portions of the two posterior regions will first be considered together, since they form a fairly complete unit, the planum basale (Gaupp). Prianum Basar. The planum basale comprises the basal portions of the two poste- rior regions of the skull. It extends from the foramen magnum forward to the posterior border of the hypophysial fossa and is perforated for nearly its entire length by the notochord. Its anterior three-fifths, 7. e., the portion belonging to the regio otica, is narrow and rod-like (Pl. I and Figs. 1, 3, 4 and 5), while its posterior por- tion is spread out into a broad quadrangular plate, the basilar part of the occipital region. The anterior portion of the basal plate in Sus differs strikingly from that in other mammals on account of its narrow, rod-like shape and also because of the character of its union with the ear-capsules. Here it is separated from the ear-capsules except for a thin connect- ing lamella of cartilage, while in the other mammals that I have examined it is firmly united with the capsules. In the other mam- mals, also, the planum basale forms a broad plate of cartilage, which passes over without sharp demarcation into the broad basal cartilage of the orbitotemporal region. Tarsius offers an exception to this statement, since in it the portion between the cochlear spheres is even narrower than in Sus. This, however, is due to the extremely large size of the ear-capsules, which approach the middle line and compress the cartilaginous plate which lies between them. Back of the auditory bulle the planum basale spreads out into The Chondrocranium of an Embryo Pig. sly a broad quadrangular plate, limited posteriorty by the foramen mag- num, anteriorly by the foramen jugulare or fissura metotica and the auditory bullx, while laterally it passes over into the pars lateralis, regio occipitalis. On the boundary between the basal plate and the N.C. b.p. Fic. 1. Section showing the broad basal plate b.p., the notochord m.c. near its dorsal surface, and the large auricular branch of the vagus nerve X r.aur. Sey VIII, nervus acusticus; m.st., stapedial muscle; j.v., jugular vein; Ls.c., lateral semicircular canal; wtr., utriculus; a.s.c., anterior semicircular canal ; l. par., lamina parietalis; Y, nervus vagus; X//, nervus hypoglossus. pars lateralis the processus paroccipitalis arises and extends forward. The basal plate is perforated just median to this process by the foramen nervi hypoglossi (Pl. I, f. hyp.). Three branches of the hypoglossus nerve have been demonstrated in a number of mammalian 172 Charles Searing Mead. embryos, but the anterior one disappears and the other two usually leave the cranial cavity through a single foramen, the foramen hypoglossi (foramen spinodccipitale). Voit found that in Lepus embryos two foramina hypoglossi are present on each side, and these may persist even in the adult. Fischer described in an embryo of Semnopithecus pruinosus two hypoglossal foramina on the left side and three on the right. On the other hand, his reconstruction of Semnopithecus maurus shows but a single foramen on each side. This shows what variations may occur within the limits of a single genus or even species. Back of the hypoglossal foramen the basal plate passes, without demarcation, into the lateral walls of the occipital region. The union which occurs between the cochlear part of the auditory capsule and the basal plate is later destroyed by the absorption of the lamella of cartilage mentioned above. At this stage this lamella is perforated on each side by a foramen (Pls. I and II, *, and Fig. 4) filled with tissue resembling precartilaginous tissue (Vorknorpel). It is probably the homolog of the fissura basicochlearis posterior, which Noordenbos (1905) has deseribed in embryos (14 mm.) of Talpa. However this may be, this lamina fails to ossify so that in the adult the foramen lacerum anterius and the foramen jugulare are united by a broad slit median to the auditory bulla. In a Tarsius embryo of a somewhat later stage this lamina of cartilage is not present and the ear-capsules are separated from the planum basale for nearly their entire !ength by a large fissure, the fissura basicochlearis. In Talpa the large foramen piercing the basis cranii median to the trabecula alicochlearis is not the foramen lacerum anterius, as Fischer called it, but the fissura basicochlearis anterior (Noordenbos). It is located internally to the foramen caroticum. No nerves nor blood vessels pass through it, as was deter- mined by examining the series of sections from which Fischer’s re- construction was made; it is filled only with connective tissue. The foramen for the facial nerve, which in the reptiles pierces the basis cranii anteriorly and ventrally to the ear-capsule, has here migrated around to the antero-dorsal side of the cochlear portion of the ear and lost completely its connection with the basal plate. This will be considered in greater detail in connection with the otic region. The Chondrocranium of an Embryo Pig. 173 Looking now at the inside of the skull, it is seen that the base rises gradually and in an almost straight line from the foramen magnum to the dorsum ephippii. From side to side the curve on the inner wall of the brain-ease is nearly uniform, thus closely simulating the half of a cylinder. On each side of the anterior end of the basal plate there is a large triangular opening (PI. I, for. abduc.). This is bounded anteriorly by the widely projecting processus clinoideus posterior, and postero- laterally by the cochlear portion of the ear-capsule. It is filled mostly with indifferent tissue, but serves also for the passage of the nervus abducens. This is especially noteworthy since such a foramen has not been described in any mammal. In the chondrocranium of Semnopithecus Fischer found the nervus abducens passing through a groove, which was closed by a band of connective tissue, but no distinct foramen was present. Meckcart: = = — —— ala orbit. ala tempor. ~~ ‘ et “eee for.optie. 4 cor.branch. I~ , ——.-. com.orbito-par. cornu hyale ~ — tt ———— — —. for.abdue. f /_! — ~~ for.spheno-par. ey man.malleus ~~ _ _.. for.facialis malleus — CUS enim = fen.cochlear. proc.paroe. for.jugul. ~~ condyle crista parotica proc.periot.sup. f post.semi.canal aA ~lam.parietal. fis.lam.par. fis.metotica _ for.petro.oce, — — for.jug.spur. tectum post. ~ ant.semi.canal THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. 7 ae A ee f : P ea p ~ ¢ i 7 ' 7 - : _ a : = } i Geran Cer ee ga . ae TL Y Pa) ere © THE CHONDROCRANIUM OF AN EMBRYO PIG. PLATE IV. CHARLES SEARING MBAD. Meck.cart. corpus hyoid. cornu branch... cornu hyale man.malleus [PHE AMERICAN JOURNAL OF ANATOMY.—VoL. IX, No. 2. “ay ee pe? 5 2 Mt) = x A el ® a = bi % bes z = he Po. aie: seal Sobe 55 THE DEVELOPMENT OF THE HEART IN SHAD (Alosa Sapadissima, Wilson). Wirn a Nore on THE CLASSIFICATION OF TELEOSTEAN EMBRYOS FrrRoM A MorPHOLOGICAL STANDPOINT. BY H. D. SENIOR. (From The Wistar Institute of Anatomy and Biology, Philadelphia, and the Department of Anatomy in the College of Medicine, Syracuse University). WITH 27 FIGURES. CONTENTS. PAGE. Introduction—A brief description of the heart and great venous chan- nelssoL alrecently WAtCheGeSha Ge ryacev. clerststelekels clelets =) ielels|n )rlielsteeRelal-tensie 212 Contrast drawn between embryos of the shad type and those in which a vitelline vessel-network occurs. Suggested classifica- tion of teleostean embryos into morphological types depending on the relation of the ventral vessel system to the yolk........ 215 Statement of the object in investigating the development of the heart in shad, and of the scope of this investigation.................. 219 Materialvands MeGnodswseaca octet cc oclere oo) ele ole) ole) ele at ele)! o) eile! o/s) cles) «le/ei steers) 219 Development of the Heart...........cc cece ccc ce cer ee cece reer cecrcceces 222 Period 1. Formation of the heart anlage................2e+esseeees 222 Review of the evidence bearing on the relation of the endo- cardium to the vascular endothelium of the head in gen- @qill! sodooo mea do pocoon ocedo oleic SUD OUD OUDO CU DIS COGnOD DOO 232 Period 2. Lasting until rhythmical contraction begins in the par- tially, formed heart-tube - 2055. 2. ee eee eee ere eeraenem = 237 Period 3. In which the heart-tube is completed, to form conus, ventricle, and atrium; and assumes the adult position. SRE Gite GA NNine cas cocoa ose qadauucue Do OGD Od FODMOOmC 0H COGS 249 Jorrelation of the stages estimated by the number of somites with those designated by the length of the embryo...... 244 Mechanism of the circulation in different stages of develop- TMYAOLF bod AO SO SOOO DOOR DIDO OO Ow a bi Oke Cc ONC DORON ae 244 SIGS OF GB nile bocaoodonouse send cedar ono caOdoUrcuOG TS Dod aor 246 Sige GE 7B itil ss gee pombe tao como aig ob DOr ao doonoEn Odeo moat 249 Stage of 8.75 mm., and a comparison of the heart with that of the SPicermescripeds in une) IMtTOOWMCLIOME rjcicte t+) ote) tctere years @linle 250 Period 4. Formation of the sinus venosus and hepatic vein.......... 252 THH AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. 212 H. D. Senior. INTRODUCTION. A Brief Description of the Heart and Great Venous Channels of a Recently Hatched Shad. Tn examining a recently hatched specimen of shad, it will be found that the heart (Fig. 1) is apparently widely open at the venous end. The atrium is separated from the ventricle in the usual way, but, on tracing the atrial wall back to where the sinus venosus should be, no sinus venosus, as such, is found to exist. The heart wall immediately succeeding the somewhat constricted venous end of the atrium represents the anterior wall of the future sinus venosus. This flares out abruptly, and its cireumference, having reached the body-wall, is reflected forward upon the latter as the parietal peri- cardium. These relations are shown in Fig. 1; the general arrange- ment of the circulation, at this period, is diagrammatically indicated in Fig. 2. i] The relations here are obviously peculiar; the peculiarity, how- ever, does not consist in the continuity of the myo-epicardium with the parietal pericardium. Inasmuch as both the myo-epicardium and the parietal pericardium are developed from the mesothelium of the lateral plates in all vertebrates, this continuity is invariable. The peculiar feature in connection with the heart, in its present state, is that the anterior pole of the yolk in a sense replaces the posterior wall of the sinus venosus. Since the yolk is entirely naked, the vascular system, closed though it is, is not completely lined by vascular endothelium, as is very commonly the case in vertebrate embryos at a comparatively early stage of development. The venous blood is returned to the heart through four veins, the jugulars and cardinals; also by means of a blood-sinus situate dorsal to the yolk, which may be called the supravitelline blood-sinus (or, for the sake of brevity, the supravitelline sinus). The supravitelline sinus is formed in the following way: The peritoneum has a ventral attachment on either side to the dorsal surface of the yolk; the peritoneal attachments involve almost the entire longitudinal extent of the yolk. Between the lines of attachment there is enclosed an arched tunnel, of which the floor is formed by the surface of the yolk, The Development of the Heart in Shad. 213 position of Fig. 21 position of Fig. 20 position of Fig. 19 | position of Fig. 18 et Spinal cord Ectoderm Notochord Dorsal aorta Peritoneal coelom L. cardinal vy. (Esophagus R. cardinal y. Supravitelline sinus L. jugular v. “Yolk-process”’ of sinus venosus Br. cleft 2 Branchial cleft | Branchial a. 4__ Branchial a. 3 Ventral aorta Yolk [in mid- sagittal sec.] Dorsal parietal pericardium Conus arteriosus Atrium Ventricle Anterior wall of sinus venosus Body-wall [cut] Ventral parietal pericardium (mostly removed) Fic. 1.—The left side of a reconstruction from the posterior gill region of a recently hatched shad, stage of 114 hours » 100 diams. Sufficient body-wall has been removed to open the pericardial cclom ventrally and laterally and to show the terminations of the left jugular and cardinal veins. The anterior pole of the yolk and the adjacent parts of the heart and pericardium are shown in mid-sagittal section. 914 H. D. Senior. and the roof by the ventral surface of the splanchnic mesoderm. The roof is entirely lined by vascular endothelium. The floor, having no endothelial lining, presents the naked periblast of the yolk to the blood-stream passing over it. Although the mid-sagittal plane on the embryo would fall (except near the anterior pole of the yolk, see Figs. 21 and 22) entirely within the tunnel, it would separate the latter into two unequal parts, of which the left would be greater than the right. The supravitellins sinus, consisting of the tunnel described above, receives blood from the short vena revehens of the liver and dis- charges it into the, somewhat roomy, chamber embracing the anterior pole of the yolk. The liver, at this time, is situated dorsal to the posterior pole of the yolk (see Fig. 2). The chamber at the anterior L. jugular va Vic. 2.—-Diagram indicating the arrangement of the principal blood-channels of the recently hatched shad of which the heart is shown in Fig. 1. x 10 diams. Arteries black; veins, and supravitelline sinus, stippled. pole of the yolk, which corresponds im position with the future sinus venosus, is bounded as follows: Posteriorly by the naked anterior pole of the yolk; elsewhere by the portion of the heart-wall im- mediately succeeding the atrium, which is later to form the anterior wall of the sinus venosus. Venous blood enters the chamber from the jugular and cardinal veins and from the supravitelline sinus, and leaves it by passing through the orifice leading into the atrium. The relation of the yolk to the vascular system is somewhat as follows: While it converts the partially formed sinus venosus and the (subintestinal) supravitelline sinus into a closed passage, capable of retaining and transmitting blood, it, at the same time, delays the formation of the posterior wall of the sinus venosus and of the vessel later to be formed by the vascular endothelium lining the supravitelline sinus. The Development of the Heart in Shad. 215 In order to identify the parts briefly described above with the structures found in the adult, it may be said that during the rapid dwindling of the yolk the piers of the arched supravitelline sinus ap- proach each other and that between them, by a process of rearrange- ment of vascular endothelium, there is formed a vein, the hepatic vein of the adult. The portion of the heart-wall connecting the venous end of the atrium with the parietal pericardium forms not only the anterior wall of the sinus venosus, but also the pericardial surface of the pericardio-peritoneal septum. The posterior wall of the sinus venosus, together with the peritoneal surface of the pericardio-peritoneal septum, is furnished by the anterior part of the splanchnic peritoneum. No vitelline vessels, other than the hepatic vein (derived from the vascular endothelium lining the roof of the supravitelline sinus) are ever developed. Contrast drawn between embryos of the shad type and those in which a vitelline vessel-network occurs. Suggested classification of teleostean embryos into morphological types depending on the rela- tion of the ventral vessel system to the yolk. The entire absence of a network of vessels on the ventral and lateral surfaces of the yolk imparts to the egg of shad (and to other eges of the shad type) an appearance strikingly different from that of the teleostean eggs (e. g., those of Salvelinus) which for some time before hatching display a vitelline vessel-network filled with corpuscles. The type of egg to which shad belongs may be called Type 1 in contradistinction to the type in which a vitelline vessel- network occurs, which may be called Type 2. Type 1 appears to be almost universal in pelagic eggs. Uran- oscopus scaber? is the only pelagic teleost of which I find it recorded The following are some examples of pelagic eggs in which a vitelline vessel-network is stated not to occur, or in which its absence has been inferred from figures depicting well-advanced stages: Elacate canada, Gadus morrhua (callarias), Chaetodipterus faber, Scomberomorus maculatus (Ryder ’82, ’84, and ’87). Hemipterus americanus, Temnodon saltator, Lophius piscatorius, Ctenolabrus (Tautogalabrus) adspersus, Tautoga onitis, Pseudorhombus oblongatus, Motella argentea (Agassiz and Whitman, ’85). Labrax lupus, Serranus cabrilla, 8S. scriba, Sargus Rondeletii, Box vulgaris, 216 H. D. Senior. that a vitelline network appears (Raffaele, ’88). Type 1 is also commonly found in demersal eggs (among which the majority of the eggs belonging to Type 2 occur) including shad? itself; and, finally, although some of the viviparous eggs belong to Type 2 (e. g., Zoarces and Gambusia) others* occur which conform to Type 1. Since the difference between the two types referred to can scarcely be said to exist in the earlier stages of development, it is well to define, as exactly as may be, what is considered to be the essential difference between them. In both types the heart pulsates prior to the appearance of free blood corpuscles, and the space between the yolk and the extra-embryonic ectoderm is occupied by circulating blood-plasma. In the type in which a vitelline network occurs (Type 2), the blood, which acquires corpuscles comparatively early, is, sooner or later, confined upon the yolk, as elsewhere, in actual vessels. In Type 1 no vessels are ever found upon the yolk, the Scorpaena, Lepidotrigla aspera, Callionymus, Mugil (capito?), Gadus minutus, Coris (several species), Merlucius vulgaris, Motella vulgaris, Solea (several species), Rhombus laevis, Arnoglossus, Clupea, Engraulis encrasicholus, and several undetermined species (Raffaele, ’88). Hippoglossoides limatoides, Rhombus (Psetta) maximus, Pleuronectes plates- sa, P.cynoglossus, P.microcephalus, P.flesus, P. limanda, Solea vulgaris, Molva vulgaris, Centronotus (Pholis) gunellus, Motella mustela, Gadus morrhua, G. aeglefinus, G. luscus. G. merlangus, G. pollachus, Lophius piscatorius, Trachinus, Clupea sprattus, Trigla gurnardus, Callionymus lyra (McIntosh and Prince, ’90). Fierasfer dubius, Stelaphorus ringens (Higenmann, 792). Also Pomolobus vernalis (pseudoharengus), Roccus americanus, Osmerus (Ryder, ’84 and ’87). Typhlogobius californiensis (Higenmann, ’92). Also Pseudopleuronectes americanus and, doubtless, many others. ®’The examples found on record are Sebastodes auriculatus and Cymato- gaster aggregatus (Higenmann, ’92 and ’94). Probably a great many more of the viviparous perches also belong to Type 1. The absence of a vitelline vessel network in both the cases mentioned has been assumed from the figures alone. Through the courtesy of Dr. J. Percy Moore I have had an opportunity of verifying the type of Cymatogaster aggregatus. ‘The arrangement of the vitelline vessels (which are invariably veins, Hochstetter, ’87) varies considerably in different species, and these variations can be again Classified into sub-types (see Ryder, ’82, Wenckebach, ’86, H. BH. Ziegler, ’87, Hochstetter, ’87, Ziegenhagen, 94 and ’96). The Development of the Heart in Shad. 217 ventral surface of the splanchnic peritoneum is, however, lined by vascular endothelium which eventually forms the hepatic vein® The hepatic vein then in Type 1, replaces the vitelline network of Type 2, and it might itself be considered a vitelline vessel, but for the fact that when it is fully formed the yolk is reduced to a very small size. The light in which the hepatic vein is regarded, however, does not affect the essential validity of the types: Hmbryos of Type 2 differ from those of T'ype 1 in that they possess, at some period prior to the disappearance of the yolk, vitelline vessels lateral to the margin of the celon. In all cases in which the site of origin of the blood corpuscles has been investigated in embryos of Type 2,° it has been found to occur in the cardinal veins, which may be separate or partially conjoined (Stammvene). In embryos of Type 1* the cardinal veins have invariably been found, when they first appear, to contain no corpuscles. At the present time, although the information at our command is rather suggestive, it appears neither safe to assume that the blood anlage is always developed within the cardinal veins in embryos of Type 2, nor that this never happens in embryos of Type 1. It would seem that more information is needed on the entire subject of blood-formation in teleosts, before a generalization of this kind can be made with safety. I refer particularly to the fact that Marcus (’05) has recorded for Gobius ecapito (Type 2) that corpuscles arise in the tail as well as in the cardinal vein region ; ‘It is neither assumed nor implied that the formation of the hepatic vein in other embryos belonging to Type 1 is similar in mechanism to that later to be described for shad; the serial sections of other Type 1 eggs, mostly pelagic, in the possession of the writer do not cover all the stages necessary for the determination of this point. *Zeigler, ’82 and ’87, Salmo salar; Wenckelbach, ’85, Perca fluviatilis, ’86 Belone and Esox; Felix, 97, salmon and trout; Swaen and Brachet, ’00, trout, 02, Leuciscus cephalus, and Exocoetus volitantes ; Sobotta, 702, Trutta fario, T. iridea and Salmo salvelinus; Marcus, ’05, Gobius capito. TWilson, ’91, Serranus atrarius (Centropristis striatus, L); Sween and Brachet, ’02, Clupea sprattus, Rhombus (?), Solea vulgaris, Pleuronectes microcephalus, Trachinus vipera, Caranx trachurus, and Callionymis lyra; Derjugin, 02, Lophius piscatcrius. 918 H. D. Senior. this is probably a fact of great importance. The blood anlage of shad (Type 1) arises as a cord of cells in the tail, which forms a direct continuation backwards of the, then, partially developed caudal aorta and caudal vein; part of the blood anlage of Opsanus tau (Type 2) also arises in a similar manner (the remainder arising in the cardinal veins). It is possible that the tail is the site of blood formation common to all teleosts, and that the cardinal vein blood anlage occurs as a further source of corpuscles in the forms which acquire numerous corpuscles at a comparatively early stage of devel- opment. Without assuming this actually to be the case, I would venture to suggest that, in connection with the origin of the blood corpuscles, the tail deserves thorough examination in all teleosts, whether or not, in the species under examination, corpuscles are found to arise in the cardinal veins. Reference to investigations setting forth the surface of the yolk as the source of blood corpuscles has been purposely omitted. It has been my experience that the yolk in eggs of Type 1 is in excellent condition for cutting after it has been fixed in formalin ; whereas formalin-fixation produces in eggs of Type 2 a yolk difficult to cut and sometimes of almost stony hardness. This is not due merely to difference in size, but seems to point to a difference in chemical composition between the yolks of the two types of egg. It is well known that the differences in structure, and in the general processes of development which occur among teleostean em- bryos of different species bear little or no relation to the structure and affinities of the corresponding adults; since, therefore, the type of embryo cannot be inferred from the systematic position of the adult, it would seem advantageous to classify the embryos them- selves according to their own structural peculiarities. The division of embryonic teleosts into the morphological types indicated above appears to be warranted by the present state of our knowledge and, since it is applicable alike to pelagic, demersal, and viviparous eggs it may prove of some service as a starting point for classification. The demand for some such division into types is, T think, indicated by the not infrequent use in the literature of the terms pelagic and demersal in a morphological connection. The The Development of the Heart in Shad. 219 following objections to the use of the words pelagic and demersal in a morphological sense will sufficiently indicate their unsuitability. (a) Their use, in this sense, is apt to entail the statement that a given egg is either demersal in habit and pelagic in structure, or vice versa, which is undesirable. (6) Neither of these terms can be used to express the structure of a viviparous egg. (c) These terms, as strictly applied, have no more morphological significance than has the term viviparous itself. Statement of the object in investigating the development of the heart in shad, and of the scope of this investigation. The development of the heart in embryos of Type 2 has received a large share of attention, particularly in Salmo and allied genera in which the process of heart-formation has been definitely made out. The heart in embryos of Type 1 differs considerably from that in embryos of Type 2, particularly in its relation to the vascular system of the yolk. The development of the heart, however, appears to have received somewhat scant notice. Ryder has given a brief account of some of the changes undergone by the heart in Gadus morrhua (callarias ), ’82, and in Clupea (Alosa) sapadissima, 785 ; Boeke has described the early development of the heart in Mureena (endocardium particularly), ’03. There are also some earlier in- vestigations, dealing with the living embryo alone, which, necessarily, leave much to be desired. It has been attempted here to give a consecutive account of the development of the heart in shad, as representing Type 1, from the earliest possible stage until the adult arrangement is recognizable. Mareriat anp Mrrnops. The material investigated, the property of The Wistar Institute of Anatomy, Philadelphia, was collected during the seasons 1905, 706, and ’07 at the hatchery of the Pennsylvania State Fish Commission, Torresdale, Pa. To the Commissioner, Mr. W. J. Meehan, I hereby tender my thanks for his many courtesies. Prior to a period of development at which the embryo is capable of 220 H. D. Senior. self-extension after removal of the egg-membrane (so that it may be fixed in the extended position) the stages have been estimated by the number of somites. Stages have been designated in terms of length of embryo (in a straight line from end to end) from the time that the embryo is approximately straight until the, somewhat arbitrarily selected, period of hatching. After hatching the age is given as being the sole guide to the stage of development; the length of the embryo, unfortunately, does not convey the required information. It is well known that development is relatively accelerated by a high water temperature. The period of development within the egg is actually shorter, however, in warm water, for the embryo is hatched in a progressively immature state in direct proportion as the water- temperature rises. ‘To mention a few examples: My stage of 114 hours, 10.5 mm. (just hatched at a low-water temperature*) is much more advanced in development than the stage of 8.7 mm., 63 hours, (hatched about twenty hours in warmer® water); the latter is only slightly more advanced than a stage of 8.3 mm., 107 hours (still in the egg, water-temperature®’ low). Direct comparison, except in individual cases, is not easily made between the hatching stages in different water-temperatures because the embryos of any one batch do not hatch simultaneously but continue to hatch over a period last- ing twenty-four hours or more; the hatching period must, therefore, be in any case somewhat arbitrarily determined. I have selected the stage of 114 hours as the just hatched stage (in preference to earlier ‘“just-hatched” stages) because at all stages prior to this the length of the embryo, from the time it is capable of self-extension, accurately indicates the period of development. Shad is anadromous; the egg, demersal and non-adhesive, is con- venient for study on account of its transparency and because it is easily removed from its roomy capsule (diameter of egg proper is about 2 mm. capsule slightly under 4 mm.). Details of the spawn- ing-habits and of the methods of rearing eggs and larvee are given in “A Manual of Fish-Culture,” published by the U. S. Fish Commis- sion, revised edition, Washington, 1900. SAverage temperature 63° F. *Average temperature 70° F. The Development of the Heart in Shad. 221 All the methods of fixation in common use for teleosts were tried. That of Sumner (saturated corrosive sublimate containing 10 per cent glacial acetic; followed by 10 per cent. formalin; see Sumner, 00) proved the most satisfactory and was generally employed; all the figures were drawn from material fixed in this way except Fig. 8 (H. Virchow’s method) and Figs. 10 and 11 (Pereny’s fluid)."° The embryos were cut into serial paraffine sections ranging from 5 to 10 microns in thickness. Sections of the earlier stages were stained with iron hematoxylin, later stages were usually stained in toto with alcoholic carmine. Eleven wax-plate reconstructions'? were made, two hundred times larger than the originals after fixation (correction having been made for shrinkage in paraffine). In making the figures the reconstruc- tions were photographed (natural size) and the outlines of the photo- graph traced. In finishing the drawings the irregularities due to the plates were omitted. All figures, representing reconstructions, have been reduced one-half in reproduction. Below follows a list of the figures with data for identification of their sources. (The numbers are from the catalogue of The Wistar Institute of Anatomy) : Fic. 1.—Reconstruction 14504, from series 14275, sections 87 to 134. Fie. 2.—Outline from embryo afterwards cut into series 14275. Fig. 3.—From series 14556; 3A section 91, 3B 120, 3C 132, 3H 142, 38F 152, 3G 181. Fic. 4.—Reconstruction 14534, from series 14533, sections 45 to 87; 4A sec- tion 58, 4B 57, 4C 62, 4H 71. Fic. 5.—Reconstruction 14535, from series 14524, sections 91 to 151; 5B section 104, 5C 111, 5D 114, 5G 148. Fic. 6. Reconstruction 14536, from series 14520, sections 63 to 110; 6B sec- tion 76, 6C 79, 6D 82, 6F 92. Fic. 7.—Reconstruction 14537, from series 14532, sections 68 to 113 section 82, 7C 86, TD 89, 7G 113. 3 7s 7 ”Pereny’s fluid, in which, unfortunately, all my material about the time of beginning heart-beat has been fixed, gives fair general results, but is extremely unfavorable for cytological study. UShown at the Chicago meeting of the Association of American Anatomists, Christmas, 1907. 222 H. D. Senior. Fie. 8.—Series 14567, section 39. Fie. 9.—From the reconstruction used for Fig. 12. Fic. 10.—From series 14667; 10B section 70, 10D 79. Fie. 11.—Reconstruction 15011, from series 14670, sections 68 to 110; 11C section 79. Fie. 12.—Reconstruction 15012, from series 14668, sections 39 to 69; 12C section 54. Fic. 13.—Reconstruction 15013, from series 14671, sections 21 to 80. Fie. 14.—Diagram from the reconstruction used for Fig. 18 Fie. 15.—Diagram from reconstruction 15014, from series 14672, sections 28 to 61. Fic. 16.—Reconstruction 15015, from series 14568, sections 20 to 56. Fic. 17.—F ig. 1 repeated. Fic. 18.—Series 14275, section 126. Fic. 19.—Series 14275, section 130. Fic. 20.—Series 14275, section 133. Fic. 21.—Series 14275, section 1386. Fig. 22.—Reconstruction 14507, from series 14588, sections 82 to 136. Fic. 23.—Series 14538, section 128. Fic. 24.—Series 14538, section 182. Fic. 25.—Series 145388, section 140. Fic. 26.—Series 15002, section 1388. Fic. 27.—Series 15002, section 148. DEVELOPMENT OF THE HEART. The process of development of the heart in shad may conveniently be divided into four periods as follows: 1. Formation of the heart anlage. 2. Lasting until rhythmical contraction begins in the partially formed heart-tube. 3. In which the heart-tube is completed to form conus, ventricle and atrium, and assumes the adult position. 4. Formation of the sinus venosus and hepatic vein. PERIOD I. FORMATION OF THE HEART ANLAGE. The myo-epicardium and the parietal pericardium are developed from the lateral plates of the mesoderm. That the endocardium is derived from mesoderm, and from mesoderm alone, has already been *1 THE DEVELOPMENT OF THE HEART IN SHAD. H. D. SENIOR. Notochord we Lateral plate “Entodermal pharynx 3B Entodermal 8 pharynx, dorsal wall 3A ventral wall Hyoid pouch 4 / & see Fog a s Lars ae sa\g £ See Somital & SR mesoderm L he Entodermal & Entodermal pharynx, dorsal wall ~ pharynx, ventral wall Fic. 5.—Transverse sections of shad, stage of 15 somites << 100 diams. For situation of sections see footnote on opposite page. THE AMERICAN JOURNAL OF ANATOMY.—VOL. EX) NOS 2: The Development of the Heart in Shad. 223 shown in other teleosts by Oellacher, ’73, H. E. Ziegler, ’87, Swaen and Brachet, ’00, and by Sobotta, 02. Nothing has been found in shad supporting the view that the endocardium arises from entoderm alone or from entoderm and mesoderm together. The particular part of the mesoderm from which the endocardium is derived is a, bilaterally symmetrical, cord of cells on either side immediately adjacent to the medial borders of the lateral plates; of this Swaen and Brachet have given a careful description (as found in Trutta fario) and to it they have applied the term “Portion moyenne du mésoblaste.” In shad at a stage of 15 somites the portion moyenne is recognizable throughout most of its, eventual, longitudinal extent; although comparison with earlier stages shows that it has been recognizable for some time but in a less advanced stage of development. In order to gain a clear conception of the portion moyenne, as it occurs in shad at the stage of 15 somites, it is necessary to enquire into the cause of its distinctness from the remainder of the head mesoderm. There are, as far as I can see, no features in the cells composing the portion moyenne which distinguish them at this stage from other mesodermal cells. The portion moyenne, as a whole, is distinguishable from the lateral plate by reason of the orientation of the cells of the latter (where this has occurred) around the, now virtual, clon; see Figs.’ 3B, 3C, 3E and 3F. The lateral plate, 219 facilitate comparison between the sections shown in figures 3, 4, 5, 6, 7, 10, 11, and 12 the same letter has been used throughout to indicate a certain region. Seven regions have been selected as follows: A is near the, eventual, anterior limit of the lateral plates. B is between A and the mandibular entodermal pouch. C is through the mandibular entodermal pouch. D is midway between C and the gill anlage spoken of in the text as the hyo-branchial. EB is a short distance in front of the hyo-branchial anlage. F is through the hyo-branchial anlage and the anterior part of the develop- ing otocyst. G is some distance posterior to the branchial region of the pharynx. In figures of sections P.M. indicates portion moyenne. The anterior aspect of the section is always represented ; the structures on the right side of the embryo will, therefore, appear on the left side of the figure, and vice versa. 294. H. D. Senior. which is now in progress of differentiation from behind forwards, is not yet clearly distinguishable at the site of Fig. 3A (q.v.) and here the portion moyenne (although it is later well developed, see Fig. 4A) is not clearly defined. The distinction of the portion moyenne from the somital portion of the head mesoderm depends on an actual separation of cells from the lateral region of the somite; or (perhaps more correctly) on an isolation of the cells situated between the somite and the lateral plate. The position of the portion moyenne, where portion moyenne occurs, bears a definite relation to the lateral margin of the entodermal pharynx. In the region of the entodermal pouches the pharynx is wide and the portion moyenne is always situated lateral to its lateral margin (see Figs. 3B, 3C, 3E and 8F). Posterior to the region of the entodermal pouches the entodermal pharynx is much reduced in width, and here the portion moyenne does not occur at all, for the lateral plate is in contact with the somite (see Figs. 3G and 5G). The transition between the wide and narrow portions of the ento- dermal pharynx occurs, somewhat abruptly, on a level with the middle of the otocyst. In the region of the entodermal pouches, the prominent lateral margin of the pharynx tends to insinuate itself between the somital mesoderm and the portion moyenne. Posterior to the entodermal-pouch region the margin of the narrow entodermal pharynx tends to remain altogether ventral to the somital mesoderm (see Figs. 3G and 5G). Swaen and Brachet suggest that the portion moyenne is separated from the somital mesoderm under the combined influence of the prominent margin of the entodermal pharynx on the one hand, and of the ectoderm and the anterior part of the otocyst on the other; the conditions found in shad are quite in accordance with this view. The appearances in Fig. 3F clearly suggests the influence of the wide hyoid pouch and of the otocyst in causing separation of the portion moyenne. In Figs. 3B, 3C and 3E the ectoderm would appear to be of little assistance in causing separation of the portion moyenne, but it is probable that the ectoderm is normally in contact with the mesoderm and that the separation of ectoderm from mesoderm, seen in the sections, is artificial and mainly due to the action of the fixative. The Development of the Heart in Shad. 225 The portion moyenne appears as a cord of mesodermal cells on either side, intervening between the somital and lateral mesoderm, which tends to remain in contact with the lateral plate. It extends from the middle of the otocyst forward to the anterior end of the lateral plate (see Fig. 4A). Small posteriorly, the portion moyenne becomes larger anterior to the mandibular pouch; here and there it comes into immediate contact with the somital mesoderm. Where contact occurs at this stage, the distinction between portion moyenne and somital portion of the head mesoderm cannot be made out because there is, as yet, no differentiation between the cells belonging to these two parts of the mesoderm. In spite of the fact that in occasional sections the portion moyenne is not quite clearly defined, it forms, as a whole, a perfectly definite structure. That the portion moyenne, as found in shad, is directly comparable to that described with such admirable distinctness in trout by Swaen and Brachet cannot, I think, be doubted; for this reason I have ventured to adopt the term employed by them rather than run the risk of confusion by the unnecessary introduction of another name. It has been said that the endocardium is derived from the portion moyenne of the mesoderm; before proceeding to trace the formation of the endocardium it may be stated that special care has been taken to determine whether the entire longitudinal extent of the portion moyenne is involved in the production of endocardium. In order to settle this question (among others) a series of four wax-plate reconstructions has been made from stages during which the cells of the portion moyenne are undergoing migration and differentia- tion to form the endocardium. The result indicates that the endo- cardvum, together with the central aorta, is derived exclusively from that part of the portion moyenne originally situated anterior to the transverse plane passing through a point midway between the man- dibular and hyoid entodermal pouches. That the posterior part of the portion moyenne takes absolutely no share in the formation of the endocardium is an important point which will be referred to later. Fig. 4 is a diagrammatic representation of the ventral surface of a wax-plate reconstruction of the pericardial region of a shad’s head. Stage of 18 somites (the embryo is one hour and a half older 226 H. D. Senior. than that used in the preceding figure). The parts shown’® are (normally) in immediate contact with the yolk, and represent the region extending from the tip of the notochord back nearly to the anterior limit of the first body somite. Formation of the ventral wall of the pharynx, by the folding ventralwards of the (originally) lateral region of the gut-entoderm on either side, is complete in the anterior region and is rapidly extending backwards. The lateral plates are following the medial margins of the ventral pharyngeal wall in their progress toward the mid-line. The outline of the entodermal pharynx presents, on each side, three prominences which require some explanation. Of these the posterior is the hyoid ectodermal pouch which has now reached, and blended with, the (very shallow) ectodermal pouch; this, for reasons stated below,’! will be referred to as the hyo-branchial anlage. The middle prominence is the mandibular entodermal (solid) pouch; this blends with the ectoderm later but (as is well known) gives rise to no cleft; it undergoes disintegration soon after the perforation of 8Axplanation of Figures 4, 5, 6, and 7. Red, lateral plates, where these are covered ventrally by endocardium their outline is indicated by a red line. Blue, endocardium. Continuous black line indicates the position of the outline of the entodermal pharynx and adjacent part of the head-fold (see text) ; interruptions in this line indicate blending of pharyngeal entoderm, or of head-fold, with the basal layer of the surface ectoderm. Broken black line, medial margin of (closing) ventral wall of pharynx. Stipple, ventral wall of the entodermal pharynx and adjacent head-fold ectoderm. Plain white, dorsal wall of (incomplete) pharynx. Arrows on each side indicate the longitudinal limits of the “descent area” of portion moyenne. “Although the (solid) hyoid pouch is alone present at this stage the branchial entodermal pouches (also solid) are about to be laid on, very rapidly, from before backward. The hyoid and branchial entodermal pouches all reach a common ectodermal anlage and are separated from one another by an ex- tremely delicate partition of mesoderm so that, at any given time, a very care- ful examination is necessary to determine the exact number of pouches actu- ally present; in order to ayoid a repeated analysis of the condition, which is unnecessary for this investigation, the entire series of compactly grouped pouches has been looked upon as a single structure. THE DEVELOPMENT OF THE HEART IN SHAD. 18 9D, SENTOR, Notochord (tip) ae aS a) Lateral plate ‘ wth Somital—.¥ mesoderm gee; * Lateral plate Entodermal a pharynx, ventral wall v Entodermal pharynx, 4B dorsal wall Mandibular pouch as We Dyer Endocardium - EY, dermal ph Hyo-branchial anlage“ WESTER 4c Lateral plate 4E ventral wall Fic. 4.—Diagram of the ventral surface of a reconstruction of the peri- cardial region of shad, stage of 18 somites & 100 diams. See explanation of figures, footnote on opposite page. THE AMBRICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2 = be a a qj ral wy The Development of the Heart in Shad. 2% bo -~I the hyoid cleft. The anterior prominence is not strictly pharyngeal ; it represents the posterior limit of the head-fold eetoderm,'’® which, growing in from either side, blends with the anterior extremity of the closing entodermal pharynx. Inasmuch as it has been found difficult or impossible to distinguish the lne of union between ecto- derm and entoderm after blending has occurred, no attempt has been made to indicate this in Figs. 4, 5, 6 and 7; there is no doubt, however, that, although the lateral prominences consist of ectoderm, the axial region (as far as shown in the figures) is truly pharyngeal. At 18 somites the anterior part of the portion moyenne is des- cending on either side, between the lateral plates and the, now closing, pharynx to gain a position ventral to these structures; the longitudinal area over which descent is occurring (“descent area”) is indicated by the space between the arrows in the diagram. Fig. 4A is a sec- tion from this region. Sections from behind the descent area (Figs. 4B and 4C) show that the portion moyenne, which can be seen in them dorsal to the lateral plate, is not descending; descent is prevented, apparently, by the close contact between lateral plate and pharyngeal entoderm. It will be seen that the portion moyenne is *The “optic region” of the head (anterior to the parts reconstructed) is separated from the yolk by a double layer of ectoderm which grows in, in this situation, from the basal ectoderm around the anterior periphery of the head. This ectoderm, which performs several functions, corresponds, in teleosts, to the head-fold of other vertebrates (see Froriep, 05). The formation of head-fold from this double layer of ectoderm occurs literally, in shad, only as far back as the hypophysial region, anterior to which no gut-entoderm occurs. There is a region, extending from some point (approximately) ventral to the hypo- plysis back to a point slightly posterior to the tip of the notochord, throughout which the ingrowing ectoderm encounters the anterior extremity of the phar- yngeal entoderm ; the ectoderm in this region, although its origin is intimately connected with that of the head-fold, has an entirely different subsequent his- tory, briefly indicated as follows: Until the head of the embryo begins to arise from the yolk the double-layered ectoderm of the region in question forms a bond of union between the anterior end of the pharynx and the surface of the embryo. As the head rises from the yolk the layers of the head-fold ectoderm proper become separated to cover the contiguous portions of the head and yolk. Shortly before perforation in the oral plate occurs, the anterior cul-de- sac of the pharynx becomes widely dilated, and the ectoderm connecting the lateral margins of the anterior end of the entodermal pharynx with the basal ectoderm of the surface undergoes, very rapid, disintegration. 228 H. D. Senior. in process of an entire alteration in its distribution; it now consists, on either side, of three parts: One part still remains dorsal to the lateral plate, a second is on the ventral surface of this structure, and a third forms an isthmus between the other two and occupies the descent area. From now on it will be convenient to speak of the part ventral to the lateral plate as endocardium (for such it really is) and to retain the original term for the part which is still dorsal to the lateral plate and for the isthmus. The endocardium now appears as two patches (colored blue in Fig. 4) ventral to the entodermal pharynx and the lateral plates. These patches are not limited to the descent area, but are spreading backwards (more so on the left side of the embryo than on the right, see Fig. 4B and 4C). The backward (caudad) movement of the endocardium, which is now beginning, is soon to become very pronounced. The asymmetry seen in this reconstruction is thought to be due to unequal growth of the embryo, rather than to faulty building up of the plates, the entire right side of the head appears to be in a less advanced stage of development than is the left. In an embryo of 22 somites (Fig. 5, one hour and a half older than the preceding stage) there has been considerable advance in development. Ventral closure of the pharynx has progressed rapidly from before backwards, and is also beginning in another place pos- terior to the gill-region. The medial margins of the lateral plates are approaching one another, and the notochord, slightly longer than before, is now fully formed as far as its anterior end is concerned. The endocardium has travelled back to a point posterior to the mandibular pouch (Fig. 5D), and the portions arising from each side have met across the mid-line. Fig. 5B (as compared with 4B) shows that the descent area has extended considerably backwards, but that descent is not yet occurring opposite the apex of the mandibular pouch is shown on the left side of Fig. 5C (right side of embryo). At a stage of 26 somites (Fig. 6, one hour and a half later than the preceding stage) the ventral closure of the pharynx is approach- ing completion ; rapidly, however, as closure of the pharynx is taking place, it has been overtaken by the backward growth of the endo- THE DEVELOPMENT OF THE HEART IN SHAD. H. D. SENIOR. Notochord (tip) pac Lateral plate ¥ /Noto- Lateral plate Now| : . P2M: Somital 624 ree descending mesoderm i £ me “SSE Endocardium = Entodermal pharynx, Entoderma! pharynx, 5B closed ventra! wall (S Fic. 5.—Diagram of the ventral surface of a reconstruction of the peri- cardial region of shad, stage of 22 somites 100 diams. See explanation of figures, footnote on page 226. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. THE DEVELOPMENT OF THE HEART IN SHAD. H. D. SENIOR. Notochord (tip) Lateral plate Nee | ateral pla Nord Lateral plate Aortic cells °F, GS Carotid cells eee Mandibular ec- “Sse tod I b re ee gs al a = P. M. descending ~ S P. M. descending “SSs5% SS fetid Entodermal pharynx, i eh esoee Fyo-branch- Ae y) 6D Endocardium Endocardium 6F ial anlage Fic. 6.—Diagram of the ventral surface of a reconstruction of the peri- cardial region of shad, stage of 26 somites * 100 diams. See explanation of figures, footnote on page 226. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. THE DEVELOPMENT OF THE HEART IN SHAD. : H. D. SENTOR. Norochora (tip) s > LaF %, Se hee eee Pericardial coelom 7B Carotid cells’ 7C BAG “at Posie = eae - . tat = ¥ P Ventral aortic cells ~~. Aortic root Aortic cells 42© Endocardium 7D 7G ; Fig. 7.—Diagram of the ventral surface of a reconstruction of the peri- cardial region of shad, stage of 32 somites « 100 diams. See explanation of figures, foomote on page 226. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. The Development of the Heart in Shad. 229 eardium. The endocardium, which now covers a considerable area, not only lies over (ventral to) the closing edges of the entodermal pharynx, but partially covers the ventral surface of the future roof as well (see Fig. 6 and 6F). The portion moyenne is no longer descending in the region anterior to the mandibular pouch, for here it has, apparently, been exhausted (see Fig. 6B). The descent area on the left side (right in Fig. 6C) is situated toward the back of the mandibular pouch, and on the right (left in Fig. 6D) is altogether behind this structure; here again there is slight asymmetry. The lateral plates, which are now much increased in width, are approach- ing the mid-line; the notch in the medial border of each foreshadows the point at which the actual borders never meet, but at which they are about to embrace the root of the ventral aorta. The lateral margins of the somital portion of the head mesoderm are, at this stage, spreading ventrally around the lateral margins of the entodermal pharynx in the region between the mandibular and hyo-branchial pouches (see Figs. 6C and 6D). The cells from the somital mesoderm, which now partially embrace the gut ventrally, will form the muscle and supporting framework of the hyoid arch. There is no difficulty now in distinguishing the head mesoderm proper from the undescended portion moyenne, with which it is, in many places, in close contact. The cells of the portion moyenne (like the endocardial cells ventral to the entodermal pharynx) are becoming plainly endothelial; they differ from the other cells of mesodermal origin in that the nuclei appear, in transverse sections of the embryo, to be small and rather flat and to be surrounded by a comparatively large amount of cytoplasm. At a stage of 32 somites (Fig. 7, three hours later than the preceding stage’®) the medial margins of the lateral plates have met and blended throughout the anterior three-fourths of the peri- cardial region except at one place foreshadowed in the preceding stage. The medial margins of the lateral plates, where they fail to *In my stage of 30 somites (exactly intermediate in time between the stage of Fig. 6 and that of Fig. 7) the conditions in the heart-region are practically indistinguishable from those found at 32 somites (Fig. 7); the later stage has been used for reconstruction on account of its superior preserva- tion. 230 H. D. Senior. blend, enclose a circular area which contains the cells about to form the root of the aorta (see Fig. 7D). Where blending has occurred (Figs. 7B and 7C) continuity is established between the somatic layers of the right and left lateral plates; the splanchnic mesoderm of the two sides becomes continuous across the mid-line in a similar manner. In the process of blending the ecelom becomes continuous across the mid-line by the apparent loss of the medial margin of each lateral plate; the medial margins together constitute the dorsal mesocardium which is, thus, early lost. The entire ecelom, paired or unpaired, occurring in Fig. 7 is pericardial. The site of discharge of the jugular veins (which later determines the points of separation, on either side of the embryo, between the pericardial and peritoneal regions of the original celom) will occur slightly behind the site of Kies 7G. The endocardium has undergone very rapid growth backwards (see Fig. 7G), and has now reached the anterior limit of the first body somite. The interval between the first body somite and the head is some little distance behind (candad from) the posterior limit of the reconstruction and the future site of discharge of the jugular veins. The ventral surface of the entodermal pharynx and of the adjacent region of the (peritoneal) splanchnic mesoderm is, therefore, in the head region posterior to the reconstruction, covered ventrally by the endocardium. As far as has been ascertained, the endocardium does not encroach upon the region ventral to the first body somite it- self; a re-investigation of this difficult point will form a part of a study of the origin of the body-vessels to be undertaken at a later date. The descent area of the portion moyenne has moved slightly backwards since the preceding stage; it has narrowed considerably (in the antero-posterior dimension), and now consists of only a narrow cord of cells on each side. The term “descent area” which has hitherto been used to designate cellular connection between the endocardium ventral to the lateral plates and the portion moyenne dorsal to them is no longer applicable, for descent has ceased; the cells between the entodermal pharynx and lateral plate (seen on each side in Fig. 7D) represent the first (transverse) part of the ventral aorta, and the portion moyenne, as such, has ceased to exist. The Development of the Heart in Shad. 3 The entodermal pharynx is now closed throughout, forming a flat tube with a horizontal (virtual) lumen; its ventral surface scarcely appears in Fig. 7 since this is almost entirely hidden by the peri- and endocardium. The heart anlage is now complete, and, although it is quite flat, its component parts can be (by comparison with later stages) already recognized. If an isosceles triangle be described, the base of which corresponds to a straight line connecting the two crosses near the top of Fig. 7, and whose, truncated, apex skirts rather closely round the (red) circle which embraces the aortic root, the area of splanchnic aes Ectoderm, superfice x Ectoderm, basal %* nee < se = Rey re \ = ieee Pericardial coelom ~SREF- Infundibulum ore \ \\ Endocardium Hypophysis N a ~ Entodermal pharynx, ventral wall Ss = as Pr Se Entodermal pharynx, dorsal wall Head-fold ectoderm a Ectoderm covering yolk Itc. 8.—Mid-sagittal section through the head of shad, 30-31 somites x 100 diams. Owing to slight obliquity the section passes through the brain mainly to the right of the ventricular cavity. Fixation by Virchow’s method. mesoderm contained within the triangle will represent conus, ventricle and atrium (in the order named, from behind forwards). The (pericardial) splanchnic mesoderm not included in the triangle will form the anterior wall of the sinus venosus and of the pericardio- peritoneal septum. All the somatic mesoderm anterior to the (future) site of discharge of the jugular veins will become parietal pericardium. The heart anlage in mid-sagital section is shown in Fig. 8. The embryo of Fig. 8 is slightly younger (30 to 31 somites) than that of Fig. 7 (32 somites). Two points in regard to the descent of the endocardial cells have been brought out by the use of the plastic method of reconstruction 232 H. D. Senior. which, it appears from the literature, have previously escaped notice. Firstly: The endocardial cells do not descend in a hap-hazard fashion; descent proceeds, in a perfectly orderly manner, continu- ously from before backwards; the last cells to descend (7. e., those in the region of the future aorta) are arrested, as it were, in the act of descent to form the first part of the central aorta. Secondly: In the region posterior to the aortic root descent of endocardial cells does not occur at all; the endocardium in this region is exclusively fur- nished by cells descending in, and derived from, the region anterior to the root of the aorta. The part of the portion moyenne, which at 15 somites was easily recognizable in the region behind the (future) aortic root, extending back as far as the middle of the otocyst( see Figs. 3K and 3F), has disappeared long before the completion of the heart anlage, apparently by blending with the adjacent somital mesoderm. It is still recog- nizable at 18 somites (see right side of Fig. 4E), but posterior to . this it has already disappeared or is very indefinite, see left side of Fig. 4E (through their hyo-branchial anlage). At 22 somites the portion moyenne, in this region, has entirely disappeared. Review of the evidence bearing on the relation of the endocardium to the vascular endothelium of the head in general. The development of the pericardial ccelom, including in this term the future myo-epicardium, may be looked upon as a subject prac- tically complete in itself which can be considered independently of that of other structures. The case of the endocardium is entirely different; the fact alone that part (at least) of the ventral aorta arises, in common with the endocardium, from the portion moyennne is quite sufficient to indicate that the endocardium cannot be con- sidered as an independent structure. It is only reasonable to sup- pose that the origin of the aorta is essentially similar throughout the head, so that separation of the aorta from the endocardium, in this connection, would be artificial and, therefore, not conductive to a clear conception of the origin and relations of the latter. During the study of the process of formation of the heart anlage several facts bearing on the origin of the vascular endothelium became The Development of the Heart in Shad. 233 evident; mention of these, since they had no direct bearing on the subject in hand, has, hitherto, been omitted. In the following notes the facts alluded to are briefly reviewed, and an attempt made to estimate their significance. The portion of the head shown in Fig, 7 may be divided trans- versely into three regions: anterior, middle and posterior; of these the middle region extends from the back of the red circle, indicating the aortic root, back to the place at which the entodermal pharynx becomes narrow behind the hyo-branchial anlage; the anterior region corresponds to the pericardial area in front of the middle region, and the posterior to the corresponding area behind it. In the embryo of 15 somites, from which sections are shown in Fig. 3, the cells immediately adjacent to the medial borders of the lateral plates are isolated from the remainder of the mesoderm to form the portion moyenne of Swaen and Brachet; this isolation of the portion moyenne occurs only in the anterior and middle regions (comparing the stages of 15 and 32 somites approximately) and not in the posterior. The endocardium, as seen in Fig. 7, is derived exclusively from the portion moyenne of the anterior region. Having recalled these points regarding the portion moyenne and endocardium, the occurrences bearing on the vascular endothelium belonging to the three regions mentioned will, as far as possible, be considered separately. The anterior region may be examined first. In Fig. 7D the lateral, now pericardial, plate is not in contact with the ventral surface of the entodermal pharynx, as is the case elsewhere (save in the two neighboring sections), contact being prevented by a cord of cells on either side. The cells of these cords were derived from the por- tion moyenne, and, like other cells known to come from the same source, they are at this stage easily distinguishable from the ordinary mesoderm. Fig. 9 is from a reconstruction of the aorta at the earliest stage in which it appears as a well defined vessel throughout the head (42 somites; the ventral surface of this reconstruction is shown as Fig. 12). The cells between the entodermal pharynx and pericardial plate in Fig. 7D correspond exactly in position with the first (or transverse) part of the ventral aorta. 234 HH. D. Senior. In Fig. 7C the entodermal pharynx and pericardium are in contact, and here cells descending from the portion moyenne have been ar- rested lateral to the line of contact between the two; reference to Fig. 9 will show that these cells correspond in position to the second (or oblique) part of the ventral aorta. This is not all; in Fig. 7C (also 7B and 7D) just dorsal to the entodermal pharynx there are one or two cells on either side which differ markedly from the sur- rounding mesodermal cells; the cells in question (which can be dis- ‘iz Surface Head-fold ectoderm ectoderm fae [ Carotid artery Mandibular arch Mandibular ec- Oblique; — todermal pouch Ventral on Transverse— Ectoderm (cut) tt Hyo-branchial ectodermal pouch Margin of lateral plate (dotted) “ Pericardial coelom Pharynx, lumen Fic. 9.—Diagram showing the course taken by the right aorta throughout the pericardial region of shad, stage 42 somites s 100 diams. The dorsal surface of the entodermal pharynx is shown on the right side, and the dorsal surface of part of the lateral plate on the left. tinguished in practically every section used in the reconstruction) are found (see Fig. 9) to invariably occupy the line of the dorsal aorta and are, without doubt, aortic endothelium. It is hoped that the position of the aortic cells has been sufficiently indicated in the figures referred to; there are rarely more than two dorsal aortic cells on each side of a section, the differentiation of these will be illustrated in a future study of the aorta itself. The Development of the Heart in Shad. DoD The aortic cells do not appear suddenly. At 26 somites (Figs. 6B, 6D, ete.) a break is apparent, in most sections, in the line of the future dorsal aorta, (the breaks together forming a tunnel) the cells about the break have small nuclei and a large amount of cyto- plasm; at 32 somites the aortic cells are not only clearly endothelial but tend to lne the tunnel. In the light of later stages the break referred to can be distinguished in many sections at 22 somites (Fig. 5), but it is questionable whether the cells about it have under- gone much differentiation. The differentiation of the cells of the dorsal aorta thus goes hand-in-hand with that of the cells known to be derived from the portion moyenne, and the dorsal aorta is recog: nizable before the ventral aortic cells have taken up their definitive position. Finally, the history of the carotid artery is similar to that of the dorsal aorta, and, at 32 somites, the cells of the ventral and dorsal aorta are continuous around the front of the mandibular pouch through the mandibular aortic arch. This entire chain of circumstances suggests very strongly that the dorsal aorta and carotid of the anterior region, like the ventral aorta and endocardium, arise from the portion moyenne. In the middle region (of the three before-mentioned) there is also isolation of the cells adjacent to the medial borders of the lateral plates to form the portion moyenne. Soon after the portion moyenne (which is here less bulky than in the anterior region) ceases to be recognizable the differentiation of the cells about to form the dorsal aorta can be followed in exactly the same manner as in the anterior region. Here are two sets of facts, the disappearance of the portion moyenne, and the gradual differentiation of the dorsal aorta nearer to the mid-line; they are, of course, not necessarily dependent on one another, but considered in connection with the circumstances in the anterior region they may be looked upon as suggestive. By reference to Fig. 9 it will be seen that the dorsal aorta in the anterior and middle regions is always situate medzal to the line of the lateral margin of the entodermal pharynx (usually very much so). Figs. 3B, 38C, 3E and 3F show that the portion moyenne is always in a position lateral to the above mentioned line. From these two facts it follows that although the conditions in the anterior 236 H. D. Senior. and middle regions suggest the origin of the dorsal aorta from the portion moyenne, any attempt to prove this supposition is met with the difficulty that the aortic cells are known to differentiate in situ. To trace a few undifferentiated aortic cells in their migration through mesodermal cells which they closely resemble would scarcely be pos- sible by ordinary embryological methods. In the posterior region the cells adjacent to the medial borders of the lateral plates are not isolated, as in the anterior and middle regions, to form portion moyenne. Fig. 9 shows that the aorta in the posterior region is situate immediately dorsal to the lateral margin of the entodermal pharynx, and, in the posterior part of this region, just dorsal to the medial margin of the lateral plate as well (sections from this part of the posterior region are shown in Figs. 3G and 5G; 7G is slightly anterior to it). The aorta is thus placed exactly in the line of the mesodermal cells adjacent to the medial borders of the lateral plates apparently arises directly from them. In the foregoing notes an attempt has been made to bring together some evidence bearing on the development of the vascular endothelium of the head in order to arrive at a conception of the nature and rela- tions of the endocardium. The evidence in question appears to justify the statement that the endocardium in shad arises from meso- dermal cells which are found, after differentiation of the lateral plates, to be placed in the region bordering on the medial margins of the latter; further, that the mesodermal cells in this situation appear to be given up exclusively to the formation of vascular endo- thelial cells of which the endocardium only forms a part. A few words may be added by way of re-examination of the “Portion moyenne du mésoblaste” of Swaen and Brachet. In the anterior pericardial region the cells which later form the endocardium must necessarily be separated from the somital portion of the meso- derm, for the former eventually take their place as endocardium ventral to the lateral plates, while the latter retains its position dorsal to them. In the middle and posterior pericardial regions, separation of the cells bordering on the lateral plates is not a neces- sity, for neither region produces endocardium. Nevertheless, separa- tion occurs in the middle region but not in the posterior; the dif- The Development of the Heart in Shad. 237 ference in behaviour of the cells in question does not necessarily depend on the nature of the cells themselves, but appears to be due to conditions occurring in the middle region which are not found in the posterior. It may be said that the portion moyenne appears to consist in the main of cells which will later form vascular endo- thelium, but it is difficult or impossible to show that in the anterior region it includes all of these, or that in the middle region it does not include more. In other words, although the portion moyenne forms a well defined group of cells which is of great assistance in following the movements of the endocardium, it does not appear, in itself, to be a structure of real morphological importance. Greil, in his recent paper on the origin of the blood and blood-ves- sels, ’08, traces the origin of the endocardium in Ceratodus, and in some amphibia and selachii, from two sources which he distinguishes as the Angioscleroblast and Angiohzemoblast. Greil does not refer to teleosts in this connection, but his statement gives additional in- terest to the description by Boeke, ’03, of a two-fold origin of the endocardium in Murena. JBoeke describes the major part of the endocardium as arising from cells developed in the head-region, but traces the origin of some of the cells lining the venous end of the heart from the region of the closing blastopore (the latter cells would seem to correspond to those described by Greil as emanating from the hemangioblast). I have looked carefully for cells corresponding to those described by Boeke as arising from the region of the blasto- pore without result, and believe that any cells which may migrate forward from this region in shad must be arrested posterior to the junction of the first body somite with the head. It is intended to re-investigate the origin of the aorta in the head, and to look for additional evidence regarding the origin of the jugular veins, and afterward to study the origin of the vascular endothelium of the body vessels. PERIOD 2. LASTING UNTIL RHYTHMICAL CONTRACTION BEGINS IN THE PARTIALLY FORMED HEART-TUBE. Between the stages of 30 and 32 somites, the heart anlage has undergone little change; at 33 somites the beginning of progress 238 H. D. Senior. becomes apparent. In mapping out the heart anlage (see page 231) it has been shown that the splanchnic mesoderm contained within a triangle drawn with its apex at the aortic region and its base near the anterior end of the pericardial plate would correspond to the future conus ventricle and atrium. By referring to Figs. 7B, 7C and 7D it will be seen that the splanchnic mesothelial cells included in this triangle are columnar in shape, while those lateral to the triangle are much flatter. Passing forward from the region 7B, the cells within the triangular area diminish in height until they become cubical, and finally, near the anterior limit of the pericardial plate, quite flat. Ectodermal and en=% todermal mandibular * . **Ventral aortic cells Columnar cells of pouches (blended) Cavity of (closing) conus arteriosus Endocardium Sex cell (future) heart-tube: 10D Fic. 10.—Two transverse sections of shad, stage of 34-35 somites ~ 100 diams. 10B passes through the mid-sagittal plane of the embyro anterior to the mandibular pouch, 10D midway between the mandibular and hyoid pouches. The left side of the figures represents a region anterior to that seen on the right. Fixation by Pereny’s method. The splanchnic mesoderm included within the triangular area is soon to form a tube, which may provisionally be called the heart-tube ; the columnar mesothelial cells will form the myo-epicardium. Before formation of the heart-tube begins, the triangular area spoken of undergoes a migration toward the left, carrying with it the underlying endocardium; this is the movement which begins at 33 somites. The sinistral movement of the “columnar area” of the splanchnic mesoderm and of the endocardium, which begins at 33 somites, is very pronounced an hour later at the stage of 34-35 somites from which Fig. 10B is taken. Reference to Fig. 10D shows that in the aortic region there has been no sinistral movement; this The Development of the Heart in Shad. 239 movement takes place around the aorta as a center, and becomes progressively greater as the base of the triangle referred to is reached. In the region of Fig. 10B (anterior to the mandibular pouch) the columnar area of the splanchnic mesoderm, being about two-thirds to the left of the mid-line, has accomplished its migration. The movement of the columnar area of the splanchnic mesoderm is not quite simple; it is accompanied, and partly brought about, by a slighter movement to the left of the entire pericardial plate (see Fig. 10B). The slighter sinistral movement of the entire pericardial plate began as early as the stage of 32 somites (see Fig. 7B). In Fig. 10D the initiation of heart-tube formation can be recog- nized. Union is about to take place between the splanchnic meso- derm in the region of the original medial margins of the right and left lateral plates. This union takes place around the lateral and posterior circumference of the aortic root. The splanchnic mesoderm to the right and left of the aortic root is seen in Fig. 10 D to be some- what prominent ventrally, so that, by uniting in the mid-line (which it is just about to do), it will enclose a small chamber, the conus arteriosus ; the latter contains a small amount of endocardium directly continuous with the endothelium of the aorta. At a stage of 36 somites (see Fig. 11, one hour older than the pre- ceding stage) the formation of the heart-tube has made considerable progress. The arterial extremity of the heart-tube has been formed by the blending of the splanchnic mesoderm about the circumference of the aortic root symmetrically in the mid-line. The remainder of the heart-tube is also undergoing formation by the blending of splanchnie mesoderm on either side. The axis about which the further blending of splanchnic mesoderm occurs corresponds to a straight line connecting the middle of the base of the triangle spoken of with its apex. The apex of the triangle corresponds in position with the aortic root; the middle of the base is now placed (owing to rotation of the triangle to the left around its apex) quite near to the left side of the anterior margin of the pericardial plate. The axis, then, along which union of the splanchnic mesoderm is pro- ceeding o>) as it passes forward it diverges to the left so as to form an acute extends from the (medially placed) aortic region forward ; angle with the mid-sagittal plane of the embryo. 240 H. D. Senior. In the formation of the heart-tube anterior to the aortic region, the splanchnic mesoderm of the right side undergoes active movement, while that of the left remains comparatively passive. The splanchnic mesoderm to the right of the heart-tube axis arises abruptly from the somatic layer to form a crest which moves over to the left; this crest becomes imminent and falls to the left somewhat in the manner of a wave breaking upon the shore (see Fig. 11C). The splanchnic mesoderm to the left of the axis rises slightly to meet the splanchnic mesoderm from the other side as the latter falls; between the two a tube of splanchnic mesoderm is formed of which the ventral wall is derived mainly from the right side, and the dorsal wall mainly from the left. Fig. 11 shows the heart-tube in process of formation, as indicated by a reconstruction of the stage of 36 somites; contact of the two sides has occurred at the posterior (arterial) end. The splanchnic mesoderm on each side (right particularly), for some little distance anterior to the contact area, shows evidence of preparation for bend- ing in the manner described above (Fig. 11C). Heart-tube forma- tion is now in rapid progress; the posterior (arterial) end now being complete, the venous end will be progressively formed, from behind forward, along an axis deviating to the left. The irregular black line in Fig. 11 indicates the outline of the endocardium; a small quantity of the latter has been included in the heart-tube. The heart shown in Fig. 11 (such as it is) is contracting rhyth- mically, and has been doing so for some 10 or 15 minutes. The heart which was quiescent at the stage of 35 somites began beating (after very little preliminary oscillation) at a rate of 52 beats per minute, about 15 minutes before the thirty-sixth somite was com- pletely marked off.” It may be questioned whether the rhythmical contraction of the “Water temperature 62° F. (July 11, 1907). Some evidence has been obtained which suggests that in higher water temperatures the heart begins to beat at an earlier stage of development (as estimated by the number of somites). In order to exclude the possibility of a miscount of somites the data on which this evidence rests require to be controlled by comparison with the results of further observations, preferably made on eggs of another species. The Development of the Heart in Shad. 241 heart anlage is of assistance in the folding over of the splanchnic mesoderm to form a tube; that it is not essential to this process is Notochord (tip) Head-fold ectoderm Mandibular 11C ~~ re pouch Hyo- branchial anlage = ventral wall] Noto- chord Hi : | Pharynx, Lateral plate Fs Cavity of (closing) heart-tube 1C. Fic. 11.—Ventral surface of a reconstruction of the pericardial region of shad, stage of 36 somites » 100 diams. The heart-tube is in process of formation, contact of the two sides has occurred along the broken line. The continuous black line encloses the area covered by endocardium. Fixation by Pereny’s method. indicated by the fact that a stage of 35 somites (half an hour earlier) the folding over has already commenced, although it is not so advanced as at 36 somites. 949 H. D. Senior. PERIOD 3. IN WHICH THE HEART-TUBE IS: COMPLETED TO FORM CONUS, VENTRICLE AND ATRIUM, AND ASSUMES THE ADULT POSITION. Stage of 42 sonutes. Fig. 12 is from a reconstruction of the ventral surface of the pericardial region of an embryo of 42 somites. The ventral wall of the pericardial ccelom is very thin and to some extent moulded upon the heart-tube, so that the outlines of the latter are clearly indicated. A portion of the splanchnic mesoderm forming the ven- tral wall of the pericardial celom has been removed over an area mainly to the right of the mid-line. In the area referred to a portion of the heart wall and of the somatic mesoderm are seen near the mid-line. ‘Toward the lateral region of this area the somatic meso- derm and some of the mesoderm of the pharyngeal floor are repre- sented as having been removed in order to uncover the ventral aspect of the entodermal pharynx and of the ventral aorta. The heart-tube, which has only just been completed, is cone- shaped; the (venous) base of the cone is directed anteriorly and to the left. There is, as yet no external indication of separation into conus, ventricle and atrium, but the wall becomes progressively thinner in passing from the arterial to the venous end. The splanchnic mesoderm, not included in the heart-tube, forms the ventral wall of the pericardial celom; it becomes continuous with the wall of the hart-tube at the venous end of the latter. In closing, the two sides of the heart-tube do not appear to blend where the splanchnic mesoderm first meets; some further adjustment occurs in order to bring the columnar area of the latter (and the endocardium in contact with this) within the limits of the tube. Completion of the heart-tube is effected by the blending of splanchnic mesoderm of the right and left sides, and continuity of the ventral wall of the pericardial ceelom is maintained by a similar process; the two processes together entail loss of the ventral mesocardium. Comparison of Figs. 11 and 12 shows there is a large amount of endocardium not included in the heart-tube; also that, after closure of the latter, the endocardium tends to move over to the left side. The Development of the Heart in Shad. 2433 Before passing from the stage of 42 somites to that of 6.2 mm. (the next one to be examined) it will be necessary to make a digres- Notochord (tip) Heart-tube Splanchnic mesoderm (cut) Head-fold Venous end Somatic mesoderm of heart tube Entodermal pharynx Mandibular pouch L E\—12C Ventral aorta Site of Hyo- branchial anlage ers Heart-tube | Pericardial coclom | Endocardium Fic. 12.—Ventral surface of ‘a reconstruction of the pericardial region of shad, stage of 42 somites x 100 diams. Over an area mainly to the right of the mid-line the ventral wall of the pericardial coelom has been removed to show heart-tube and somatopleure ; also, laterally (where somatopleure has been removed), the pharynx and ventral aorta. The continuous black line encloses the area covered by endocardium. sion; firstly, to correlate the stages as estimated by the number of somites with those designated by the length in millimeters; secondly, to mention some points in connection with the general circulation. 944 H. D. Senior. Correlation of stages. The embryo becomes sufficiently straight to yield a satisfactory end-to-end measurement when it has about 51 somites; its length is then 3.6 mm. From this time on the stages are designated by the length in millimeters; it may be mentioned that the last somite (59th or 60th) is formed at the stage of 5.2 mm. Mechanism of the circulation at different stages of development. Prior to the stage of 42 somites there has been no true circulation, because the aorta opposite the first three somites, late in being formed, is still wanting. At 42 somites the aorta is practically com- plete as far back as the anus; posteriorly it bifurcates, and the two vessels form a loop encircling the gut and join the subintestinal vein. The blood plasma (for there are no corpuscles) flows from the heart into the aorta, turns forward near the anus, into the sub- intestinal vein, which, in turn, discharges it on to the posterior pole of the yolk. The plasma flows ventral and lateral to the yolk in a wide channel between the ectoderm and the yolk-periblast and, following the contour of the yolk, enters the venous end of the heart-tube. The plasma is not in contact with the periblast ventral to the peritoneum, for it is excluded from this situation by the at- tachment of the lateral margins of the latter to the yolk; as far as can be determined the periblast elsewhere is bathed in plasma. The caudal aorta and vein are as yet unformed, but a cord of cells ventral to the notochord in the tail represents these vessels together with the blood-anlage. The jugular and cardinal veins are not yet developed ; there is no liver. The jugular veins have reached completion at a stage of 4.4 mm., and discharge their blood upon the yolk immediately posterior to the vagus ganglia. It may be mentioned that the endothelial cells of the jugular veins have been recognizable for some time prior to the complete formation of the veins themselves, certainly as early as the stage of 42 somites. The place of termination of the jugular veins is a point of great interest, for here the celom is separated into its pericardial and its peritoneal portions. The orifices of discharge of the jugular veins are placed laterally to the lateral The Development of the Heart in Shad. 245 plates in a situation approximately corresponding to the lower end of Fig. 12. The celom anterior to the orifices become pericardial ; posterior to them it becomes peritoneal; the ccelom between the two orifices ceases to exist.'* Since separation of the pericardial from the peritoneal portion of the celom occurs at a situation in which the lateral plates are still separate, the posterior end of the peri- cardium extends farther backward on each side than in the mid-line. At the stage of 6.2 mm. the caudal aorta and caudal vein replace the anterior end of the cord of cells found in the tail at 42 somites, and the blood contains a very few corpuscles. Blood passes from the dorsal aorta through the short caudal aorta and then forward through the caudal vein. The caudal vein meets the cardinals” near the anus, and from the point of junction two veins pass ventrad (embracing the gut) to join the subintestinal vein; through these two veins most (or all) of the blood from the caudal vein enters the subintestinal. The subintestinal vein is now involved in the rapidly growing liver; its extreme anterior end (vena revehens of liver) is free, and discharges its blood ventral to the peritoneum. JBlood is now re- tained in the-space ventral to the peritoneum (supravitelline sinus) by the very agency which formerly prevented its flowing there, 7. e., by the lateral attachment of the peritoneum to yolk. At the site of discharge of the jugular veins the blood from these meets that flowing from the supravitelline sinus; and the blood from these two sources enters the venous end of the heart. There appears to be no special mechanism for retaining the blood in the space between the ventral wall of the pericardial ecelom and the yolk, for the pericardial plate is attached peripherally to the ectoderm and not to yolk. The condition of the circulation in the stage described in the in- troduction differs from that in the stage just described in that the cardinal veins are fully formed. The blood from the, now practi- *Conditions at the site of discharge of the jugular veins are much compli- cated by the fact that the mesenchyme of the pectoral fins is arising from the somatic mesoderm in this region. A more thorough study will be undertaken later in connection with the veins themselves. *The cardinal veins, at this stage extend only from the anus as far as the anterior end of the liver. I am unable to determine their functions in connec- tion with the general circulation. 246 H. D. Senior. cally complete, caudal vein is received entirely by the cardinals. The subintestinal vein has lost all connection with the caudal and the cardinals, and is now the portal vein. Stage of 6.2 mm. Fig. 13 represents the ventral surface of a reconstruction of the pericardial region of an embryo of 6.2 mm.; the reconstruction extends further forward than those shown in the previous figures; it, in fact, includes the whole of the anterior part of the head which remains at this time in contact with the yolk. The ventral wall of the pericardial ccelom has been partially removed to show the con- dition of the heart-tube. The conus, ventricle and atrium are now quite distinct, but there is no prominence on the ventricle correspond- ing to its future apex. There is evidence, at this stage, that the ventral wall of the pericardial celom is attached, rather extensively, to the yolk just to the right of the venous orifice of the atrium. Over the area of attachment no endocardium is present; the exact distribution of endocardium over the remainder of the ventral wall of the pericardial ccelom is difficult to make out owing to the ex- treme tenuity of the latter. The venous end of the atrium has moved forward and is now placed ventral to the posterior half of the left eye (it was altogether pos- terior to the eye in the preceding stage); the anterior end of the pericardial plate has moved forward even more than the atrium and now appears to have reached the anterior limit of the head-fold. There are several changes going on, however, which tend to com- plicate matters by shifting former landmarks; these changes can be partially appreciated by reference to Figs. 13 and 14. In the first place the head is rising from the yolk: this is accomplished by forward growth of the head, by shrinking of the yolk, and by a horizontal separation of the head-fold into its original two layers. The dorsal and ventral layers of the head-fold are now being added to the surface ectoderm (basal layer) of the continuous regions of head and yolk respectively. In the second place the, now separating, head-fold is moving bodily backwards so that it approaches the man- dibular pouch; the latter shows evidence of antero-posterior com- The Development of the Heart in Shad. 947 pression and is, in its turn, crowded back so as to approach the hyo- mandibular anlage. The backward movement of the above mentioned structures produces an effect of advance in the tip of the notochord, this is relative only and not actual. Having found that the heart-tube represents conus, ventricle and atrium, it remains to be seen how the different parts of the wall of the pericardial ccelom attain their definitive positions; this can be most conveniently studied from the left side. Ectoderm cover- ing yolk Ventral ecto- derm of head ~Head-fold ectoderm Position of tip of notochord Vv d i enous en of atrium Ventricle Atrium Mandibular pouch ( Conus | Hyoid cleft Hyo- + Pericardial branchial coelom pouch anlage ea ee Pharynx Tlic. 13.—Ventral surface of a reconstruction of the pericardial region of shad, stage of 6.2 mm. x 100 diams. Part of the ventral wall of the pericardial ccelom has been removed to uncover the conus, ventricle, and atrium. To make a satisfactory drawing of the heart at this, and the following, stage, from the left side would be very difficult; a com- plete view of the organ is obstructed partly by the dorsal and partly by the ventral wall of the pericardial celom. The parts considered essential to the elucidation of the changes about to occur are, there- fore, shown diagrammatically and the less important parts elimi- nated altogether. 248 H. D. Senior. In Figs. 14 and 15 the heart has been almost entirely omitted, the position of the venous orifice of the atrium (as it lies near the surface of the yolk) is indicated by a broken line; the other struc- tures shown are indicated as they would appear in a mid-sagittal section of the embryo. Fig. 14 is a diagram made from the reconstruction used for Fig. 13, the left side is represented. The relations of the head-fold ecto- derm to the surface have been approximately determined by com- parison with earlier stages, and are indicated in the diagram. The line of separation of head from yolk is indicated externally, in the Ventral ecto- derm of head Hypophysis Notochord ee my Ectoderm co- Pericardial coclom XN vering yolk Site of Ent- rance to atrium ic. 14.—Diagram made from the reconstruction shown in Fig. 15 as seen from the left side, x 100 diams. The yenous end of thé atrium is indicated by a broken line; other structures are represented as cut in the mid-sagittal plane. Ectoderm belonging to the original head fold is shaded. The yolk is stippled. embryo, by a U-shaped groove; the anterior part of this groove (cor- responding to the cross-piece of the U) appears in section in Fig. 14. The anterior margin of the pericardial plate is attached to the head-fold ectoderm and is, at present, dorsal to the groove referred to (see Fig. 14); the lateral margins of the pericardial plate are attached to the surface ectoderm just ventral to the groove. The pericardial ccelom is, as in previous stages, dorso-ventrally compressed ; three points in its wall will require to be noticed in the following stages. The points referred to are: the anterior end, the posterior end and the aortic root (apex of conus arteriosus) ; these are indicated, respectively, by the letters X, Y and Z, The Development of the Heart in Shad. 249 Stage of 7.3 mm. Fig. 15 is a diagram, made in the same way as Fig. 14, from a reconstruction of the pericardial region of an embryo of 7.3 mm. The head fold proper no longer exists, having been absorbed into the basal ectoderm of the surface. The pharynx is dilated, and the oral plate is soon to be perforated. Antero-posterior compres- sion of the head is still more marked than in the preceding stage, and is accompanied by the formation of a head-bend of the mid-brain Notochord Ventral ecto- _ ae derm of head Oy. RE Se 7 it S=7N Site of entrance Ectoderm cover- 4-::.": >. * to atrium ing yolk ---_- Fie. 15.—Diagram made from a reconstruction of the pericardial region of shad, stage of 7.5 mm. & 100 diams. The left side is shown, structures indicated as in the preceding figure. and of a curve, in the ventral direction, of the anterior end of the notochord. There is an ectodermal band connecting the lateral margin of the anterior end of the entodermal pharynx, on either side, with the basal ectoderm of the surface. The ectodermal bands re- ferred to represent the posterior margin of the original head-fold ectoderm, and have a developmental history similar to that of the head-fold proper (see footnote on page 227.) These ectodermal bands and the mandibular anlage (combined entodermal and ecto- dermal mandibular pouches) are now undergoing disintegration. The heart differs from that of the preceding stage in that the apex of the ventricle is well-marked. Fig. 15 shows (compare with Fig. 14) that the ventral layer of the head-fold, in completely separating from the dorsal layer, has 250 H. D. Senior. carried with it the (attached) anterior periphery of the pericardial celom. The point X has, therefore, moved in a ventral direction, and is now placed between ectoderm and yolk some distance below the head of the embryo. (The point Y, which has not altered its position, is not shown.) The groove between the head and yolk is now deep and, anteriorly, very narrow. The ectoderm bounding the groove impinges upon the dorsal wall of the pericardial ecelom and constricts it. Accompanying this constriction there is a diminu- tion in the area of the ventral wall of the pericardial ccelom, and the venous orifice of the atrium is brought nearer to the mid-line. The, somewhat dome-shaped, part of the pericardial ccelom dorsal to the constriction (and, therefore, on the side of the groove towards the head) contains the conus and ventricle. The lateral periphery of the pericardial ccelom, together with the venous orifice of the atrium, is ventral to the constriction (and, therefore, on the side of the groove towards the yolk). Stage of 8.75 mm., and a comparison of the heart with that of the stage described in the introduction. Fig. 16 represents a reconstruction from an embryo of 8.75 mm. The reconstruction, which is shown from the left side, was made from sagittal sections. The last section (on the left side) passes through the yolk on a level with the left margin of the pericardial ccelom, and fails to complete the left wall of the pharynx. In the preceding stage the groove between the head and yolk was narrow and nearly horizontal; the periphery only of the peri- cardial celom was ventral to it. The groove in question at this stage has become, by reason of the recession of yolk from head, oblique and much wider. Practically the entire pericardial eelom is now on the yolk side of the main axis of the groove, and radical changes have taken place in the arrangement of its walls. The point X has moved so far back that the somatic mesoderm between X and Z has been stripped from the ventral wall of the pharynx and will form the ventral parietal pericardium (compare Figs. 16 and 17). The somatic mesoderm between the points Z and Y has remained stationary and will form the dorsal parietal pericardium. The The Development of the Heart in Shad. 251 splanchnic mesoderm between the points X and Y, which has hitherto formed the ventral wall of the pericardial ccelom, has undergone considerable contraction and is stouter than before; it will form the anterior wall of the sinus venosus (compare Figs. 16 and 17). Between the stage of 8.75 mm. and that of 114 hours, noticed in the introduction (see Fig. 17), the heart is brought into the adult position; the arterial end is fixed (by the ventral aorta) to the floor of the pharynx, while the venous end follows the retreating yolk. Common trunk of Left branchial arteries 3 and 4 Branchial cleft 1 Hyoid cleft Br. cleft 2 Somatic mesoderm (pericardial) / Body-wall it Mouth .J Supravitelline ~ blood-sinus Atrium Site of entrance to atrium L. Mandibular art. L. Hyoid art. Ventral aorta Conus arteriosus Pericardial coelom, Ventricle Splanchnic mesoderm (pericardial) Yolk [section] Somatic mesoderm (pericardial) Ectoderm [cut] Yolk [surface] Fig. 16.—The left side of a reconstruction from the gill region of shad, stage of 8.75 mm., « 100 diams. The pericardial colom and adjacent parts have been laid open to the left of the mid-sagittal plane. The main axis of the ventricle (approximately transverse at 8.75 mm.) becomes longitudinal; the venous orifice of the atrium moves from left to right until it reaches the mid-sagittal plane of the embryo. On comparing the parts of the pericardium and heart, as described in the introduction (Fig. 17), with those of the original heart anlage (Fig.-7), it will be seen that the entire parietal pericardium has been derived from somatic, and the myo-epicardium from splanchnic 252 H. D. Senior. mesoderm. Part of the original splanchnic mesoderm was employed in the formation of the tube which has become the conus, ventricle and atrium; the remainder (hitherto referred to as the ventral wall of the pericardial eelom) is now about to take part in the formation of the sinus venosus. PERIOD 4. FORMATION OF SINUS VENOSUS AND HEPATIC VEIN. Fig. 17 is from an embryo 10.67 mm. in length, designated (for reasons already stated) the stage of 114 hours. The struc- tures shown have been briefly mentioned in the introduction; it is now necessary to describe them more fully. The portion of the reconstruction posterior to the venous orifice of the atrium may be divided into two regions by a vertical trans- verse plane passing through the orifices of discharge of the cardinal veins. The region anterior to the plane mentioned is the site of the future sinus venosus and may be called the sinus-venosus region; the region posterior to it corresponds to the anterior end of the supravitelline sinus and may be called the hepatic-vein region. In the sinus-venosus region the peritoneal ecelom has extended forwards dorsal to the pericardial ceelom. The peritoneal ccelom is here small (and remains so) being practically confined to the region dorsal to the gut. The splanchnic mesoderm extending between the points X and Y is to form the anterior wall of the sinus venosus. The part of this immediately to the right of the venous orifice of the atrium is known to have been attached to the yolk since the stage of 6.2 mm. The anterior wall of the sinus venosus is now, at the site of attachment, drawn out into a long process referred to below as the “‘yolk-process” of the sinus venosus. The posterior surface of the anterior wall of the sinus venosus is in contact with (and possibly attached to) jhe yolk around its ventral and lateral periphery. Endocardium lines the posterior surface of the anterior wall of the sinus venosus where the latter is not in contact with yolk, and, at this stage, is beginning to migrate from the yolk process on to the yolk itself (see Fig. 18). Elsewhere the yolk is entirely destitute of endocardium (see Figs. 19, 20, 21 and 22). The yolk process, extending obliquely up- The Development of the Heart in Shad. 253 ward from the anterior wall of the sinus venosus to a projection on the yolk (dorsal to the anterior pole of the latter) forms an position of Fig. 21 position of Fig. 20 position of Fig. 19 | position of Fig. 18 jf =], Spinal cord Ectoderm Notochord Dorsal aorta Peritoneal coelom L. cardinal v. (Esophagus R. cardinal v. Supravitelline sinus L. jugular v. “Yolk-process”’ of sinus venosus Br. cleft 2 Branchial cleft 3 Branchial a. 4_ & Branchial a. 3 Ventral aorta Yolk [in mid- sagittal sec. | Dorsal parietal pericardium Conus arteriosus Atrium Ventricle Anterior wall of sinus venosus Body-wall [cut] Ventral parietal pericardium (mostly removed) Vic. 17.—The left° side of a reconstruction from the posterior gill region of a shad, stage of 114 hours. X 100 diams. Explanation as in Fig. 1. Norr.—The warping of the posterior plates of this reconstruction (concavity forwards) was not noticed before drawing ; otherwise it would have been rectified. The warping has to be taken into consideration in estimating the correct positions of Figs. 18, 19 and 20. incomplete septum between the right and left sides of the sinus venosus. The two sides of the sinus venosus are in communica- 254 H. D. Senior. tion both anterior (dorsally) and posterior (ventrally) to the yolk process. Conditions in the hepatic-vein region become more readily in- telligible after preliminary examination of a section from 0.02 mm. posterior to the reconstructed part of the embryo. Here (Fig. 21) the peritoneal mesoderm (in which the celom is to a large extent virtual) covers the yolk laterally and dorsally and is generally in contact with it. Contact between the yolk and peritoneum is interrupted on the left side by the interposition of ie iz > f Xs Ziy { | kA penes =U ZY yy WY coelom (> Boe if / Ys é eritoneal }://4 LG Oo, Dorsal gotta coelom : Gy Fin-muscle Splanchnic q| , mesoderm - \ (pericardial) Somatic WA mesoderm ” (pericardial) Pericardial coelom | Dre ‘\ Ectoderm Mesoderm 18 19 (peritoneal) Fies. 18 and 19.—Two transverse sections of which the positions are marked on Kiedy. <50) diams: Yolk horizontally shaded, voluntary muscle obliquely shaded. The distribu- tion of vascular endothelium, including endocardium, is indicated by large dots. the supravitelline blood-sinus (see introduction). In passing tail- ward in the series from the position of Fig. 21, the yolk increases in size and the supravitelline sinus becomes more dorsally placed with regard to the yolk, but is always mainly to the left of the mid-sagittal plane. In Fig. 20, from the hepatic-vein region of the reconstruction contact between the peritoneum and yolk is interrupted on both right and left sides. The interruption on the right of the figure (left of embryo) corresponds to the future left hepatic vein and The Development of the Heart in Shad. 255 is a direct continuation forward of the main supra-vitelline sinus. The interruption on the left side of the figure (right of embryo) corresponds to the future (smaller) right hepatic vein, which is not at present in direct communication with the supravitelline sinus. The future right hepatic vein communicates anteriorly with the future sinus and posteriorly ends blindly. The dorsal mesentery in this stage, as in the adult, is absent in the region anterior to the stomach. Hig. 22 is a left lateral view of a reconstruction of the posterior / Z\ Peritoneal ; G7 SALSA : ; / coelom R. cardinal v. —— LL: L. cardinal vy. = Mes R. Hepatic v. { (future) L. Hepatic v. (future) Mesoderm (peritoneal) 20 21 Fies. 20 and 21.—Two transverse sections of which the positions are marked on Fig. 17. xX 50 diams. Yolk and voluntary muscle shaded and vascular endothelium dotted as in the preceding figure. Mesoderm (peritoneal) gill region of a shad of 166 hours (9.46 mm. in length). At this stage the peritoneal ccelom in the hepatic-vein region is so large that it can be opened by merely removing the body-wall; the anterior pole of the yolk is shown in situ. The plane of separation between sinus-venosus and hepatic-vein regions is indicated, as before, by the point of discharge of the left cardinal vein. It will be convenient to consider the hepatic-vein region first. In this region the peritoneal ccelom has undergone great expansion by reason of the extensive separation of splanchnic from somatic meso- 256 H. D. Senior. derm (compare Figs. 20 and 24). The shrinkage of the yolk has had an effect on the shape of the future hepatic veins; the latter are now semilunar rather than crescentic in section. The ventral position of Fig. 25 position of Fig. 24 position of Fig. 23 Ectederm Left jugular vein See a L. cardinal v. (Esophagus Posterior wall of sinus venosus Arrow in L. hepatic vein (head of arrow rests on yolk) Branchial cleft 2~~.# Left wall of L. hepatic vein Wena Parietal peritoneum [cut] aorta Branchial a. 3 Conus yh | Ventricle sane Ectoderm Dorsal parietal pericardium Atrium Anterior wall of sinus venosus Ventral parietal pericardium (mostly removed) Fic. 22.—The left side of a reconstruction from the posterior gill region of shad, stage of 166 hours. > 100 diams. Sufficient body-wall has been removed to open the pericardial ccelom laterally and ventrally and the peritoneal coelom laterally. The anterior pole of the yolk is intact, the ventricle and atrium have been opened. The vein imme- diately ventral to the left jugular is the left inferior jugular. part of the pericardial eelom has extended backward, so that the endocardium lining the anterior wall of the sinus venosus assists in the formation of the hepatic veins (see Fig. 24). The expansion The Development of the Heart in Shad. 257 of the peritoneal ccelom affects only the hepatic-vein region (Fig. 22). It ends so abruptly that the splanchnic peritoneum, passing peripherally from the anterior ends of the hepatic veins to become somatic, forms a partition between sinus-venosus and hepatic-vein regions. This partition of splanchnic peritoneum represents the posterior wall of the sinus venosus, and has been labelled accordingly in Fig. 22. In the sinus-venosus region shrinkage of the yolk has brought about marked diminution in the vertical dimension of this part of Peritoneal |; —— coelom __| 4/7 * Ectoderm Splanchnic [Splanchnic = eden Peritoneal\/ > x sete dial) pericardial) cocina Pp rdia Pericardial ~ i \ \L. Hepatic v. coelom | Ventri R. Hepatic vy. Pericardial : entricle (future) coelom 4 Supravitelline, Somatic mesoderm (pericardial) Somatic mesoderm (pericardial) ieaaes blood-sinus Peritoneal) =e 24 25 Fics. 23, 24, and 25.—Three transverse sections of which the positions are marked on Figs. 22. »X 50 diams. Yolk, voluntary muscle, and vascular endothelium indicated as in Figs. 18 and 19. the embryo (compare Figs. 22 and 17). The yolk-process has become thicker and much shorter; the space between its posterior surface and the yolk (which constituted the posterior communica- tion between the right and left sides of the sinus venosus) is now obliterated. The most striking change taking place at this stage occurs, alike, in the sinus-venosus and hepatic-vein regions; it is a process of re- arrangement of the vascular endothelium. The vascular endothelium is rapidly spreading from the splanchnic mesoderm, both pericardial and peritoneal, on to the yolk so as to exclude the latter from the 258 H. D. Senior. vascular system (see Figs. 23 and 24). This process has already progressed so far that the sinus venosus has a complete lining of vascular endothelium, which is true also of the adjacent part of the left hepatic vein. The yolk is still uncovered in the region of Fig. 25. The sinus venosus, since the yolk has been consigned to a posi- tion definitely external to it, may now be looked upon as a complete structure in which the achievement of the adult condition is merely a matter of detail. The layers of splanchnic mesoderm, pericardial and peritoneal, which have been called the anterior and posterior walls of the sinus venosus are, to some extent, in mutual contact just behind and above the apex of the ventricle (see Fig. 22). Where these two layers are in contact the terms anterior and pos- terior wall of sinus venosus are not strictly applicable, for they together form the anlage of the pericardio-peritoneal septum. During the process of disappearance of the yolk, the pericardio-peritoneal septum undergoes further increase, and later gowth produces the extensive structure of the adult. A few words may be added with regard to the completion of the hepatic vein. The conditions indicated in Figs. 24 and 25 are tend- ing in a direction which ends in the complete formation of the hepatic vein, as shown in Figs. 26 and 27. The latter figures come from sections of an embryo eight days old, and correspond in position to Figs. 24 and 25. In order to understand the condition of the hepatic vein at eight days it is necessary to appreciate the follow- ing facts: The yolk is not only smaller at its equator but the dis- tance between the poles is also much diminished. The liver, which has grown forward so as to keep pace with the posterior pole of the yolk, is now very much nearer the heart than before. Redis- tribution of vascular endothelium, excluding the yolk from the vas- cular system (see Figs. 26 and 27), has now occurred throughout the entire extent of the yolk (1. e., from heart to liver). The hepatic vein is, at this time, rather a long vessel and consists of a main stem bifureating anteriorly into short right and left branches ; both stem and branches replace the original supravitelline blood sinus. The left branch formerly transmitted all the - blood The Development of the Heart in Shad. 259 passing from the liver to the sinus venosus; this function is now shared by the right branch. At eleven days the yolk has entirely disappeared ; the final disappearance of the (latterly torpedo-shaped) yolk leaves the anterior pole of the liver still some distance posterior to the pericardio-peritoneal septum. The liver now grows forward (somewhat slowly) into the space formerly occupied by the yolk and, therefore, receives its peritoneal covering from the splanchnic meso- derm formerly enveloping the yolk and supravitelline sinus. The advancing liver receives into itself the entire main hepatic stem and the adjacent parts of the right and left branches. Finally, the anterior pole of the liver reaches the posterior surface of the peri- Dorsal aorta Peritoneal Ectoderm P » L. cardinal v. R. cardinal v coelom Splanchnic “| — Peritoneal R. Hepatic v. mesoderm coelom (peritoneal) Ectoderm i i BS i Common Hepatic v. Pericardial coelom Splanchnic mesoderm Splanchnic mesoderm Pp 3 : (peritoneal) / : Somatic mesoderm (pericardial) (pericardial) suse SN SN) 26 P37} Fics. 26 and 27.—Transverse sections of shad, stage of 8 days. X 50 diams. The transverse diameter of the sections is diminished from a tendency of the notochord to collapse. Yolk, voluntary muscle, and vascular endothelium as in Figs. 18 and 19. cardio-peritoneal septum and protruding from its anterior end are the right and left hepatic veins of the adult which transmit blood into the sinus venosus. The vascular endothelium lining the heart and the hepatic veins has been derived from two sources: Firstly, from the vascular endo- thelium (of which the origin has been described in the section deal- ing with the formation of the heart anlage) which has been referred to as endocardium. Secondly, from the vascular endothelium originally lining the roof of the supravitelline sinus, of which the origin has not been studied. 260 H. D. Senior. The endocardium originally lined the ventral surface of the splanchnic mesoderm anterior to the junction of the head with the first body somite. Of the splanchnic mesoderm lined ventrally by endocardium the (larger) part anterior to the site of discharge of the jugular veins became pericardial and the (smaller) part between the site of discharge of the jugulars and the first body somite became peritoneal. The disturbance of the original relations between the structures in the posterior head region which occurred between the stage of 7.2 mm. and that of 166 hours has been so extensive that it is very difficult to determine the exact amount of splanchnic peri- toneum which was originally situated anterior to the line of junction of the first body somite with the head; nevertheless the account given below of the eventual distribution of the endocardium is probably not very far from the truth. The endocardium lining the ventral surface of the pericardial splanchnic mesoderm was partially, in- cluded in the conus, ventricle and atrium to line these cavities; the remainder formed the lining of the anterior wall of the sinus venosus. The anterior wall of the sinus venosus assists in the formation of the anterior ends of the right and left hepatic veins (see Fig. 24). The vascular endothelial cells, migrating from the anterior wall of the sinus venosus on to the yolk to furnish the ventral lining of the hepatic veins, are, thus, derived from endocardium. The splanchnic peritoneum forming the posterior wall of the sinus venosus is so close to the site of discharge of the jugular veins that the vascular endothelial cells lining the posterior wall of the sinus venosus are undoubtedly (like those lining the remainder of the heart) endocardial in origin. Since the ends of the hepatic veins im- mediately adjoining the sinus venosus are lined ventrally by endo- cardial cells migrating from the anterior wall of the sinus venosus, there is a great probability that the cells migrating from the splanch- nic peritoneum to provide their dorsal lining (see Fig. 24), are also endocardial in origin. The main stem of the hepatic vein is composed of vascular endo- thelium which originally lined the roof of the supravitelline sinus, and it is not at all unlikely that the adjoining roots of the right and left hepatic veins (eventually contained within the liver) are of similar origin. : yes The Development of the Heart in Shad. 261 There seems to be nothing unusual in the method of development of the heart valves. The sinu-atrial valves (two, right and left) are developed very late. I have serial sections of an embryo of 114 hours (No. 14,994) in which the heart has apparently ceased to beat during atrial systole. In this specimen the (valveless) venous orifice of the atrium is tightly contracted as by a sphincter. There is little doubt that this specimen indicates the normal mechanism of atrial systole prior to the formation of the sinu-atrial valves. The true circulation does not begin with the initiation of rhyth- mical contraction in the, partially formed, heart-tube (36 somites), but with the completion of the aorta (42 somites), which occurs at a time when the heart-tube has been completely formed. Prior to the division of the primitive heart-tube into conus ventricle and atrium, the mechanism of systole of the entire tube is, in all proba- bility, similar to that described for the atrium. Syracuse, N. Y., November 2, 1908. LITERATURE CITED. AGASSIZ and WHITMAN, 1878. Development of Osseous Fishes. 1. Pelagic stages of Young Fishes. Mem. of the Museum of Comp. Zool. Har- Varden Vio lee xeloVe BorEkE, J., 1903. Beitraige zur Entwicklungsgeschichte der Teleostier. Petrus Camper. DI. II. DeERJuUGIN, K., 1902. Ueber einige Stadien in der Entwickelung yon Lophius piscatorius. Traveaux de la Société Imp. des Naturalistes de St. Petersburg. T. XXXIII. EIGENMANN, Cart H., 1892. Fishes of San Diego. Proc. U. S. National Museum. Vol. XV. , 1894. On the Viviparous Perches of the Pacific Coast of North America. Bull. U. S. Fish Commission. Vol. XVII. FELIX, W., 1879. Beitrage zur Entwickelungsgeschichte der Salmoniden. Ana- tomische Hefte, Arb. a. Anat. Inst. Bd. VIIT. Froriep, A., 1905. Lehrbuch der Entwickelungslehre der Wirbelthiere, O. Hertwig. Bd. II, Theil 2, s. 171. GreIL, A.,; 1908. Ueber die erste Anlage der Gefiisse und des Blutes bei Holo- und Meroblastiern (speziell bei Ceratodus Forsteri). Verhandl. d. Anat. Gesellsch. Berlin. Hocustetter, F., 1887. Beitriige zur Vergleichenden Anatomie und Entwicke- lungsgeschichte des Venensystems der Amphibien und Fische. Morph. Jahrb. Bd. XIII, 1888 (Heft I appeared in 1887). 962 H. D. Senior. Marcus, H., 1905. Ein Beitrag zur Kenntniss der Blutbildung bei Knochen- fischen. Arch. f. Mikr. Anat. Bd. LXVI. OELLACHER, J., 1873. Beitriige zur Entwickelungsgeschichte der Knochen- fische nach Beobachtungen am Bachforellenei. Zeits. f. Wiss. Zool. Bd. XXIII. RAFFAELE, F., 1888. Le uova gallegianti e le larve dei Teleostei nel golfo di Napoli. Mitth. a. d. Zoolog. Stat. zu Neapel. Bd. VIII. Ryver, J., 1881. Development of the Spanish Mackerel. Bull. U. S. Fish Commission. Vol. I. , 1884. Embryography of Osseous Fishes. U. S. Fish Commission, Report for 1882, Washington, 1884. , 1885. On the Development of Viviparous Osseous Fishes. Proce. U. S. National Museum. Vol. VIII. , 1887. On the development of Osseous Fishes. U. 8. Fish Commis- sion. Report for 1885, Washington, 1887. Soporra, J., 1902. Ueber die Entwickelung des Blutes, des Herzens, und der grossen Gefiissstiimme der Salmoniden. Anatomiscke Hefte. Arb. aus Anatom. Instit. Bd. VIII. Summer, F. B., 1900. Kupfer’s Vesicle and its Relation to Gastrulation and Concrescence. New York Acad. of Sciences. Vol. II, Part 2. SWAEN ET BrRACHET, 1900. Etude sur les premiéres phases du developpement des Organes dérivées du Mésoblaste chez les poissons Téleostéens. Premiére partie. Arch. de Biologie. T. XVI. , 1902. La méme, Deuxiéme partie. Archiv. de Biologie. T. XVIII. Unitep STATES COMMISSION OF FISH AND FISHERIES, 1900. A Manual of Fish Culture. Revised edition, Washington. WENCKEBACH, K. F., 1885. The development of the Blood-corpuscles in the Embryo of Perca fluviatillis. Journ. Anat. and Physiol. London and Cambridge. Vol. XIX. , 1886. Beitrige zur Entwickelungsgeschichte der Knochenfische. Arch. f. Mikr. Anat. Bd. XXVIII. Witson, H. V., 1891. The Embryology of the Sea Bass (Serranus atrarius), Bull. U. S. Fish Commission. Vol. IX. ZIEGENHAGEN, P., 1894. Ueber das Gefiaisssystem bei Salmonidenembryonen. Verhandl. d. Anat. Geseilsch. Strassburg. , 1896. Ueber die Circulation bei Teleostiern, insbesondere bei Belone. Verhandl. d. Anat. Gesellsch. Berlin. ZIEGLER, H. E., 1882. Die Embryonale Entwickelung von Salmo salar. Diss. inaug. Freiburg. ———, 1887. Die Entstehung des Blutes bei Knochenfischembryonen. Arch. f. Mikr. Anat. Bd. XXX. HISTOGENESIS AND HISTOLYSIS OF THE INTESTINAL EPITHELIUM OF BUFO LENTIGINOSUS. BY MARY A. BOWERS. WITH 4 PLATES AND 1 TExtT FIGURE. The various phenomena associated with metamorphosis present many problems which are of great biological interest. They have attracted a large number of investigators, and the literature, includ- ing all phases of the subject, the macroscopic and microscopic changes which occur at the time of transformation and the experi- mental and theoretical work on the causes of metamorphosis, has become most voluminous. However, previous to 1906 but two papers had been published on the modification of the tadpole intestine, Ratner, 91, and Reuter, 1900. Ratner describes the changes which occur in the subepithelial layers; Reuter, using Alytes obstetricans, gives a very full account (Part I) of the macroscopic changes of the alimentary tract; (Part IT) the degenerative and regenerative modifications of the epithelium. I should mention that Kingsbury, 99, published a brief abstract of work which he had begun on Bufo. Since my work was begun another paper has appeared, by Duesberg, 96, but as his observations and interpretations are not in accord with those of Reuter and as his material, Rana fusca, is dif- ferent from mine, it seems worth while to state briefly my results. This investigation was undertaken at the suggestion of Professor E. G. Conklin, and has been carried on in the laboratories of the University of Pennsylvania. I wish to express my sincere appre- ciation of Professor Conklin’s valuable assistance and his kindness to me throughout my, work. I am also greatly indebted to Mr. Herbert G. Kribs, who most carefully prepared the photomicro- graphs and kindly aided me in all my photographic work. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. 264 Mary A. Bowers. MarTeriaAL AND Metuops. The material used for this investigation has been our common toad, Bufo lentiginosus, supplemented by the examination of many series of the bullfrog, Rana catesbiana, and of the green frog, Rana clamata. The most satisfactory fixing fluids were Flemming’s and Zenker’s. Paraffin embedding was employed and sections cut 6 2/3 microns thick. Considerable difficulty was experienced in cutting the first series, especially the young stages, owing to the diatoms and sand in the intestine. This trouble was avoided by feeding the tadpoles for a day before killing, on fine yellow corn meal, which replaced the gritty contents. A long series of substances, flour paste, egg (yolk and white), beef juice gelatine, and meat were tried, but were not satisfactory. The Flemming material was stained with Heidenhain’s iron hematoxylin and orange G. Delafield’s hematoxylin (in toto) and eosin (on the slide) were used for the larve preserved in Zenker. All figures were drawn with the aid of an Abbé camera lucida. The photomicrographs were taken with violet light. Living tadpoles were used for all X-ray photographs except Figs. 3-10; these were taken from preserved specimens whose alimentary tracts contained only the normal food. Larve were prepared for all other photographs in the following way: subnitrate of bismuth, a non-irritating powder and insoluble in water, was placed in a flat-bottomed glass dish and the tadpoles allowed to feed on it for four or five hours. Larve which are not transforming feed almost con- tinuously, by night as well as by day, so the whole canal is kept well filled. They were then placed in a 5 per cent solution of ether and when quiet (usually in about 15 minutes) they were taken out and arranged on a thin piece of paraffin paper, to protect the X-ray plate, and exposed to the rays for thirty seconds. The animals were immediately replaced in pure water where they revived in from 5 to 10 minutes. Macroscopic CHANGES.. Reuter, in the first part of his work, describes the gross changes which occur during metamorphosis, in the alimentary tract of Alytes Intestinal Epithelium of Bufo Lentiginosus. 265 obstetricans, and Duesberg reports that Rana fusca shows complete agreement. with these conditions. The phenomena are essentially the same in Bufo lentiginosus. I shall refer only briefly to a few points brought out by the method used in studying Bufo. The employment of subnitrate of bismuth and the X-rays was sug- gested to me by Cannon’s paper (’02) in which he describes its use in his physiological work upon the movements of the intestines of the cat. By employing this method with Bufo it has been possi- ble to figure successive changes in the same individual from day to day, and the danger of displacement by dissection is done away with. Also the exact time at which the larve begin to take food is easily determined. With animals reared under normal condi- tions (20° C.), this occurred on the sixth day after hatching (Fig. 1), which corresponds with the time of rupture of the stomodeal septum. This series, exposed first upon the sixth day, was subjected to the X-ray eight times. These rays had no effect upon development or metamorphosis, as could be determined by comparison with control larvee. The coil of the small intestine increases in length until a maxi- mum is reached at a time when the hind legs are well developed, but not yet drawn up on the body (earlier than Fig. 3). The short esophagus is followed by the stomach and duodenum, which lie on the extreme right side and dorsal to the large coil. The rectum lies on the left, also dorsal in position. As Yung and Babak have shown experimentally, the extreme length of intestine in larval Anura is an adaptation to the plant- eating habit—a difference of 58.15 per cent was obtained by Babak (706) in the length of intestine of two sets of larve, one fed upon plants, the other upon meat. As Ratner points out, the Urodeles are meat eaters throughout life, and in Anura this plant-eating habit is secondary. The change in adult Anura to meat eating and the short intestine is a return to the normal. In the early differentia- tion of the alimentary tract, two or three days after hatching, the anterior part which is to form the stomach, lies in the normal position for this organ in Vertebrates, on the leff. There appears to be a 266 Mary A. Bowers. passive crowding of this anterior portion to the right side by the rapid differentiation of the large coil from the posterior yolk mass. This conclusion seems justified from experimental work, which will be described later. When the coil has reached its maximum length, the liver, lying anteriorly on the right, is very small, but it increases rapidly in size. This growth of the liver on the right, plus a decrease in size of the intestinal coil on the left, and a slight growth in length of the stomach, probably combine to effect the interchange in position of stomach and coil. This is clearly represented in the X-ray series, Figs. 3 to 10. In the twenty-four hours immediately following the breaking through of the fore legs, the shortening of the coil is usually completed (Figs. 7-9) and stomach and coil pass each other in the median line (Fig. 9). At the time the tail begins to shorten (usually on the second day), the change has as a rule been made (there is some individual variation), and the stomach has taken the adult positon on the left side, the reduced coil lies on the right (Fig. 10). This transformation of the intestine is described by Reuter and Duesberg as occurring in Alytes obstetricans and Rana fusca before the breaking through of the fore legs. Reuter says the rectum changes position slightly or not at all in Alytes. In Bufo it follows the coil from the left to the right side, or, in some cases, only to the median line. It becomes shortened and widened. Besides the shortening of the small intestine, there is also a marked diminu- tion in the diameter,—compare Fig. 18 (before reduction of coil), and Fig. 20 (after reduction), noting the increase in thickness of the circular and longitudinal muscle layers. In striving to get a full bismuth-X-ray series, extending through this critical change of shortening and narrowing, it was incidentally determined at exactly what time the larve stop feeding in prepara- tion for the renewal of the intestinal epithelium. Figs. 11 to 13 indicate that feeding continues to the day before the appearance of the fore legs. At the time when the fore legs break through, X-ray plates of many different series show that the bismuth has been eliminated from the rectum (except in a few cases, as Fig. 14). Although the normal food seems to be not so freely eliminated as the bismuth, Intestinal Epithelium of Bufo Lentiginosus. 267 the examination of a large number of alcoholic specimens was required before the tadpoles in Figs. 7-10 were found. q After the appearance of the fore legs and the coincident shortening and narrowing of the intestine, there remains one more marked change before metamorphosis is completed, the absorption of the tail. This shortening usually begins about 48 hours after the fore legs have come through and is completed in from 2 to 4 days. Feeding is resumed on the third or fourth day after metamorphosis is completed. Microscopic CraNGESs. I. Historical. As stated in the introduction, there have been but three papers on the modification of the intestine of Anura during metamorphosis,—those of Ratner, ’91, Reuter, ’00, and Duesberg, ’06. Ratner’s work does not consider the epithelial changes; it is con- cerned with the subepithelial layers. Reuter, using Alytes obstet- ricans, and Duesberg, working with Rana fusea, disagree on the main points of the process. Reuter recognizes that the epithelium of young larve is composed of two forms of cells, first, cylindrical, and second, basal round cells, the latter relatively few in number. These ‘round cells” show fine granular contents and might be mistaken for leucocytes. During de- generation of the epithelium they become more numerous, large and may be multinucleate. They contain great masses, food sub- stance in process of absorption, which are colored brown with Flem- ming. He considers them special absorbing cells. Somewhat later “oiant cells” appear, also basal in position and multinucleate, and differing from the “round cells” only in not having the brown masses and in their fate. Reuter claims that both round and giant cells originate by amitosis from the cylindrical epithelium. At the beginning of metamorphosis the larvee stop feeding, the round cells receive from the cylindrical cells the last absorbed food, but they do not pass it on into the lymph channels,—they cease secret- ing, become overloaded, die, and with the degenerating cylindrical cells are cast off into the lumen when shortening of the intestine occurs. The giant cells remain, gain the ability to divide mitotically, and form the definitive epithelium. 268 Mary A. Bowers. Duesberg describes the epithelium of the coil at a time before the appearance of the hind legs, as composed of principal cells and, be- tween their proximal ends, scattered basal cells. Later, as the hind legs appear, other basal elements are noticed, which clearly cor- respond to Reuter’s round cell. Vacuoles and granules appear in the. protoplasm, neighboring cells become confluent and form a single cell, the brown masses become more numerous, the nuclei go through chromatolysis, and the final stage in this process is the typical “round cell.” These cells present none of the characteristics of active ab- sorbing cells, on the contrary they, show marked degenerative phe- nomena. They are cast off as described by Reuter. Duesberg figures the definitive epithelium as forming from the basal cells of the larval epithelium, which are at first not distinguished by any peculiar structure, but later show the characteristics of Reuter’s “giant cells.”” He has not seen in any of his preparations the formation of round or of giant cells by amitotic division of the prin- cipal cells. My conclusions, which agree in the main points with those of Dues- berg, were formed independently before the reading of his paper. II. Description. The histological differentiation of the small intestine of Bufo, 2 e., the degeneration and regeneration of the epithelium, takes place progressively from the anterior to the posterior end. Therefore, for clearness of comparison in successive stages, one particular region has been selected for description, the duodenum near the entrance of the bile duct, and all figures except 16 and 44 are from this region of the intestine. At the time of hatching the stomodeum and proctodeum are already formed and the rest of the alimentary canal is a mass of yolk, through the dorsal portion of which runs an irregular lumen. Anteriorly, ex- tending back to the level of the pronephros, the lumen is definite, and in the dorsal and lateral walls may be distinguished scattered nuclei and very faint irregular cell walls. Fig. 22, five days after hatching, shows the earliest differentia- tion of the duodenal epithelium into a definite layer of columnar cells. The cell walls are very faint, the ciliated border is not formed, the cells are solidly packed with yolk spherules and often contain two nuclei. Intestinal Epithelium of Bufo Lentiginosus. 269 The stomodeal septum is usually broken through on the sixth day (larvee 11 mm. long). On the seventh day the coil is completely formed and fills the whole ventral part of the abdominal cavity. Figs. 23 to 25 show this stage in cross section. There is a low, ciliated, columnar epithelium, having large nuclei at the proximal end. ‘The yolk is mostly absorbed, only an occasional cell being well packed with small spherules. Rarely one finds a basal cell (Figs. 23 and 24), and in the distal border a mitotically dividing cell (Fig. 25). The sub-epithelial tissue forms a very delicate layer. — Figs. 26 to 31 (two weeks after hatching) show the ciliated columnar epithelium thrown into folds. Many mitotic figures appear at the bases of the folds. These cells as a rule are clearer than those that form the folds,—their appearance is suggestive of special activity, perhaps of a grandular character. Bataillon (’91) noticed in the epifhelium of Alytes obstetricans, “a curious localization of karyo- kinetic figures” at the bases of the folds. He suggests that perhaps the irritation of compression causes activity at these points. Figs. 26, 27 and 28 show resting, spireme and anaphase stages of basal cells, the first differentiation of the giant cells of Reuter and Duesberg. ‘This stage is characterized by many mitotically dividing cells in the distal zone of the epithelium (Figs. 29 to 31). A few round cells appear (Fig. 31). Fig. 32 (three weeks after hatching) again shows the activity and relative clearness of the protoplasm of cells at the base of a fold. The other cells are well filled with fat, stained black with Flemming. Figs. 32 and 83 were made from larve which correspond approximately to individuals I and II of Fig. 2, the hind legs having appeared. Histolysis. For the sake of clearness the histolytic phenomena which now begin to appear in a marked degree, will be followed through succeeding stages; histogenesis will be considered later, al- though the two processes go on side by side. Figs. 18 and 35 to 37 are cross sections of No. III, Fig. 2. The cytoplasm still shows the fine mottled appearance of the preceding stages, only rarely (Fig. 34) showing small vacuoles and products of degeneration, brown and yellow granules (Delafield and eosin stain). Fig 84 shows also one of the many “round cells” which have now made their appearance. 270 Mary A. Bowers. It is filled with degenerating material. Its nucleus, like those of the columnar cells, shows an early stage of chromatolysis. There is a pale nuclear groundwork, the chromatin is in deeply stained clumps, the membrane is slightly thickened and is beginning to be irregular in outline. The structure marked ch. appears to be a mass of chromatin in an early stage of degeneration (compare Figs. 38 to 41, showing later stages of chromatolysis). The chromatin must undergo chemical change, for increasingly large areas fail to take the hematoxylin. These unstained areas, in a later stage, are filled with small yellow granules, and in a final stage the whole mass has a clear straw color (Delafield and eosin stain). Figs. 35 to 37 show the characteristic appearance of the cells at this stage (No. III, Fig. 2). There appears to be fragmentation of the nuclei, also a breaking down of cell walls and a clumping to- gether of nuclei (Fig. 37). No. V (Fig. 2) has pushed one fore leg through. Feeding has ceased, the muscular contraction 1s pronounced (see Figs. 19 and 42 for cross sections). The cytoplasm has now become vacuolar, and throughout the cells are scattered globules and granules of brown, yellow and black degenerating substance. The nuclei have become more irregular. In No. VI, Fig. 2 (both fore legs through, tail not absorbed), con- traction has been completed and the organs of the alimentary tract are in their adult position (Figs. 20 and 43). Degeneration of the old cells is nearly completed, the cell walls have become indistinct, the cilia have disappeared. Marcelin (’03) in his tabulation of the histogenetic changes of the intestinal epithelium of Rana esculenta, shows that the disappearance of the cilia occurs at the time when the intestine is at its maximum length, that is, relatively much earlier than in Bufo. He believes that the reason for this disappearance of the cilia is to be found in the fact that their function, the propelling of the food, is now usurped by peristaltic contraction of the muscles, which have grown stronger. In Bufo the cilia remain throughout the larval life, although there is a strong peristaltic movement, even in very young larve. Figs. 16 and 44 (from the posterior end of the small intestine of Intestinal Epithelium of Bufo Lentiginosus. 271 No. VII, Fig. 2) show the appearance of the final stage of degenera- tion of the columnar and round cells and the manner in which they are pinched off into the lumen when the final muscular contraction oceurs. The debris is given off through the anus. Duesberg suggests that some of it may be absorbed as nourishment. Histogenesis. At the stage represented by No, III (Fig. 2) many giant cells can be distinguished in the duodenum (Figs. 18 and 35 to 37). Owing to the fact that a giant cell, as it now becomes active, takes a diffuse deep blue stain, with hematoxylin, it can be easily distinguished, even when it has but one nucleus, and more readily when there have been repeated mitotic divisions and a syncytium or a cyst has been formed (Fig. 18, g. ¢.). Figs. 35, 36 and 42 show stages in the formation by mitosis of the syncytium and the characteristic appearance of the nuclei, large and plump, with fine chromatin network and large nucleolus. These nuclei and the small amount of deeply staining cytoplasm in which they are irregularly massed (Fig. 42) soon form a cyst, a hollow sphere, which breaks open on the side toward the lumen of the alimentary tract (Fig. 43). This condition corresponds to that found in Alytes obstetricans. In Rana fusca the developing epithelium does not go through a cyst stage,—the syncitia appear as scattered patches of new epithelium, which meet and fuse when contraction occurs. This broken cyst opens more widely (Fig. 44), and at the same time the nuclei become oriented, with their long axes parallel to one another. Cell walls appear and a cuticular border is formed. Eventually these isolated crypts of new epithelium become joined edge to edge (Fig. 16) ; thus the continuous, definitive epithelium is formed (Figs. 17 and 45). As the process of histogenesis is progressive from stomach to rectum, it was possible to find these two last stages (Figs. 44 and 45) in the same individual (No. VII, Fig. 2). The new epithelium forms much earlier, in comparison with the progress of degeneration, in Bufo than in Alytes (compare Fig. 43 with Reuter’s Fig. 38). This account of the histological changes which occur in Bufo agrees, I believe, in the main with that given by Duesberg for Rana fusca. It has been limited chiefly to a statement of the facts, because 272 Mary A. Bowers. the arguments have already been fully set forth by him, also because it is believed that the figures given in this paper offer their own argument. It is probable that the significance of the histolytic and regenera- tive phenomena which have been described in the three Anurans, Alytes obstetricans, Rana fusca, and Bufo lentiginosus, will be more clearly seen when more extended work has been done upon other forms, upon amphibia in general. It appears from the comparative work which has already been done that in the tadpole of Anura we have merely a temporary adap- tation, and, as in the case of the larve of those insects which undergo complete metamorphosis, that these conditions have no phylogenetic significance. EXPERIMENTAL WorK. I. Mechanics of the early differentiation of the alimentary canal. Text figure A shows the successive changes which had occurred in one individual of Rana palustris by the second, third, fourth and sixth days after hatching. Rana palustris was used because the lack of pigmentation at this stage allows one to see clearly, without dis- section, the changes in the alimentary tract. Dissection of different individuals of Bufo on successive days indicates that the changes are the same as in Rana palustris. Text figure A, I, shows the esophagus, stomach, anterior part of the duodenum and the rectum formed; the rest of the tract is an un- differentiated mass of yolk from which the coil will be formed. The stomach lies on the left side, in the normal adult position. Further growth, 2. e., differentiation of the coil, takes place in the region marked x, and because of this vigorous growth, the stomach and duo- denum are crowded into the temporary larval) position on the right. Theoretically this transfer from the left to the right side would appear to be due to a passive crowding, not to an active movement of the anterior part of the digestive tract, for the cellular structure of this region is already laid down in the stage shown in I. To test experimentally this question of crowding, that is, whether the stomach would remain on the left if the coil did not usurp its Intestinal Epithelium of Bufo Lentiginosus. 273 place, a small cut was made through the abdominal wall of several Bufo larve, one day after hatching. In some cases these healed so rapidly that development went on normally, but in several indi- Fic. A. Rana palustris. Ventral. x 10. I. Two days after hatching. II. Three days after hatching. III. Four days after hatching. IV. Six days after hatching. e., coil; dm., duodenum; oe., oesophagus; rect., rectum; st., stomach; x., point at which the differentiation of coil from yolk mass takes place. viduals as the coil developed it pushed through the opening in the body wall, forming a hernia outside the body. In these cases the stomach, which was not crowded by the coil, remained on the left. Of these individuals none lived over four days. 974 Mary A. Bowers. II. Carmine injection test for leucocytes. Reuter speaks of the superficial resemblance of the round cells of the epithelium to the round cells of the blood, viz., the leucocytes. Bizzozero (’02) and others have considered them to be leucocytes, but Reuter strongly maintains that the two should not be confused, —those of the epithelium arise as epithelial cells and remain epi- thelial cells throughout life. He says that they may migrate to the outer zone of the epithelium and undergo division. The round cells of Bufo do not have the appearance of leucocytes, and as they are degenerating cells, it is improbable that they migrate to the distal zone and divide. It was not possible to identify as leucocytes the numerous mitotically dividing cells of the distal zone (Figs. 29-31), in fact none were distinguished in the epithelium. To test their presence, and to determine, if possible, whether phago- eytosis plays any part in the histolysis of the epithelium, the method employed by Mercier (’06) in his study of phagocytosis in the tail of Rana temporaria, was tried. Mercier injected powdered sterilized carmine into the dorsal lymph sacs of tadpoles, and the animals were killed 24 hours later. He found that the leucocytes had taken up the carmine granules and wandered with them into the muscular tis- sue of the tail. It was hoped that leucocytes might be traced by this method into the degenerating epithelial tissue of the intestine. Tadpoles just ready for metamorphosis were etherized, and ster- ilized powdered carmine was injected under the skin of the back. On the first, second and third days after the injection tadpoles were killed in corrosive-acetic, stained with Delafield’s hematoxylin, and imbedded in paraffin. Several series have been carefully examined, with but negative results thus far, no carmine having been found in any of the layers of the intestine. Ill. Contraction of the Intestine. Reuter and Duesberg refer to the contraction which results in the shortening and narrowing of the intestine as a peristalsis, moving in a cranio-caudal direction. Duesberg states that this peristaltic con- traction is like true peristalsis with the exception that it is permanent. The reduction in size of the intestine, he says, always occurs Intestinal Epithelium of Bufo Lentiginosus. 275 during the period between the appearance of the posterior and the anterior limbs and takes place in a relatively short time. According to Reuter 48 hours is the maximum time for contraction in Alytes obstetricans, Bufo, at a stage corresponding to No. I (Fig. 2), often has a mid- ventral longitudinal strip of the body wall which is free from pig- ment. Beneath this unpigmented area the normal peristaltic move- ment of the intestine can be clearly seen. The food is pushed toward the anus by the typical slow, rhythmic, wavelike contraction. Older larvee, in which the whole ventral surface was deeply pigmented, were etherized, a longitudinal cut was made in the ventral body-wall, and they were then placed in normal salt solution. The normal successive contraction and expansion was observed as before. Many series of tadpoles, corresponding to the different stages represented in Fig. 2, were examined by means of this method. Among one lot of especially large strong tadpoles, two individuals were found whose whole bodies remained abnormally free from pig- ment throughout their development. The intestinal movement could thus be observed without opening the body cavity. These two tad- poles were etherized and observed daily until the end of meta- morphosis, one individual for a week, the other for twelve days. In no case was anything like a “permanent peristalsis’ observed. The records obtained from the unpigmented individuals show that the contraction of the coil is a slow process, extending over the week or ten days previous to the appearance of the fore legs. I should say that X-ray photographs of the same individual on successive days also prove this to be true. From observation of gross conditions, the shortening and narrow- ing of the intestine in Bufo would appear to be accomplished by a very gradual, even contraction of all the muscles, longitudinal and circular, of the intestinal wall. Bataillon (’91) ascertained by the measurement of dissociated muscle fibres, that their contraction corresponds exactly to the total shortening of the intestine. Although individual variation occurs, this slow contraction of the coil has, as a rule, been completed at the end of the first day after 276 Mary A. Bowers. the breaking through of the fore legs. In two individuals, opened at this stage, the reduced intestine was observed to move slowly and steadily, but without visible action of the muscles of the alimentary tube, from its larval position on the left to its adult position on the right. The loop, which had traveled across the body, then made a double coil. It is not possible to draw conclusions with certainty from the observation of two cases, but this whole movement appeared to be due to contraction of the mesentery; the intestine seemed to be passive. The mesentery usually contains a large amount of pigment. The fact that aggregations of pigment are often found on the ali- mentary tube after it has passed to the right side of the body, and not before, would seem to add evidence to the view that the contrac- tion of the mesentery is the agent in this movement. Bataillon has tested experimentally the question whether the modi- fications of the coil are localized. He curarized tadpoles several days before the appearance of the anterior legs, opened the abdominal cavity and marked equal distances on the small intestine by means of fine pieces of silk thread, which were held in place by the mesen- tery. The cavity was then closed and the animals soon recovered. At the end of metamorphosis examination showed that the modifications are not localized, except that the shortening is slightly more marked at the summit of the coil. ' The cause of the contraction of the intestine may be involved in the puzzling question of metamorphosis, which has never been satis- factorily- answered, although many interesting theories have been advanced. Reuter suggests that the contraction of the intestinal muscles is perhaps in response to a stimulus derived from the degenerating epithelial cells. In Bufo the first appearance of epithelial degenera- tion and the beginning of the slow muscular contraction are coin- cident, and they may be causally related. Bataillon holds that the changing conditions of respiration and circulation which result in partial asphyxiation, are the causes of metamorphosis. This could hardly be the cause of the contraction, since epithelial degeneration and muscular contraction begin several days before these changes Intestinal Epithelium of Bufo Lentiginosus. QT7 in respiration and circulation take place. Likewise they are initiated before there is any change in the feeding habits. It appears probable that the muscular contraction is in some way dependent upon the epithelial condition, perhaps both chemical and mechanical changes, which accompany degeneration. Why the degeneration of all but the giant cells of the larval epithelium should take place seems to me to be a question of life cycles, which cannot be answered until we know more of the laws of growth and senescence. My observations of the histological con- ditions of the Bufo larva lead me to believe that there is an early differentiation of the two sets of epithelial cells, one set, the principal and round cells, destined to function through the larval existence, to run its whole life cycle within six or eight weeks, the other, the giant cells, becoming active only after a latent period of four or five weeks, and functional as absorbing and secreting cells only after meta- morphosis is completed. SUMMARY. I. Between the time of hatching and the end of metamorphosis the alimentary tract of Bufo undergoes striking changes, both macro- scopic and microscopic. II. The macroscopic changes consist of : 1. The differentiation, from the posterior part of the yolk mass, of a huge intestinal coil, which crowds the stomach and duodenum to the right side of the body. 2. This intestinal coil reaches its maximum size at a time when the hind legs are well developed, but not yet drawn up on the body. 3. A gradual shortening and narrowing of the intestine then takes place, by means of a slow, even contraction of the longitudinal and circular muscles. This contraction oceurs during the week or ten days previous to the breaking through of the fore legs and is usually completed in the following 24 hours. 4. With the completion of the intestinal reduction the stomach resumes its original position on the left, and the intestine moves, ap- parently by means of contraction of the mesentery, to its adult posi- tion on the right. 278 Mary A. Bowers. III. Miscroscopic changes: 1. From earliest differentiation of the larval epithelium two kinds of cells may be distinguished, the principal cells and the basal giant cells. 2. The principal cells form the temporary, larval epithelium. Degenerative phenomena begin to appear in these cells about two weeks before the breaking through of the fore legs; they are coin- cident with the shortening and narrowing of the intestine. 3. As degeneration begins in the principal cells, the giant cells become active, increase in size, divide mitotically, and form syncitia. 4. These syncitia form cysts, hollow spheres. The nuclei then become oriented, their long axes radiating from the center, and cell walls form, making a single layer of columnar epithelium. 5. The cysts break open on the side toward the degenerating epithelium, unite with each other and form the definitive epithelium. 6. With the final muscular contraction, the degenerating epithelium is pinched off into the lumen of the alimentary tract and eliminated through the anus. PAPERS REFERRED TO. 1. Basak, E. Ueber den Einfluss der Nahrung auf die Lange des Darm- kanals. Biolog. Centralbl., Bd. 23, 1903, pp. 477-483. Bapak, E. Experimentelle Untersuchungen tiber die Variabilitit der Verdauungsrohre. Arch. f. Entwickl.-mechanik d. Organismen, Bd. XXI, Heft 4, 1906, pp. 611-702. 8. BararILLon, E. Recherches anatomiques et experimentales sur la méta- morphose des Amphibiens Anoures. Ann. Univ. Lyon, T. II, Fasc. I, 1891, pp 1-128, 6 pls. 4. Brzzozero. Ueber die schlauchférmigen Driisen des Magendarmkanals und die Beziehungen ihres Epithels zu dem Oberflachenepithel der Schleimhaut. Arch. f. mikr. An., Bd. XL, 1892, pp. 325-375, taf. 18-19. 5. Cannon, W. B. The Movements of the Intestines studied by means of the Réntgen Rays. Amer. Journ. Physiol., Vol. 6, No. 5, 1902. 6. Duvesspere. Contribution a Vétude des phénoménes histologiques de la métamorphose chez les Amphibiens Anoures. Arch. d. Biol., T. XXII. Fasc. I, 1906, pp. 163-228, pls. 10-11. 7. Kinessury, B. F. The Regeneration of the Intestinal Epithelium in the Toad (Bufo lentiginosus Americanus) during Transformation. Trans. Amer. Micr. Soc., XX, 1899, pp. 45-48. rw) 10. ti, 15. 14. Intestinal Epithelium of Bufo Lentiginosus. 279 MARCELIN, R. H. Histogénése de l’épithelium intestinal chez la Grenouille (Rana esculenta). Rey. Suisse Zool., T. XI, Fasc. II, 1908, pp. 369- 392; pl. 12. MekrcriER, L. Les processus phagocytaires pendant la métamorphose des Batraciens anoures et des Insects. Arch. d. Zool. Exper. et Générale, 1. V, No: 1, 1906; pp- 1-151, 4 pls: RATNER, G. Zur Metamorphose des Darmes bei der Froschlarve. Inaug. Diss., Dorpat, 1891, 34 pp., 1 taf. ReEvuTER, K. Ueber die Riickbildungserscheinungen am Darmkanal der Larve von Alytes obstetricans: I Teil. Anat. Hefte, H. XLV, 1900, pp. 483-445, taf. 19-20. Reuter, K. Id., 2 Teil, Micr. Untersuch. der Organenverinderungen. Anat. Hefte, H. XLIX, 1900, pp. 625-675, taf. 52-56. Yune, E. De Vinfluence du régime alimentaire sur la longueur de l’intestin chez les larves de Rana esculenta. Comp. Ren. Acad. Sc., T. 139, 1904, pp. 749-751. Yune, E. De la cause des variations de la longueur de l’intestin chez les larves de Rana esculenta. Comp. Ren. Acad. Sc, T. 140, 1905, pp. 878-879. LIST OF ABBREVIATIONS. b. c., Basal cell. c., Coil. ch., Chromatin. d. e., Degenerating epithelium. e., Epithelium. g. c., Giant cell. m., Mitosis. ms., Muscles. n., Nucleus. n. e., New epithelium. p. ¢., Principal cell. r., Rectum. r. c., Round cell. st., Stomach. vac., Vacuole. y. g., Yellow granules. y. s., Yolk spherules. z., Detail shown in Fig. 22. PEATKMe Fic. 1. X-ray photographs. Four tadpoles, sixth day after hatching. The intestinal coil filled with subnitrate of bismuth. Dorsal view. Itc. 2.—Photograph. Seven stages in the development of the tadpole, from 3 weeks after hatching to 3 days after the appearance of the fore legs. Ventral view. Fics. 3-10.—X-ray photographs. Stages in the development of the tadpole alimentary tract, from about 10 days before to 2 days after the appearance of the fore legs. Alcoholic material. Normal food in the alimentary tract. Ventral view. Fies. 11-15.—X-ray photographs. Living material. Subnitrate of bismuth in the alimentary tract. The same individual photographed on June 10th (Fig. 11), June 14th (Fig. 12), June 15th (Fig. 18), June 16th (Fig. 14), just after the appearance of the fore legs, June 16th (Fig. 15), a few hours later than Fig. 14. Ventral view. Fic. 16. Photomicrograph. Cross-section of the posterior part of the intestine of No. VII, Fig. 2. Detail (ne) shown in Fig. 44. > 165. Fic. 17. Photomicrograph. Cross-section of the duodenum of No. VII, Fig. 2. Detail shown in Fig. 45. 165. INTESTINAL EPITHELIUM OF BUFO LENTIGINOSUS PLATE I, MARY A. BOWERS. I 3 4 5 6 THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. PuiateE II. Fig. 18. Photomicrograph. Cross-section of the duodenum of No. III, Fig. 25° S<65. Fic. 19. Photomicrograph. Cross-section of the duodenum of No. V, Fig. 2. X< 165. Fie. 20. Photomicrograph. Cross-section of the duodenum of No. VI, Fig. 2. > 165. INTESTINAL EPITHELIUM OF BUFO LENTIGINOSUS. MARY A. BOWERS. THe AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 9 PLATE Il. PLATE ITI. Fic. 21. Cross-section of Bufo tadpole, at the level of the pronephros, 5 days after hatching. Detail (z) shown in Fig. 22. x 80. Fic. 22. Duodenum, 5 days after hatching. Cross-section. x 990. Figs. 23-25. Duodenum, 1 week after hatching. Cross-section. x 990. lies. 26-31. Duodenum, 2 weeks after hatching. Cross-section. x 990. Fie. 32. Duodenum, 3 weeks after hatching (like No. I, Fig. 2). Cross- section. Xx 990. Fic. 33. Duodenum, from a tadpole like No. II, Fig. 2. Cross-section. x 990. Fic. 34. Duodenal epithelium from tadpole No. III, Fig. 2. Cross-section. Micr. Leitz. oc. 4, obj. im. 7/;6. Fics. 35-37. Duodenum. Tadpole No. III, Fig. 2. Cross-section. x 990. Fics. 38-41. Stages of chromatolysis. Nuclei from duodenal epithelium of tadpole No. IV, Fig. 2. Oc. 4, obj. im. */2. oa Z of a me be”) i inal Epithelium of Bato. Mary A Bowers Journal of Anatomy. Vol IX. The American PLATE LY. Fic. 42. Duodenum. Tadpole No. V, Fig. 2. Cross-section. X 750. Fic. 43. Duodenum. Tadpole No. VI, Fig. 2. Detail of Fig. 20. Cross- section. X 750. Fig. 44. Cross-section of the posterior part of the small intestine of tadpole, No. VII, Fig. 2. Detail of Fig. 16. x 750. Fic. 45. Duodenum. Tadpole No. VII, Fig. 2. Detail of Fig. 17. Cross- section. xX 612. Intestinal Epithelum of Buto. Marv A. Bowers PLIV. MAS. del The American Journal of Anatomy. VOLIX. ON THE EARLIEST BLOOD-VESSELS IN THE ANTE- RIOR LIMB BUDS OF BIRDS AND THEIR RELATION TO THE PRIMARY SUBCLAVIAN ARTERY.* BY HERBERT M. EVANS. From the Anatomical Laboratory of the Johns Hopkins University. WITH 20 FIGURES. CONTENTS. PAGH PF ALUM UELO MU CCOLY KO5 é are | [ea] pea Left. | Right. 1 .. | 24 | Opposite | 0 10) |: eae 9th | ee: 2 | she 30 14th 0 1 (13th intersomitic | space : 3 60 32 20th 4 10 14th-17th cP Period of pri- 4 60 Pe ss 5 11 |14th-18th ‘“ mary subcla- 5 65 31 21st 5 6 15th-19th ‘“* vian capillary 6 60 a3 ee 4 6 15th-19th ‘“ plexus. uf 66 | 33 e 3 4 |15th-18th “ Period of mul- 8 72 | aie 4 4 che ie y tiple segmen- 9 72 | as Completely 3 4 /|16th-18th ‘“ tal subcla- 10 72 34 Ss 2 4 18th-19th es vians. : 11 78 36 se 2 2 |18th-19th “ Period of pri- 12 80 | 45 1 2 |18th-19th mary subcla- 13 84 | Se fa 1 1 Sts ee ay ce ee ee ee oe ee ee 16 116 te : 1 1 VS ehaeey mes 17 70 38 21st 5 6 |16th-20th ‘“ if It will be seen by referring to the table that the definitive primary subelavian artery is at the level of the eighteenth inter-somitic space. It is a branch of the dorsal segmental vessels of that interspace. My injections, controlled by careful study of serial sections, show that in the embryos embraced by the table, 7. e., in chicks possessing from twenty-four to forty-eight somites, the first interspace (7. e., Earliest Blood Vessels in Anterior Limb Buds. 291 that between the first and second myotomes) is not occupied by a dorsal segmental artery. The series of dorsal segmental arteries in these embryos begins with the vessel present in the interspace between the second and third somites. Consequently the vessels present in the eighteenth interspace are really the seventeenth pair of the series actually present, and I have so labelled them in all the drawings. It is to be borne in mind, then, that the seventeenth segmental vessels of the figures are in the eighteenth inter-somitic septum. Inasmuch as the first four somites of the chick are to be considered cephalic, rather than cervical, in their ultimate fate, and as the third actual seomental artery later courses near the first cervical nerve, and is hence the first cervical artery, the primary subclavian artery arises from the fifteenth cervical segmental artery, a vessel occurring in the eighteenth interspace and the seventeenth of the series actually present. There are five periods in the history of the bird’s subclavian and not four as Rabl maintained. These may be briefly enumerated as follows: I. Period of capillary outgrowth from the aorta forming a pri- mary-limb plexus, not influenced in its arrangement by metamerism. Il. Period of multiple segmental subclavians, a condition result- ing from the atrophy of all capillaries in the preéxisting plexus not at segmental points. III. Period of the establishment of the primary subclavian artery from the persistence of one of the pairs of segmental subclavians— v. e., that of the eighteenth segment. IV. Period of double arterial supply through contemporary ex- istence of dorsal and newly arisen ventral subclavians. V. Period of enlargement of the permanent channel, the sec- ondary subclavian, and coincident atrophy and disappearance of the primary vessel. The last three periods or phases were described by Hochstetter and Sabin, the second period, in which segmental subclavians exist, by Rabl and Miller, the earliest or first period for the first time in the present study. 992 Herbert M. Evans. It will be seen that the embryos included in the table presented comprise only those in the first three periods or stages of the devel- opment of the subclavian artery Embryos 1 and 2 show some early steps in the downgrowth of the primary body-wall plexus, the system forming the latter, umbil- ical vein. Embryo 1, with twenty-four somites, shows this mesh of eapilla- ries extended caudally in the somatopleure to the level of the ninth inter-somitic space. In embryo 2, with thirty somites, these capillaries have reached a point opposite the fourteenth interspace. In addition, from the aorta itself several capillaries have now grown out, one of which has joined the main plexus at a point near the thirteenth inter- space and its dorsal segmental vessels. These apparently unim- portant endothelial sprouts are but the first of a considerable series to grow from the lateral aortic wall, and though there is as vet no external indication of a limb bud, they are probably to be regarded as the first limb capillaries. In the next succeeding stage, embryo 3, of thirty-two somites, a slight swelling of the somatopleure constitutes an infinite, yet un- doubted, limb bud, and we find a considerable row of these aortic or rather subclavian capillaries. A glance shows that these vessels are not segmentally arranged (Figs. 2a and 2b). In such injected specimens one may look down on the prepara- tion as a whole and examine carefully any area. No reconstruc- tion is necessary; no doubt about relations exists, for the entire picture is spread out before one. The dorsal segmental vessels stand out sharply and it is easy to determine the relation of these to the new subclavian capillaries. It is impossible to say that the latter vessels are determined in position by the former for the whole appearance given is of a profuse irregular outgrowth of capillaries which form a simple plecus. On the right side, as the figure shows, Fic. 1.—Chick embryo of 30 somites (embryo 2 of table). Showing downgrowth of primary wall plexus, x 53%. Ant. Card. V., anterior cardinal vein. Ext. Jug. V., external or inferior jugular vein (linguo-facial vein). Post Card. V., posterior cardinal vein. P. b. w. p., primary body wall plexus. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS. HERBERT M. EVANS. AnveirdV. Extdue V : € Duet of Cuvier : Umbilical V. Post Gard V- , PTiersth, Teter Fig. 1. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. - me rt wh - i) q ro i = - i. i - aa _ ; = - ys 7 Le we Vee >» ‘ . re Nery Pe: ve , te, fey

mi a: ‘ Ug. a Post Gard Vein) —------>--+ ~ [5 ede eee Ree: BEE ne * ‘ Ges Dorsal Interseq. Vein. iG SS 13 Aortic Capillary. \. —csnnnee-o- eae Sh s ¢. r) Or Subc aie = ™) eN no [6 2D, | Yorn YF nde Plexus OF OriginGt - Xaae ee ES | R* 38 &F4Svbclavans. ©. 17% Dorsal Interseg Vein o% /8* Dorsal interseg. Vein. ee 7 Venvies Opening,._____----—---- pe fe eee ae into Post Card. 2 | (9% Dorsal Interse g.Vein-—------- --4 G Vic. 7.—Right wing bud of chick of sixty hours incubation (embryo 6 of table), showing primary subclavian capillary plexus, x 531%. in the development of the subclavian, however, for the majority of the vessels are at unsegmental points. With the existence of so large a proportion of the vessels out of harmony with the segmental plan, I think we can hardly classify this embryo as in the period of segmental subelavians. Of the four subclavians present on the right side, the first arises opposite the sixteenth dorsal segmental vessels, the second midway between the sixteenth and seventeenth segmentals, the third exactly opposite the seventeenth, and the fourth Earliest Blood Vessels in Anterior Limb Buds. 299 about half-way between the seventeenth and eighteenth dorsal seg- mentals. Thus two of the subclavian arteries on the right side are true segmental vessels—those opposite the sixteenth and seventeenth dorsal segmental arteries—but an equal number, two, are completely out of harmony with the segmental plan. On the left side, the first subclavian is opposite the seventeenth dorsal segmental vessels, the second about midway between the seventeenth and eighteenth, and Spinal chord: Myolome. Notochord Ps PostCard.V, Umbilical Vein! Fie. 8.—Cross section of chick of 38 somites in region of anterior limbs and midway between the sixteenth and seventeenth intersegmental vessels. The section shows the fourth right and the second left subclavian. ‘The section shows clearly the peripheral limit of extension of the limb capillaries and the nonvascular, marginal zone. U. V., umbilical vein. Post. Card. V., Posterior cardinal vein. the third subclavian considerably below the interspace belonging to the eighteenth pair. Here, then, only one of the subclavians is a segmental vessel, and of the total of seven vessels but three are segmentally placed. The umbilical vein has been appreciably ex- tended considerably below the region opposite the limb bud by a still further caudal downgrowth of the primary body wall capillaries. Opposite the limb, it receives on each side about ten distinet tribu- taries, the upper ones, especially, being no longer capillaries in size 300 Herbert M. Evans. but small venules. Thus the earliest drainage channel for the limb does not lose this function during the next succeeding stage, but instead becomes increasingly important as the chief vein of the limb. : I have presented two typical sections through this embryo in the arm region since these will answer well for the relations thus shown in all the embryos belonging to the first subclavian period. Fig. 8 shows a cross-section through the embryo at the region of origin of the fourth right and the second left subclavians. Both vessels are unsegmentally arranged and hence the dorsal segmental vessels do not appear in the section. While on the right side the subclavian arises from the dorso-lateral angle of the aorta, the left vessel emerges from the true lateral side of the aortic wall, and only a short distance above the origin of the nephriec capillaries, one of which is shown in the section. Thus in the early stages the place of origin of the subclavians from the aortic circumference varies considerably, and Fig. 4, showing the first left subclavian in this embryo, indicates how far laterally the early vessels of the subclavian series may arise. In this instance the subclavian is almost a mid lateral derivative of the aorta. Such vessels must curve dorsally in crossing the posterior-cardinal vein to reach the tissue of the limb, but the early dorso-lateral branches all course in a straight transverse line.* The character of the dorsal-segmental vessels and their capillaries has already been mentioned. A review of the table which has been presented, shows two features of interest in connection with these earliest stages in the vascular- ization of the limbs. I refer to the high position of the first sub- “Rabl has emphasized this straight course of the early subclavians, pointing out that Sabin missed it, for the stages which the latter studied were all old enough to show the dorsal bend which the subclavians then take in reaching the limbs. This dorsal bending is assuredly a secondary bending of an original straight vessel; but my own specimens have disclosed a number of the very earliest subclavians arising from so low a point on the lateral aortic wall that a primary arching course is necessary to reach the limb tissue. It is not unlikely that these subclavians disclose the more primitive place of origin of the subclavian series, for they do not occur in even slightly older embryos. Good justification thus exists in considering the subclavians as primarily true lateral branches of the aorta. Earliest Blood Vessels in Anterior Limb Buds. 301 clavian capillaries—their origin in the neighborhood of the twelfth and thirteenth segments—and to certain differences in the vascular- ization of the right and left limbs. Rabl’s studies indicated that the subclavians of later stages arose at successively lower levels from the aortic wall. The injec- tions here reported, however, indicate a more cephalic extension of the subclavians than had been previously suspected, and so extend even more the “wandering” of the upper limb and its vessels. It appears that the right limb bud is the first to receive capil- laries, and that this limb in the early stages always possesses a greater number of subclavian capillaries than the left limb. Apparently the two limbs are identical in their relations to the body and in the conditions with which they have to deal in their development save in one respect, namely, that at the time of origin and earliest stages of the limb buds, the embryo is always resting on the left side. This causes a slightly more flexed position of the under or left limb-bud with reference to the body wall and permits a some- what extended, freer projection of the uppermost right limb. This may be related to the greater speed and profusion with which the first vessels grow into the right limb. But in summing up the condition found in the five embryos illus- trating the first period in the development of the subclavian, noth- ing is more striking than that we have to deal here merely with an w- regular plexus of true capillary vessels which are in no way related to a segmental plan. Thus if the chance arrangement of any irregu- lar capillary plexus obtains here, it should happen that as many of the vessels arise from non-segmental as from segmental points, and this is actually the case. Empryos oF THE Seconp Periop. The first embryo classed in the stage of segmental subelavians (Embryo 8), has almost as high a proportion of non-segmental ves- sels as has Embryo 7, but two significant changes have occurred. These consist in the purely rudimentary character of the non-seg- mental subclavians and the enlargement of that pair of segmental subclavians opposite the eighteenth pair of dorsal segmental ves- 302 Herbert M. Evans. sels. The figure (Fig. 9) plainly indicates this. On the right side, the segmental subclavian opposite the nineteenth pair of dorsal segmentals is somewhat larger than the uppermost atrophying, non- segmental subclavians of that side. Some influence, then, favors the subclavians at strictly segmental points (7. e., opposite the inter- somitic intervals) and is inimical to the growth of those not so situated. Of the segmental subclavians, one, doubtless for purely hydrodynamical reasons, begins to be the chief supply of the limb. Embryo 9 of the series shows a most interesting condition (Fig. 10). Here all but one of the subclavian series persisting are approximately in harmony with the segmental plan. On the left side, excepting the main vessel, the only subclavians which have survived are those at true segmental points. Thus the dorsal seg- mertal vessels have opposite them at the sixteenth and seventeenth interspaces, two delicate segmental subclavians. The main subcla- vian artery on this side docs not arise at an exactly segmental point and wt is formed by two frequently anastomosing vessels, arising some- what in front of the eighteenth interspace. Doubtless this channel is to be shifted by unequal growth and be incorporated with the eigh- teenth dorsal segmentals in a later stage. The right side has two vestigial vessels, which no longer reach the limb tissue, and a larger channel opposite the eighteenth segment and constructed here also not from one but from several preéxisting capillaries. Embryo 10 possesses thirty-four somites, and has some six sub- clavians, four on the right and two on the left side. On the former side, two of the persisting subclavians are segmental vessels, exactly opposite the eighteenth and nineteenth dorsal segmentals, but the remaining two are non-segmental and occur in the interspace be- tween the former two. These non-segmental subclavians are, slrangely enough, large trunks, equally as large as the true segmental subclavians, plunging into the core of the limb and being important Fic. 9.—Dorsal view of anterior limb buds and their vessels in a chick of seventy-two hours incubation, « 531%. 15th D. I. V., fifteenth dorsal intersegmental vein, 7. e., that of the sixteenth interspace. Trans. Subel. Art., transitory subclavian artery. Chief Prim. Subcl. Art., chief primary subclavian artery. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS. HERBERT M. EVANS. ----Umbbilical Vein Actes ~.. 19% Dorsal InferSeg. Ves Poat Cara. Vein. Fie. 9. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS. THERBERT M. EVANS. 18D.1LN=-----= Fic. 10. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. Earliest Blood Vessels in Anterior Limb Buds. 303 arterial sources in the limb’s circulation. On the left side, both of the subclavians existing are true segmental vessels, at the eigh- teenth and nineteenth segmental points. The cross section (Fig. 11) shows the pair of segmental subcla- vians corresponding to the eighteenth segment, and the correspond- ing dorsal segmentals. The subclavians arise at the dorso-lateral angle of the aortic circumference and, indeed, in a slight local bulging of the aortic wall, from which the dorsal segmental vessels also take origin. The limbs do not project laterally as before but are bent in more, parallel with the main body axis. The aorta is elongated dorso-ventrally with a slight compensatory lateral nar- rowing more marked ventrally so that in section the whole vessel now appears triangular. The dorsal segmental vessels are still con- fined in distribution to the spinal cord and chiefly to its lateral aspect. Neither the dorsal nor the ventral surface of the cord are yet suppled with capillaries though these vessels have begun to extend over both of these surfaces. The highest tributaries of the segmental veins are thus now somewhat above the dorso-lateral angle ot the cord. The remaining embryo of the second period—the period of seg- mental subclavians—is Embryo 11, with thirty-six somites and two segmental subclavians on each side, those of the eighteenth and the nineteenth segments; but the latter vessels are now mere ves- tigial rudiments. The common origin of the dorsal segmental ves- sels and the subclavians is somewhat more pronounced. I need not dwell longer on the four embryos which belong to the period of multiple segmental subclavians. The accounts of FIe. 10.—Dorsal view of anterior limb buds and their vessels in a chick of seventy-two hours incubation, x 5314 (embryo 9 of table). Trans. §. A., transitory subclavian artery, here opposite the sixteenth dorsal intersegmental vessels. The figure shows an interesting stage in the evolution of the limb’s vessels. The original subclavian capillaries are now chiefly represented by those at intersegmental points, 7. e., the so-called ‘segmental subclavians.”’ But even here there are atrophying and the chief primary subclavian arteries remain. The latter vessels happen to be constructed from several contiguous subclavian capillaries rather than from a single one as is usually the case. 304. Herbert M. Evans. Rabl and of Miiller have already sufficiently emphasized this inter- esting stage in the limb vessels. We have seen that most of the subclavian capillaries arising from the aorta at non-segmental points eventually atrophy, and there now remain only the vessels which stand opposite the segmental interspaces. Thus are produced the segmental subclavians, a truly metameric arrangement of the limb vessels. Two features of some importance in these stages have been pre- viously overlooked. ‘These are: 1. Abundant traces of the earler capillary plexus stage of sub- clavians occur in the period of segmental subclavians. These con- sist in several smaller or atrophying vessels of the subclavian series which do not stand at segmental points. Such vessels are often present to complicate the picture of the segmental subclavians, espe- cially early in this period. When the stage is reached in which the multiple segmental subclavians are carried up as common branches with the dorsal segmental vessels, these non-segmental rudiments rarely persist longer and we have at length a perfect picture of mul- tiple true segmental subclavians. Most of the non-segmental subclavians of this stage are delicate vessels, but it occasionally happens that some of them are larger sturdy channels of equal value with the segmentals. This was the ease, for instance, in Embryo 10 of the series. It was thus sur- prising to me that Rabl had not found such vessels, but a careful rereading of his descriptions shows that he doubtless saw some in- stances of them. He attached a peculiar significance to them, how- ever, conceiving that they came about through a splitting of a pre- existing single segmental vessel, thus forming a double vessel whose roots wandered apart! It was quite impossible to him that the subclavians should arise at other than segmental points. Even the eases of “insel bildung’ he would make come through a similar splitting of single vascular channels. 2. The second point which I wish to make is that it must ocea- sionally happen that even the vessel most favored in the row of sub- clavians may not be at first at an exactly segmental point as Fig. 10 plainly showed. Earliest Blood Vessels in Anterior Limb Buds. 305 The effect of the intrusion of a metameric influence in the plan of the limb’s vessels is as plainly marked in the case of its veins as in the arteries, for of the row of venules entering the posterior cardinal vein, those at segmental points are often definitely larger than the remainder. Thus segmental veins as well as arteries exist. Fic. 11.—Cross section of injected chick embryo of 34 somites in the region of the anterior limb buds (embryo 10 of table), D. 8S. V., seventeenth dorsal intersegmental vein. Sp. G., spinal ganglion; N. Ch., notochord; P. C. V., posterior cardinal vein ; S. C. V., subeardinal vein; U. V., umbilical vein; D. S. A., seventeenth dorsal intersegmental artery (7. e., that of the eighteenth interspace) ; Subcl. Art., subclavian artery; N. V., nephric vein. The fact that the veins draining more ventrally into the umbilical vein are not effected by the segmental plan would indicate that a metamerism does not pervade the entire limb tissue. One feels that the segmental arrangement of the arteries and more dorsally placed veins is the direct result of the influence of the adjoining myotomes. I must comment here on the embryo listed as No. 17 in the series. 306 Herbert M. Evans. Post Card.V. Primary Sub. A. e eX Fic. 12.—Right wing bud of chick of 45 somites, x 538%. thDor Subcl Art Boat SBbSE i8Dor Seg Art. Fic. 12b.—Right and left wing buds of a chick of the fifth day. Earliest Blood Vessels in Anterior Limb Buds. 307 I have placed it there since it is the only one which does not fit well into the series, for though its age and number of somites would indicate a more advanced scheme in the vessels of the limb, I found here no less than six vessels of the subclavian series. The embryo may be viewed, as an instance, in which the limb and its vascular system has run slightly behind the normal for this age, or as a case of the persistence of multiple non-segmental subclavians. I have drawn the embryo, as a whole, since it shows splendidly the typical relations of the early limb capillaries and those belonging to the dorsal segmental series (Fig. 3). Prriop oF Primary SUBCLAVIAN ARTERY. Embryos 12 to 16 all illustrate stages in the growth of the pri- mary subclavian artery. The relation of this trunk to the preéxist- ing segmental subclavians, has already been clearly indicated in Embryo 8; even there we saw the early exaggeration of one of the members of the subclavian series. The processes of vascular atrophy and death which early eliminate the original non-segmental sub- clavians, destroy also eventually the segmental subclavians with the single exception of that vessel destined to become the primary sub- clavian artery. It is of interest that even in these late stages, there sometimes persists a non-segmental artery. Fig. 12 gives an instance of this. It is the Embryo 12 in the series and possesses some forty-five somites. The large primary subclavian artery has below it and near the middle of the adjoining somite, a narrow rudimentary vessel which has persisted from the primary subclavian series. True segmental vessels, other than the main one, may likewise persist in limbs of this age. This is possible through the early proximal anastomoses between subclavians. The use of several-of these paths will give several segmental roots of origin to the primary subela- vian trunk. Fig. 12b, isa striking instance of this. In embryos of eighty-four and ninety-six hours incubation, the primary subclavian artery has attained a large size. The dorsal segmental vessels have also increased in caliber. The capillaries belonging to the latter system have surrounded the spinal cord com- 308 Herbert M. Evans. pletely and grown out as a loose plexus over the outer surface of the myotomes. Fig. 13, from the embryo of 116 hours (No. 16) show the fur- ther growth and elaboration of these changes. The common trunks of the subclavian and dorsal segmental vessels, are themselves being shifted toward the mid-dorsal line, soon to arise from a single com- mon trunk. The segmental arteries and veins have each two main systems of branches which alternately supply and drain the cord at successive points around its circumference. Penetrating arte- ries extend from the ventral arterial tract into the cord substance at the boundary zone of the neuroblasts and ependyma. They are drained by delicate transverse venules. The subclavian arteries have two small branches before supplying the limb proper, a dorsal branch which supplies the outer capillary plexus over the myotomes and a ventral twig to the Wolffian duct. The subclavians are large vessels and control the blood supply to the limb. They must be considered now at the height of func- tional activity, and with this stage in the history of the primary subclavian the present account. closes. IV. OsserRvATIONS oN THE ConpbiITIONS PRESENT AT SIMILAR Stages In EmsBryos or THE Duck. Rabl’s research on the development of the subclavian artery was conducted ‘entirely on ducks. In it he failed to find stages earlier than the period of strictly segmental subclavians. It was conse- quently of some importance that these forms be investigated to see if the early subclavian capillary plexus which was present in chicks was not of fundamental value and hence present here also. Of a series of ten of these duck embryos I shall describe carefully only two typical ones, one, an embryo of thirty-three somites in the stage of an irregular subclavian capillary plexus, and the other, Fic. 13.—Cross section of chick of one hundred and sixteen hours incubation, in region of fore limbs. My., myotome; Dor. Vein, dorsal branch of the segmental vein; Post. Sp. Art., branches of the segmental artery which con- tribute to the formation of the posterior spinal artery; Pen. Art., penetrating artery; P. C. V., posterior cardinal vein; Se. V., Seitenrumpfvene, thoraco- epigastric veins; Rad. Art., radicular artery. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS. HERBERT M. EVANS. MYNGe aniies ier oer oy BONG Se ae Post Sp Arf——_— ——— ——§ ie en, Artie -—-P. Sp Art - Vent Vein --= = -— == — fe — 2 Rad Art Seg Art-— 5 ey : Fia. 13. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS. HERBERT M. EVANS. Fia. 14. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. a = meena oe Prim Cap.Plex of Limb. Earliest Blood Vessels in Anterior Limb Buds. 309 an embryo of thirty-eight somites, belonging to the period of seg- mental subclavians. Both embryos illustrate strikingly the facts observed in these stages for chicks. Duck Empryos. Number of Subclavians’ present. No. Somites present. Left. | Right. i Be Eola ic's 0 5 CUO ene 6 | 6 2 OS OE. teeta eie no onda, e Ay ceat eee CIP omer ger 5 6 | The duck embryo of thirty-three somites was drawn carefullly from several aspects before being cut into serial sections. A gen- eral view of the region of the anterior limb is shown in Fig. 14. One sees clearly the irregular venous channel through the remains of the primary body-wall plexus, leading now from the capillaries of the limb to the duct of Cuvier. The umbilical vein in the duck has an exactly similar origin as in the chick, and is here also the primary drainage channel for the limb. Between the sixteenth and twentieth dorsal segmental vessels an outgrowth of limb capillaries from the aortic wall occurs. There are six of these vessels. They anastomose promptly and form a continuous irregular capillary plexus extending into the limb, which is as yet a mere swelling of the somatopleure. No one capillary of the subclavian series is larger than its neighbor. The series is in no way arranged in a segmental plan. Cross sections through the embryo show the topography of the limb region. JI have drawn one which shows strikingly the hith- erto undescribed origin of these earliest subclavian capillaries from the mid-lateral region of the aortic wall. They are compelled to bend dorsally in growing into the limb bud. The section (Fig. 15) shows the fourth left and the sixth right subclavians. Neither vessel happens to arise at a segmental point. Iie. 14.—Upper body wall of duck embryo of 32 somites. Lettering as in previous figures. The wall of the aorta is concealed behind the vein. 310 Herbert M. Evans. There are six subclavians on either side in this embryo. The first subclavian on the right side occurs midway between the six- teenth and seventeenth dorsal segmental vessels, the second subcla- vian opposite the seventeenth segmentals, the third somewhat beyond this point, the fourth just in front of the eighteenth dorsal segmen- tals, the fifth somewhat beyond this point, and the sixth midway between the eighteenth and nineteenth segmentals. SpCh--—- : >”) Vein N.Ch- Fg —--Subcl. Art Suecl. Art---- , > =-— Nep Art Fic. 15.—Cross section of the duck embryo shown in Fig. 14 in the region of the anterior limb buds. One notes the midlateral origin of the subclavian capillaries from the aortic wall. On the left side, the first subclavian arises opposite the seven- teenth segmental vessels, the second midway between these and the eighteenth vessels, the third just in front of the eighteenth seg- mentals, the fourth midway between the eighteenth and nineteenth segmentals, the fifth at the level of the nineteenth segmentals, and the sixth midway between the nineteenth and twentieth segmen- tals. It is impossible, here also, to see in the arrangement of the sub- clavians any influence of metamerism. There are as many vessels out of segmental alignment as are in accordance with it and this because, again, in the origin of the typical plexus here as many Earliest Blood Vessels in Anterior Limb Buds. St capillaries should chance to be opposite the intersomitic spaces as are opposite the somite masses and vice versa. Duck embryo 2, possessing thirty-eight somites, happens to have almost as many subeclavians as occurred in the younger embryo, but in the older stage, besides being larger, these vessels are almost all at segmental points, so that the embryo belongs clearly to the period of multiple segmental subclavians. On the right side, there are six subclavian vessels arising from the aorta. The first subclavian occurs just in front of the six- teenth segmental vessel, the second and third at the level of the seventeenth segmental vessel, the fourth and fifth at the level of the eighteenth segmentals, and the sixth halfway between the eighteenth and nineteenth segmentals. On the left side, the first subclavian stands opposite the sixteenth segmentals, the second opposite the seventeenth vessels, the third opposite the eighteenth segmentals, and the fifth opposite the nine- teenth segmentals. Thus there are on the left side as many as four segments represented by subclavians. However the study of even this embryo, with such a complete series of segmental subclavians, shows that here also there persist some vessels not in segmental alignment. The last subclavian on the right side is such a vessel, for it occurs midway between the eighteenth and nineteenth dorsal segmental vessels. The cases of two subelavians existing opposite a segmental point are easily ex- plained by the chance origin of two of the early capillaries opposite one of the inter-somitic clefts. In such cases both vessels are equally favored, and both persist to the stage of segmental subcla- vians, where they increase the number of vessels to be expected. 1 have no doubt but that the condition in this Embryo 2, was pre- ceded by a stage of some ten or twelve subclavian capillaries, similar to those seen in chicks 3 and 4, but these interesting stages are so transitory in character that it is only rarely, that we have the good fortune to see them. Some capillaries, here as elsewhere in the developing vascular system, push out, function slightly and die in a surprisingly short time. ole Herbert M. Evans. V. CoMPARISON WITH THE PostTERIOR Limzg Bup. It was of great interest to ascertain whether the leg bud in the embryo had a similar capillary plexus from the aorta in its earliest stages. Such was actually found to be the case. Fig. 16 shows the hind limb buds in a chick embryo of thirty-two somites, No. 3 of the series. One may see distinctly the dorsal segmental vessels and in addi- tion, independent lateral offshoots from the aorta. At this time in the leg, the posterior-cardinal vein has not yet extended there, and both the dorsal segmental vessels and the lateral capillaries anasto- mose in the tissue of the limb and furnish its primary plexus. This plexus is dorsal to a more ventral plexus which arises very early; in fact, with the formation of the lower aorta, and is not to be con- fused with the latter. The injections demonstrate the later origin and growth of the sciatic artery from this mesh of capillaries, so that we have to do here with an exactly analogous condition as occurs in the upper limb’s vessels. In both cases, the chief axial vessel of the limb is preceded by, or may be said to exist in the form of, a simple capillary plexus arising directly from the aortic wall. VI. OssERVATIONS ON THE Earty MammMatian Arm Bop. At the present time we have the most complete history of the — earliest limb vessels in the birds, but it is naturally of the greatest interest to compare these findings with the conditions obtaining in mammalian embryos. Little is known of stages preceding the single axial subclavian in the latter class, save the two human embryos listed by Keibel and Elze, and the instance of early segmental human subclavians de- scribed by the writer. I accordingly undertook a series of injections of young mammalian embryos, choosing on account of the abundance of the material, Fic. 16.—The caudal end of a chick embryo of 32 somites (embryo 3 of table), showing the primary capillary plexus in the posterior limb buds. 26th Dor. Seg. Vein, twenty-sixth dorsal segmental vein, 7. e., that in the twenty-seventh interspace. EARLIEST BLOOD VESSELS IN ANTERIOR LIMB BUDS. HERBERT M. EVANS. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. Earliest Blood Vessels in Anterior Limb Buds. 313 embryos of the pig. Embryos young enough to show the earliest conditions in the arm bud are not common, and to supplement this material I have been fortunate enough to examine several perfect series of rabbit embryos from the Harvard Embryological Collec- tion through the kindness of Professor Minot. The latter embryos are all the more interesting since they were the types chosen in the compilation of the “Normal Plates on the Development of the Rabbit”? by Minot and Taylor.’° Thus the stages of development ee (th G oraz | Zt ANC, ——. . er oule oe o> SS = sab a ‘ey nay Cine / { Fic. 17.—Reconstruction of the position and course of the segmental sub- clavyian arteries present in a rabbit embryo of the tenth day, No. 559 Harvard Embryological Collection. may be accurately known from the various details listed opposite them in the latter, work. They comprised embryos designated as Nos. 562, 559 and 556 and in the “Normal Plates” are given the table numbers, 10, 11 and 12. I shall describe these very briefly. Embryo 562 has the limb buds as mere swellings of the soma- topleure. On the left side, one could not be certain of the existence of any subclavian capillaries, but on the right side, a subclavian is present midway between the sixth cervical and the seventh cervical segmental vessels. Minot and Taylor. “Normal Plates on the Development of. the Rabbit.” In the series edited by Keibel. 314 Herbert M. Evans. Embryo 559 has three segmental subclavians on the right side and but one on the left. The right subclavians arise from the seventh and eighth cervical and the first thoracic segmental arteries. The left subclavian is a branch of the seventh cervical segmental vessel. Fig. 17 shows a reconstruction of the subclavians in this embryo. Embryo 556, slightly older, possesses only a single subclavian on the right side, that of the seventh cervical segment, but two segmental subclavians on the left side, those of the seventh and eighth segments. In both the latter cases (Embryos 559 and 556) the subclavian arteries are already branches of the dorsal segmental vessels, but in the earlier case (No. 562), in which a single non-segmental sub- clavian existed, this was obviously not the case. There is every reason for believing that this youngest embryo is in the first stage of development of the subclavians, and one feels that the study of more mammalian limb buds at this stage will show more segmental and non-segmental subclavian capillaries.'® Not only in its arterial but also in its venous system does the early mammalian arm bud agree strikingly with that of the bird. In mammals, also the first and most important drainage channel for the arm is the umbilical vein. Figs. 18 and 19 show the position and character of the venules which drain the early mammalian arm bud into the umbilical vein. The uppermost or cephalic portion of the mammalian umbilical vein has long been known to persist for a considerable time as a much attenuated channel, still connect- Since these observations were made, Goppert has published an account of the early blood vessels in the arm buds in white mice and his reconstruc- tions bear this out. He has shown striking instances of a segmental sub- clayian series, with segmental and non-segmental members, though he does not realize the significance of the latter vessels. Goppert is disposed to view this merely as an evidence of variability in the embryonic arterial system. He has missed the key to the solution, however, for we are dealing here, as my injections show, with the persisting members of an early ir- regular, capillary plexus. In such fleeting phenomena as the outgrowth and regression of many of these capillaries, we must expect to see embryos from the same uterus in slightly different stages of development. ‘There is as good reason for this interpretation, surely as there is for his of vari- ability. Goppert, BE. ‘“Variabilitiit im embryonalen Arteriensystem.” Verhandlungen der Anatomischen Gesellschaft, Anat. Anz., Bd. XXXII, 1908, pp. 92-108. Earliest Blood Vessels in Anterior Limb Buds. 31 Or ing with the duct of Cuvier after the main vessel has established connections through the liver. Yhe reason for this persistence of the old upper portion of the umbilical vein is now clear, for at still furnishes an important drainage channel for the arm bud. Thus in pig embryos 714 millimeters long this cephalic part of the umbilical vein still receives some seven or eight tributaries from 5h. Jaaves, ieSy ad et ea Fie. 19. Fic. 18.—Lateral view of pig embryo 6 mms. long, showing drainage of arm bud into umbilical vein, < 11.2. Fic. 19.—Detailed view of venules draining the left anterior limb bud of the pig embryo shown in Fig. 18, 331/.. the arm bud as shown in Fig. 20. The mammalian arm bud is also drained by a series of venules opening into the posterior cardinal vein. Summary or Resvtts. The chief facts brought forward in the present investigation may be summarized as followed: 1. The first blood-vessels supplying the limb buds are capillaries which grow from multiple irregular points of the lateral aortic walt 316 Herbert M. Evans. and anastomosing, often even before they reach the root of the limb, form a simple and quite typical plexus. In the arm bud, this capil- ‘lary plexus constitutes the earliest stage of the subclavian artery, in the leg bud, of the femoral artery. The first subclavian capil- laries, partaking of the character of any irregular capillary plexus, are thus never arranged in a truly segmental plan. 2. The subclavian capillaries join another plexus of capillaries, which has grown down in the body-wall from Cuvier’s duct—the Persisting Cephalic Portion mo rieNel n. ubcl Art. Fic. 20.—Lateral view of pig embryo 714 mm. long, showing the persisting cephalic portion of the umbilical vein still receiving tributaries from the arm bud. The embryo is drawn at the same magnification as that figured in Iie aera, chy SC TELA, primary body-wall plexus. The consequent establishment of a cir- culation from the aorta to Cuvier’s duct converts the subclavian capillaries into arterioles and certain of the primary body-wall capil- laries into a vein—the umbilical vein. In the birds, the drainage of the early wing bud is thus the sole primary function of the um- bilical vein. In the mammals, although the development of the umbilical vein in connection with the chorionic circulation precedes the formation of the limb buds, nevertheless, when the arm buds arise, their capillaries establish, here also, a drainage into the um- Earliest Blood Vessels in Anterior Limb Buds. 317 bilical vein. This drainage of the mammalian arm bud into the upper portion of the umbilical vein persists after the latter vessel has established its chief circulation through the liver and is doubtless one of the chief causes delaying the atrophy of the upper or cephalic portion of the umbilical vein. 3. The occurrence of a period of multiple segmental subclavians is brought about by processes of atrophy and doubtless slight shifting. Thus most of the primary subclavian capillaries which are not at segmental points, 7. ¢., opposite the interspaces between the somites, eventually atrophy, leaving as functioning vessels only those members of the early series which are fortunately situated in accordance with this plan. 4. Even during the period of true segmental subclavians, how- ever, there often persist some members of the first subclavian series which are out of segmental alignment. ‘These may indeed get to be vessels of some size. The chief primary subclavian artery itself may not at first happen to lie at exactly a segmental point. The chief determining factors in the persistence of vessels are doubtless hydrodynamical and only secondarily the influence of metamerism. 5. A purely segmental character in the arm vessels is finally secured at the time of inclusion cf the subclavian vessels as common trunks with the dorsal segmental vessels. This union is not a process of active fusion of the subclavian and dorsal trunks but is effected by processes of unequal growth which occur in the expansion of the aortic wall. Dorsal and subclavian arteries are carried out together by a local bulging of the aortic wall, which becomes a common trunk. 6. The primary subclavian artery, represents the exaggeration of one of the pairs of segmental subclavians, which is most favorably situated as the principal circulatory channel for the limb. VIII. Apprication or THEse Facts To THE GENERAL Em- BRYOLOGY OF THE VASCULAR SYSTEM. Two conceptions have arisen regarding the method of develop- ment of the vascular system. According to the one, arteries and veins grow out to their end beds as development proceeds, but accord- ing to the other, vascular activity is always initiated by capillaries 318 Herbert M. Evans. which tend everywhere to form a mesh-work or plexus; arteries and veins are always subsequent formations from such capillary plexuses due to the transforming influence of the circulation. The former conception would appear to be held by most of the workers in angiogenesis, though most of the descriptions of the devel- opment of vessels are so worded as to avoid a lucid statement on this fundamental point. I may refer, for instance, to the many admirable researches of Hochstetter, where, though many important facts concerning the chief embryonic vessels are clearly given, one may look in vain for anything bearing on this point. In the case of the limb vessels, for instance, we must imagine from his descrip- tion that the single axial vessel grew out into the core of the limb. Very recently Curt Elze'’ has ranged himself with those who would recognize such a process as the means of development of all the body’s vessels and as definitely opposed to the idea of a capillary plexus anlage for any of them. On the other hand, Hans Rabl and Erik Miller have supported vigorously the latter idea, the foundation for which had been laid in the great paper of Thoma on the origin of the chick’s yolk vessels. There is plenty of morphological evidence in the adult body for a preéxisting plexus or net-like condition of all the vascular trunks. The remarkable number of variations in the position, system of branching and anastomoses cannot be explained as satisfactorily on any other basis. Thus, without knowledge of conditions in the em- bryo, Aeby'® and Baader’? promulgated such a plexus origin for blood-vessels many years ago. Professor His did not hesitate to state that the main vessels in the embryo were derived from net-like anlagen, but it remained for Thoma”? to make the meaning of all this very significant. Thoma observed that in its early stages the system of the vitelline vessels in the chick formed a strikingly uniform simple plexus of “Elze. “Beschreibung eines menschlichen Embryo von zirka 7 mm. groésster Liinge.” Anat. Hefte, 1-35, 1907. *®Aeby. “Der Bau des menschlichen Korpers.” 1871. “Baader. “Ueber die Varietiiten der Armarterien des Menschen.” Inaug. Diss. Bern, 1866. “Thoma, R. ‘Untersuchungen iiber die Histogenese und Histomechanik des Gefiisssystems.”’ 1893. Earliest Blood Vessels in Anterior Limb Bud. 319 irregular capillaries. He observed that the fortuitous position of some of these capillaries with respect to the aortee and venous ostia of the heart gave them a more constant and rapid circulation than occurred in other capillaries of the mesh. Later, stages showed these capillaries became arteries and veins respectively. As the vitelline vascular system grew, Thoma saw the same laws at work from center to periphery, that the further elaboration of the arterial and venous trees was the result of successive incorporation of adjoining por- tions of the general capillary plexus. If these processes are at work everywhere in the development of the vascular system, they furnish us with a better understanding of angiogenesis, for the development of a given artery or vein to any portion of the body cannot be due to miraculous predestination but to the definite action of quite definite physical laws. Capillaries first invade a region and the rela- tion of these capillaries to the nearest arterial and venous channels determines always the manner in which the new veins and arteries shall arise. Then elaboration of arteries and veins is always the result of hydrodynamical forces involved in the circulation. The application of Thoma’s work to the development of the blood- vessels in the body of the embryo has never been adequately tested. The method of injecting completely the embryonic vascular sys- tem has furnished much evidence that the capillary plexus anlage can be demonstrated for all the body’s vessels. The preceding ac- count of earliest circulatory conditions in the limb bud gains much significance in this light, for before there can be said to be limb arteries or veins, a primitive plexus of capillaries grows into the limb tissue. From this plecus in later stages, artertes and veins are formed. Rabl has shown the origin of several arteries in the fore-limb region from capillary nets, but one must leave this interesting story, the development of the later vessels, to another time. In the present study, we have been able to see that in the limbs, the main vessels themselves—the femoral and the subclavian arteries—eaxist originally in the form of a capillary plexus. In conclusion I beg to speak with gratitude of the many sugges- tions and helpful interest in the present investigation which I owe to Professor Mall. ie 2 _— f ¢ nis x , hee e . t i . Or, ae ae ‘i * ms, _> A pis { - f 1 THE CUTANEOUS GLANDS OF THE COMMON TOADS.* BY EFFA FUNK MUHSE. WitH T PLATES. CONTENTS. PAGH Generale tOMUE OM: 56:5 ceceke, slate reve esse Sao orev OPeNGe alinte tet cretion ome eve NCGS TS elles cre 322 Rurposerand: plan Of MAPEr (5 vas cretoweerssetele otorek or cholerae tacts czas cyenoke Bye MM EOTRT EMTs Pesraver oe: ae: Os Wiaieis ooh: eb. Fh Becca SN ae te DR PONG oP eal oc Reena -are fer rare ae 322 IMGT OMS sis store G sosne > oe avers.s a ee Dk RI ee Oh REE Cones Serer 323 1 Dal SENET UTS ene ECR MRE Re AEE RMN Teese ete a As cease ah acne carey DIC road OFC 324 GeneraleDescription, of they Skint feacae naar seis eee eee ceil aerials 324 WMEVEMMESS Bi ste ceci sors. o.c:ei ore di cuatausleneyere lela wuemaieltesevnreve, schersbremeveis sielegeve afelorteys 324 CAMS OS REF Ae end bsc siete ahs sohe caitel eo MET ene nate Peet raxete eichss se PR Ea cere nae one ate hetane 324 CIASSES” OF Wills sc csdeG acolo crore ate eveltoy oon een they ral oaks MCT ont onsn es etstn acai 325 GCOLOPATTOM Tas Sicsce a arene yer ares aie oer chs ala acs aco eters ls Gy alae ol lett ererevate: scorer CV omeeneeae 326 COLO T Mate oh cet letshas esate cg Cae ag ie ITA el ole Gr OCR aor eta ere 326 ATTLAN SEMEN OL SOUS). sie yscccescews (orersbelie opetere lap ciave) skeyobeel siretehss ounues otehetorciere 326 Relation Of warts nOuspotsr 2 ato4 ses eects aiens ova cua sv atote casei ouelic suenewe.ee 327 CVS ES OIG CLIMIS ap to syasoge se renee ct teele ii oho Suck gie cue) a; tate aerehieue jason, Rie Wieuwee oheteiekenetets aval SUTRA ew ras rote exSucte Tore he fats tol Coo ew: Secs ler syeraeare ns oP ONES ie ete eae ree) oh Sosee Sten Oe eae ee onl IBGaleOnuGOll Se. fesse ster e atedeversraes chacw opel Wore, ciertererae eget onevesers sie due ttavncw ae wile acevedave 328 Hpidermis relative to the regions of the bodiyeaerer it otoaeticsasnaees 328 dD) aesel Cold EST on eee oC otc chop renee Pon hc ker Cp OSCR Ee REI Ree iy cae aceite eMe ae eee hia tec 329 SET AL LEU rete cafarret cae crevel oracles tire eosin Taha or oeek aura ate taaeatil ood Rabel oteuclam bis lotie w Gece 329 Culisyaccordin eso NESlON See cr.ces sereieneva evo ores exerciser cisieres ae eloleieioeiaa’s 330 Cutaneouss GlandsVorsthe foad—Imtroduchiony esses oeeeicice c+ cee ee 331 BAVET AUT OW cctevectca cuca eaters iar avobensvsk trea chan. sie Saray suet chuiere aetna woeia arorereteale co ailers 332 Secretionvandnwihenunexpelledire.cvcysor eras sielerge set ettols sisters cteis: eke eles wreuereie ere 334 Orieinwor- sland Si imeem aval vases < cis ches’. crshe le “oye 61.6. crovererons) suey avers oraloiene ae 334 Gradationsofvelands inthe va duly = 2yeet). viset ale a © crereovel so icles se clevertelas 335 IB AbbeLleSnOt SAMS rs o.«chetcrsissve.c 1s lelehs\ cnciere uate ee wieke wie Megevoke,s sloreretets . 000 hes Malte eG lenny Gis csees yer cus sacs hetero oa si erie 0) wiley ole eos i8Vey Gre aired eee. er ioveureney abo eveks 336 VV SO tes tINe MANCHU GS: tar paveueverels Ya uehs cheer ciate orth eo see. Sclerpneter sa feleem wy one 337 IVES Cle wiI CT: Sees iegets ye pare cecreo et craye rarer tcl arene orto euevene.e or os, Sosa sansa sickens 337 TOME) UIN Nese ar ce hcp once cceeeek «cartes 6 oie lie iiss ai00. 0 ey sisi oe eta oubo ei aceravepaneie: osreteks 338 S CELE IO MMe rer rarer sea cies wn as aie oheyolel oie) cma iol czevernie-shreliet alievery.castranovetouets, sy srononerees 339 GLI SOU trae. rowers ene custose colic: Ss suSos eee olvena) al el vite ve/(e eirosvereel- caer evenel susteneneta rs 340 ; (CON COMETEICHZOMES vet toa loos evaveveiers Der eus sie Diels eo reherete oie Eee ore 340 Cogewireeliker SEW CCUTEr s,s. tis siete: «1/0, ovenens (MGs oi oatallere. te tekedeee ee onatens te 341 IDIKER AG & coptateno Bio teriec cea een nce een Oar GS, Ci is stckatelevereatescell: welationvof outlet to) epidermis and! Cutis; <1). --1 cei ee ate 342 ihemmature lands of different regions <5. 0:40 as + selec u oe acess 342 OUST OMe Ole LCE SCEREM ON! |F sx. /5)ci s\che susie) aie chome ea oeieieiaeictonsroieneey noe 3438 *Contribution from the Zodlogical Laboratory of Indiana University, No. 96. being a thesis accepted as in part fulfilling the requirements for the degree of Doctor of Philosophy, June, 1908. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. 2329 Effa Funk Muhse. Stages of the Gland, other than the Mature ......-.-:5. 3. os. se. 345 Developing StaVEs eaeye re setts ays cle steko. #)e) eel otek hsfelettetarnete i eteete ete 345 i ar: ad) OPA Geet a ee ices ECR OD coe momo dood Shos oOo A Swe 345-346 18 Caine Alans action opied EE aaCUOODOUOOdoD oC FosMe San oooSe 347-348 Derenerate! LOMMS yy ere oc @ 6 —clclo ree) hehe lela eledoh tel eer ne 348 One Kind of Cutaneous Gland—Its Purpose and History ............ 348 IE big NOs) Iepile DomMlohid oa coco cous oOnOdUCUOOON GooaooODadonOgba4aCS 348 Production of secretion in relation to age .................... 350 Life history of the one kind of gland .................-:.+-+00+-= 352 Iie N ADU? mmntce U55.6 Hoo Ad 6 PUIG OOO oo momo onde Hone bos 352 How glands are replaced in the toad ...............-+.20-. 354 Gradatiom) a proot of one Kim 5c... «cco nierel = «)ehelstelslel om ese 354 IDWS TAIN CHE SMGWIVEIS,, BY TORDOIE Soa ncoocasnob0booHeaasesoouc 354 Proofs from warts with degenerative glands and from grow- ATL WVINES gape neeeae Pete te tavern ome sowe rey suede (oc volfel ote cielo) oe inenetersiistetel ats 355 @OMEMISIOMS sage cies eicisus oye sale ees oro Sree Bear ste ye ciohnn'e arms elo eioTs [asin ote caine iefein le eae) 305 PST utied v2) 0th Ae ar ia enc Aer eet eanemidr Saat etiC Saar. fo a 358 Plates Sc elaliehee. sve) se wteta) eS 6)/e ere ievel'e)e e1ein, se) 6) eee, (6, eee 06 ae ss/ellele 6 ea anne Gee ele. le sie ele ae GENERAL INTRODUCTION. The purpose of the present paper is to show that there is but one kind of gland in the cutis of the common toads. The writer is con- vinced that the several kinds of glands described by authors are but different stages in the development of the same gland. Throughout the paper, the reference is to the general integument of the body, exclusive of modification in the region of the head, feet and cloaca. It is principally the conditions in the adult toad that are under consideration, but references are made to conditions in certain stages preceding the adult, when a point may thereby be more clearly dis- cussed. The structure of the epidermis and cutis in Batrachians is more or less a matter of general knowledge, and the writer has considered them very briefly, merely for ready reference while deal- ing with the glands. I wish to express my grateful appreciation of the facilities placed at my disposal by the Department of Histology and Embryology of Cornell University, where the first material for, this investigation was prepared. The work was finished in the Department of Zodlogy in Indiana University, and I am greatly indebted to Dr. C. H. Eigenmannn and Dr. Charles Zeleny for valuable suggestions, and criticism of the final report on the material examined. Material. Toads have been collected during this investigation from three different localities. The first specimens (Bufo ameri- canus) were taken at Ithaca, N. Y., principally at the breeding The Cutaneous Glands of Common Toads. WH bo Oo season late in April." Others (Bufo fowleri Putnam) were col- lected during the summer and autumn in Washington, D. C. Ad- ditional toads (Bufo fowleri Putnam) were collected at Blooming- ton, Ind., at the breeding season in the middle of April, and dur- ing May.? The two species differ somewhat in the coloration and in the shape and arrangement of the warts. The brief description of the color pattern given in this paper, and the relation of the wart distribution to the same, applies definitely to fowleri. On the other hand, the detailed description of the skin and its glands has been derived for the most part from the study of sections of americanus, whose skin is thicker and whose warts are more massive. I have noticed no essential differences in the minute structure of the parts and do not deem it necessary to limit my statements to either of the two species. All drawings and photographs of sections from adult toads, except those horizontal to the surface, 7. ¢., transverse of the glands (Figs. 1, 16, 52, 69-76), and except Figs. 55 and 77, are from americanus. Methods. Pieces of skin from different parts of the body have been killed in the more common fixing agents, Perenyi’s, Zenker’s, Gilson’s, Flemming’s, mercuric chloride and 4 per cent. formalde- hyde. The last has proved the most satisfactory for the purpose of this investigation. Many stains have been used. Delafield’s hematoxylin with Fischer’s eosin, and Mallory’s stain have been quite sufficient for general detail; Tanzer’s orcein has been used to differentiate elastic fibers. The material has not been prepared with the view to study- ing the distribution of the nerves to the glands, or the nature of the nerve endings. . Photographs give the genera! relations which are essential to the elucidation of this paper. Drawings have been made when finer detail was desired. "The specimens of americanus found in the National Museum were kindly placed at my disposal by Dr. Stejneger, for observation of external features. 7I am indebted to Miss Mary C. Dickeson for the identification of a typical specimen, one from Washington and another from Bloomington, as Bufo fowleri Putnam, 324 Effa Funk Mubhse. Interature. ‘The skin and the skin glands of Batrachians have been written about very extensively since 1840, when Ascherson dealt with the skin glands of the frogs. The family Ranidae and the families of Urodela have since furnished the favorite species on which work has been done. The skin of the common toad, or the glands contained therein, has in only a few instances been made the subject of a structural study. The toad has been dealt with alone (Eckhard, ’49, Rainey, 755, Calmels, ’83, Weiss, ’99, Bristol and Bartelmez, ’08) or incidentally as one of a series of Batrachians (Bolau, ’64, Leydig, ’67, Schultz, ’89, Seeck, 791). With but very few exceptions, the many investigators have been of the opinion that there is present in the cutis of the species of Batrachians dealt with, more than one kind of gland. There is also a diversity of opinion regarding the structure of what is known from the description or classification to be an equivalent type of gland. GENERAL DESCRIPTION OF THE SKIN. Unevenness. As compared with other Batrachians, the adult toad is very warty over its dorsal surface, and there is a general un- evenness over the whole body. Microscopic examination of mounts of the molt, and of prepared sections from various regions of the body, further emphasize the great diversity of elevations in the surface covering of the animal. Causes. Numerous gland sacs are located in the cutis, each with a duct passing to the surface of the epidermis, where a distinct opening is visible. At many places in the dorsal skin of the toad, the glands are grouped, producing the so-called warts. (Figs. 9, 12-14, 21-24.) The skin les on a more or less even, unimpression- able surface, the muscles and bone or cartilage, and since the glands can make little or no impression on them, most of the expansion due to these many gland sacs, is outward. The unevenness of the skin is further caused by the presence of elastic fibers in the cutis. The part played by these fibers is evident from two observations. If a piece of skin is cut from any region of the body, the piece becomes smaller and the hole much larger than the original incision. The opposite effect occurs when a toad is frightened. The skin of The Cutaneous Glands of Common Toads. 35 bo Or the body becomes inflated and is then stretched much beyond its normal size. The grouping of glands on the dorsal surface quite overshadows the wrinkles caused by the elastic fibers; but on the ventral surface the wrinkles are of nearly equal importance with the glands, in producing the unevenness. To the naked eye, the ventral skin has a rather uniformly flaked or blocked off appearance. In one individual the blocks may be conical in form, in another rounded eminences, while in others they appear as flattened pavement. At the areas of transition into dorsal skin, the flakes increase in size, and the uniformity is gradually broken up by the occurrence of warts. Moreover, the skin at the bend of the limbs does not have the blocked-off appearance. It either lies in creases or is smoothly stretched. In the area of the ventral skin at the union of the thighs, the blocks are larger and quite unequal in size and shape. Sections from the lower side show that the skin between the elevations is relatively very thin, and the great variation is confined largely to the outer cutis layer. The glands are sparsely scattered through the elevations (Fig. 15). They are sometimes grouped but never have the close definite ar- rangement that is found in the warts of the dorsal skin (Fig. 14), nor do they ever attain the large size of those above. Classes of warts. The warts of the dorsal surface may be divided into three classes: (1) The most prominent elevations which occur on the dorsal surface are the so-called parotids (Fig. 9). They are a pair of low, elongated, relatively smooth masses. These are located on the back, each in line with and a short distance behind either eye, the cranial crest alone intervening. The outer forward end of each is in close proximity to the tympanum. The two parotids of a given individual are quite alike in shape, color and dimensions. These warts are constant for the species and are, as a rule, proportionate to the size of the animal. The parotids of an average sized adult measure about 6 by 13 mm. The remaining warts vary and can be described in general terms only. (2) Next to the parotids the largest warts occur on the back posterior to the parotids, on either side of the median line or groove, 326 Effa Funk Muhse. which extends often without interruption from just behind the snout to the tip of the vertebral column. The skin over the dorsal tibular portion of the legs bears warts of a similar nature. ‘This class of elevations measures from 1.5 to 4 mm. in diameter, and there oc- casionally occur elongated mound-like masses which reach a length of 5or6 mm. The warts are often in groups of two, three or more. These warts or groups of warts are not bilaterally symmetrical in their arrangement, nor is there any constancy in the number present. (3) The remaining warts, those which measure less than 1.5 mm. in diameter, may be put in a third class. The warts on the upper eye-lids, cheeks, flanks, outer surfaces of the thighs, distal edges of the hind feet and in the spaces intervening between the larger elevations previously described, constitute this class. A row of these warts extends along either flank. It begins at the outer posterior edge of each parotid and extends to about the point reached by the knee of the hind leg when folded against the body. Coloration. Color. The toad follows a general rule of animal coloration. The ventral surface is light and the dorsal some shades darker. The transition region is at the line which separates the exposed from the unexposed part, when the animal lies stretched out on its ventral surface. The color of the lower surface differs with individuals and is either buff, dull white, faint yellowish or pale green. Between the fore limbs a few dashes of pigment occur. Individual toads differ greatly in the appearance of the dorsal sur- face. All are more or less conspicuously or inconspicuously spotted. In some cases the spots are but slightly darker than the remainder of the surface and may be very few in number. In others there is the greatest contrast possible with the colors present. The ground color of the dorsal surface is, as a rule, in darker shades of the ventral color. The spots appear as if superimposed in still darker shades of the same general color. The spots are, however, often so dark as to appear seal brown or even black. Frequently the spots, many of them at least, are encircled by a very narrow band of jet black, red, yellow or white, which makes the contrast greater. Arrangement of spots. The more distinct spots occur on either side of the median line of the back in a more or less regular series The Cutaneous Glands of Common Toads. 327 of six or eight pairs (Fig. 9). The first occurs over the eye bulges, a second and third close to either end of the parotids. Others are present on back to near the tip of the vertebral column. There exists no uniformity between the shape and size of the spots which occupy a somewhat corresponding position. On either side of the median line of the same individual, an approximately equal area is occupied by spots, even though their number may not be equal. Some animals possess small and numerous spots, others fewer but larger ones. Spots which appear as a continuous band across the legs when they are folded against the body occur with considerable constancy over the thigh and tibular portion of the hind limbs. Darker bands are also present over the cheeks and ulnar portion of the fore limbs. Minor, less distinct spots are found on the remain- ing dorsal surface of the flanks and limbs. Relation of warts to spots. The parotids are, as a rule, of the same or of a slightly darker shade than the ground color of the dorsal surface. Only occasionally is one or both of these warts covered by a decided spot. Quite often, however, the darker pigment spreads very faintly over a part of the surface. Far the greater majority of warts forming the second class [(2) p. 325] are grouped in those spot areas of the dorsal surface, which are most decided in color and in the dark band across the tibular portion of the legs. The warts are of a lighter shade than the spots and this fact further increases the complexity of the color pattern. Except for the one or two days of each year spent in the water, at the breeding season, the toad passes its time on, or burrowing into dry land and to this end its outer covering is adapted. The smooth slimy skin of the tadpole, during metamorphosis and growth into ‘the adult, gives place to a relatively dry and extremely warty skin. This admirably fits the toad, even aside from its characteristic colora- tion, to harmonize with the loose soil of the garden or road side. Tur Eprmpermis. Strata. The lower, layer of cells, those in contact with the cutis, constitute the germinating stratum. As the cells pass outward from this stratum, they show a gradual change in their shape and character. 328 Effa Funk Muhse. After having occupied a place in the two or more cell layers of the transitional stratum, they become lifeless material, the molt, which is periodically cast off. (Figs. 2-4, 19, 25, 33, 48, 49.) Beaker cells. One celled glands, called beaker cells, aid in this process of molting, as was early stated by Schultze (67) for Ba- trachians in general. In the adult toad these beaker cells are the only glands located wholly within the epidermis. They are situated at intervals in the upper part of the transitional stratum and differ from surrounding cells in shape and appearance. (Figs. 1, 2, 3, 48.) The bodies of these cells may lie at any depth among the two or three outer layers of the transitional stratum They connect with the surface of this stratum by means of a more or less slender neck, which has an opening at the upper end. The body of the cell is almost entirely filled by a nucleus whose chromatin is collected in a few masses. ‘Two nuclei in a cell have been observed. ‘The eyto- plasm of a beaker cell is much clearer when stained than that of surrounding ones. These cells have been met with in all regions of the epidermis, ventral skin, ordinary skin of the back and the epidermis over the parotids and other warts of the back and legs. They are often so slender that in cross section, the body of such a cell is but little larger than the nucleus of an ordinary cell. They may lie between two cells or at the angle of several. Their distribution varies. A section horizontal to the epidermis, from a parotid, shows them to occur in about the proportion of one beaker cell to three ordinary cells if we consider but one layer of cells. (Fig. 1.) In another individual, a vertical section of ordinary skin showed eighteen such cells within a distance of 5 mm. (Fig. 3.) At the opening of a beaker cell, an accumulation of granules is frequently noted lying between the transitional and molt strata. Occasionally at these points the molt stratum is raised and freed for a considerable distance. Everything points to the conclusion reached by Schultze and later by Schultz (’89) that these cells are one-celled glands, whose secretion loosens the molt from the transitional stratum. Epidermis relative to the regions of the body. The epidermis reaches its maximum thickness over the parotids. This is due both to the larger size of the cells and to the greater number of cell layers. The Cutaneous Glands of Common Toads. 329 The epidermis is here very uniform and even, as compared to other regions. (Figs. 2, 14, 28, 25, 57, 58.) The epidermis is usually thickened and dips down to form a pit about the mouths of the ducts from the large glands. The thickening is due to an increase of cells in the transitional stratum. The ducts of the small glands do not, as a rule, cause a depression. The epidermis over the larger warts of the back and legs cor- responds in the general structure to that over the parotids, except that its thickness varies somewhat with the size of the wart (Fig. 21). The epidermis in the areas between the warts has a thickness equal to only from one third to a half the thickness over the parotids. Each stratum is here proportionately decreased through a change in the size of the cells and in the number of layers. The transi- tional stratum consists of two or three layers as compared to from four to seven in the same stratum over the warts (Figs. 2, 3, 21). The average thickness of the ventral epidermis is intermediate between that over the warts and that of the areas between them on the dorsal side of the animal (Figs. 4, 15). Tue Curis. The cutis is far more varied than the epidermis. It is subject to rearrangement occasioned by the increase or decrease of intrusive elements such as the glands, nerves and bloodvessels. Its depth is always greatest where the largest gland sacs are located (Figs. 14, 23, 24). It is least where they are entirely absent (Figs. 17, 18, 21-0.s.). In the latter areas of the back, the cutis is most typical and uniform. Strata. ‘The primary element of the cutis is the connective tissue fiber. Apparent difference in the strata of the cutis is due to the arrangement of the fibers for a specific purpose. I shall speak of the outer loose, compact and inner loose strata, which are de- scriptive terms (Figs. 17, 18). The compact stratum consists in large part of bundles of fibers. These are closely associated and ex- tend in different directions. In some areas the bundles occur in successive horizontal sheets, approximately at right angles to each other (Fig. 17). Other regions or individuals show these bundles 330 Effa Funk Mubhse. somewhat interwoven. ‘he essential feature in the arrangement of the bundles is their compactness. At certain points bundles from the horizontal compact layer turn both inward and outward, to form part of vertical strands which pass through the compact stratum; these bundles subdivide, and together with bundles which branch off from either surface of the compact stratum, form the inner and outer loose strata of the cutis. In either case the fibers are very loosely arranged in a network. Fibers from the outer loose layer terminate on the side toward the epidermis, in very fine numerous branches. These are closely associated with the processes of the cells of the germinating stratum of the epidermis. This gives the appearance of a very thin homogeneous stratum, which for the most part follows intimately the lower border of the epidermis (Fig. 77). The union between the compact layer and the muscles is ef- fected through the inner loose stratum. Here, as in the outer stratum, the fibers are loosely interwoven. Elastic fibers of considerable length and of very uniform diameter are also present. Throughout the three strata they occur widely scattered and without definite arrangement. The blood-vessels pass from the deeper tissues into the inner loose stratum of the cutis. They form a coarse network parallel to the surface. From this network branches pass directly to the large glands (Fig. 55). Others pass through the vertical strands to the outer loose stratum (Fig. 17). Here, likewise parallel to the surface, is formed a fine network of capillaries, which is just beneath the pig- ment cells. From this network capillary loops pass outward and lie at the base of endbulbs (Fig. 20). Other loops project for some distance into the epidermis (Fig. 19). The branches previously men- tioned as passing from the inner network of blood vessels to a gland break up into a dense capillary mesh. This intimately surrounds the gland acinus, lying at many points in direct contact with the gland wall (Figs. 44, 47, 51-58). Cutis according to regions. The cutis varies greatly in depth wherever gland sacs occur, according to the number and size of the same. The gland sacs lie, as far as their sizes permit, in the outer loose stratum, but the larger ones lie deeper, for, the most part in the compact stratum (Figs. 14, 25, 40-42, 58). Often the latter The Cutaneous Glands of Common Toads. 33 sacs are surrounded by a very thin sheath of loose connective tissue which is continuous with the upper loose layer. About the saes and also the gland outlets, the finely divided fibers may form a homo- geneous layer similar or even continuous with that appearing at the inner border of the epidermis. A definite network of elastic fibers surrounds the large gland sacs. That this is not the result of pressure of the gland wall against the cutis is shown by the fact that the fibers about the neck and collar form a stronger, more definite network than about the sacs (Fig. 76). Here pressure could count for very little. In the warts the bundles forming the compact stratum are always more or less interwoven (Figs. 25, 40, 61). Transverse strands are present, but they curve about the glands or are visible as vertical strands through only a part of the depth of the compact stratum. Nothing indicates that they take any special part in the support of the gland sacs. Wherever warts occur, there is a change in all the strata of the cutis proportionate to the extent and height of the wart. Greater demands are made on the blood vessels, nerves and elastic fibers, and there is an increase in the depth of the inner loose and, to a less extent, in the outer loose strata. A great change likewise takes place in the compact layer, because of the increased need of support. The ventral skin is decidedly, but quite uniformly, uneven. The inner loose and compact layers are relatively uniform in depth, and in great part it is the outer loose layer which varies (Fig. 15). In the ordinary skin of the dorsal surface, especially at the edge of the warts, papillze may occur. These are produced by the projec- tion into the epidermis of an end bulb of spirally arranged cells, at the base of which is a blood loop (Fig. 20). In the ventral skin and over the warts are found what I shall call rudimentary bulbs. In this case a small amount of cutis projects into the epidermis for a considerable distance. It bears at its summit a single large cell, the long axis of whose nucleus is perpendicular to the surface. Curanrous GLANDS OF THE Toap. Introduction. The cutaneous glands with which we are dealing consist of three parts: neck, collar and acinus (Fig. 42); each is made up of many 332 Effa Funk Muhse. cells. Such a gland is a differentiated part of the epidermis. As the acinus of a gland enlarges, it pushes farther and farther away from the epidermis into the cutis, with the elements of which it has no direct connection. The strata of the cutis increase in depth and are rearranged and adapted to the bodies of the glands. These epidermal glands go by the name of cutaneous glands to distinguish them from the one-celled glands of the epidermis, the beaker cells. The present investigation has convinced the writer that there exists in the cutis of the toad only one kind of gland. All cutaneous glands, however different they may appear, are developmental stages of the one kind. The climax of the complex, graduated series is reached by the large sacs that hold in readiness a great quantity of granular secretion. Those Batrachians in which the parotids occur present the greatest differences in the size and structure of the glands, and in the location of their sacs in the cutis. Iiterature. Many authors, whether considering the glands of Ba- trachians from a histological, embryological or physiological point of view, have proceeded on the theory that at least two kinds exist. The conclusions of those who have discussed the question of the number of kinds of glands differ. The majority believe that two, even three, or four kinds of glands are present. Many of the earlier writers made their classification largely on the basis of size into large and small glands, or according to the shape of the gland acinus. (Ascherson, ’40, Eckhard, ’49, Rainey, 755, Hensche, 56, Szczesny, 67, Ciaccio, °67, Leydig, ’67.) The more recent classification is into mucus and poison, nuclear, or granular glands, based on a dif- ference in the epithelial structure and in the secretion produced. (Englemann, ’70, Schultz, ’89, Seeck, ’91, Drasch, ’92, Weiss, ’99, Phisalix, 00, Esterly, ’03, Bristol and Bartelmez, ’08.) Esterly speaks of the large poison glands and of the mucus variety. He finds that in every large gland there is the fundament of a new gland, which resembles glands of the mucus variety. He, however, shows no relation between the separate mucus glands and the large poison glands. Several investigators (Calmels, ’83, Leydig, ’92, Vollmer, 93, Nicoglu, 798, Junius, ’96, Ancel, ’01) have given evidence or expressed their belief in one kind of cutaneous gland. The Cutaneous Glands of Common Toads. 333 Calmels is the only one of this group who has dealt with the toad. He has evidently excluded from his discussion the glands of the ventral surface, for he makes the statement that the poison glands which occur, only on the dorsal surface differ from those of the ventral side, in that they contain a milky secretion produced by the poison cells. He makes no mention of smooth muscle fibers about the glands. It is evident from his description that he has seen the muscle fibers, but has mistaken them for epithelial cells. Recognizing but one kind of gland, he establishes with the epithelium as a basis four types. According to him, the youngest epithelium is found about the lumen of the largest glands, but he has mistaken the muscle fibers in slightly longitudinal view for young epithelium. What he describes as daughter cells of the epithelium in the other three stages of epithelium found in the small glands are probably the transverse view of muscle fibers. I have not had access to Leydig’s article nor to Nicoglu’s. Nicoglu and Vollmer are referred to by Esterly as having stated that they saw regenerating forms of glands. Both worked on sala- manders. Vollmer figures a small gland arising from the epidermis, but does not give intermediate forms. His study was largely an experimental one. Nicoglu and Heidenhain each seem to have pub- lished an article in 18938, as the results of the same investigation. If this is correct, the two reached different conclusions, for Heiden- hain divides glands into three kinds—mueus, poison and double. Esterly refers to Nicoglu as an investigator who believed in one kind of gland. Junius, dealing with the structure of the cutaneous glands of the frog, describes what he believes to be the young and old forms. He, however, merely expresses it as a belief that the forms he de- scribes are developmental stages of the same kind of gland. He did not observe intermediate stages. He further expresses it as his opinion that in the renewal of glands, the process of their embryonic development is probably, repeated. He offers no evidence for this opinion. Ancel deals with the development of cutaneous glands especially in the salamanders. He states that all glands arise in the epidermis, but that certain among them are arrested sooner in their evolution 334 Effa Funk Muhse. than others; that the large poison glands are more completely dif- ferentiated toward a special function. Secretion and when expelled. Under proper conditions, the secre- tion, which is produced by the cutaneous glands of Batrachians, comes to the surface of the animal. The skin of a toad, which has in no way been disturbed, is dry. If it is roughly handled, some of the cutaneous glands expel a colorless fluid and the skin becomes moist. Under a powerful stimulus a milky secretion oozes through the duets to the surface in the form of small drops. It is probable that in nature, only the most severe shock or torment by a natural enemy causes the expulsion of the milky secretion. The toads, which have been dealt with in this investigation, have in no case expelled it short of chemical, electrical or severe mechanical stimulus, or only after the head has been severed from the body. If the stimulus, e. g., electrical, is localized, only the glands from the stimulated region are discharged. The relation of the transparent and the thick milky secretions to the glands will be discussed later. As far as con- cerns this investigation we will accept it as an established fact that at least the milky secretion has an irritating effect on mucus mem- branes. Further, that on certain small animals of other species it is poisonous and even fatal in its results. Investigations of a chemical and experimental nature have shown this to be true of the secretion from a number of Batrachians. (Gratiolet and Cloég, 752, Calmels, ’82, Phisalix, ’00.) Origin of glands in the larva. The development of glands in the larva has been followed in a few Batrachians, notably the sala- manders. With one exception (Phisalix, 00) the more recent writers (Maurer, 795, Ancel, ’01) agree that they come from the ectoderm. Here and there in the lower cell layer of the primitive epidermis of the tadpole, a single cell becomes differentiated. Rapid cell division follows and a gland bud, 7. e., a small solid mass of cells is soon established. This cell collection pushes down into the cutis, retaining a connection with the epidermis. At the point of connection the future duct is developed and a lumen soon arises in the acinus. T have observed gland buds in the toad tadpole, whose body length was 8 mm. (Fig. 6). The hind legs had at this stage reached a 29K The Cutaneous Glands of Common Toads. 335 length of 2 to 3 mm. and the fore limbs, while well formed, were not yet visible on the surface. The buds occurred in both the ventral and dorsal epidermis of the body and limbs. A younger series, whose body length was 6.5 mm. and whose hind limbs were merely buds, showed no indication of gland formation (Fig. 5). It is in- teresting to note that the gland buds arise just after the animal has passed its most typical aquatic form, and appear at the same time with other adult structures. Gradation of glands in adult. I have found buds of a similar nature in the epidermis of the adult toad (Fig. 26). From this point on a very completely gradated series of glands may be selected (Figs. 27-41). The climax of the series, beyond which point degen- eration may set in, we will term the mature gland (Figs. 14, 42, 58-a). The size of the neck, collar and walls of the acinus (ex- cluding for the present the epithelium) reach, in the mature type, their highest development. The elements which constitute the parts are all present in the younger glands. They are developed in pro- portion to the size and age of the glands. Throughout the series a gradual change in the epithelium and in the secretion can be fol- lowed. The production of poison granules in the secretion completes its highest development. Batteries of glands. The great variation in the size and shape of the glands and differences in location in the strata of the cutis incident to their size, make it difficult to give a general description. I shall, therefore, for convenience designate three strata or bat- teries of cutaneous glands: (1) inner, (2) outer and (3) transitional (Fig. 25, a, ¢, b). The glands occur singly in the ordinary skin of the back, and singly or somewhat closely arranged in the ventral skin. In warts, where they reach the largest size, they are closely grouped. The glands in a given battery vary greatly according to the regions of the body. (1) The glands in any region, whose bodies reach almost to the inner loose stratum of the cutis, constitute the inner battery (Figs. 14, 25-a, 58-a). The lumen of such a gland is completely filled with seeretion and is irregularly lined by naked nuclei rather than by a cellular epithelium (Figs. 43-47). The extent of each wart is gov- 336 Effa Funk Muhse. erned largely by the size and number of glands of the inner battery. Authors have given to these glands in the toad, and to corresponding glands in other Batrachians, the name of large, contractile, granular or poison glands. (2) Among or just beneath the pigment cells are found small glands, whose bodies lie wholly in the outer loose cutis, and which make up the outer battery of glands (Figs. 12, 14, 23, 25-c, 58-c). The bodies of these glands never reach a deeper level than the collars of the glands of the inner battery. A lumen is present which is lined by a definite epithelium (Figs. 28-35). These have in general been called small, non-contractile, or mucus glands. (3) Those glands which show an intermediate condition, both as regards the structure and the location, comprise the transitional bat- tery (Figs. 28, 25-b, 36-41). Such glands are not always present where those of the inner and outer batteries occur. It is probable that when these glands have occurred, most authors have classed them with the mucus, small or non-contractile. In a few instances a separate category has been established to include these glands. I am convinced that the glands of these three batteries are all different stages in the life history of one kind of cutaneous gland. The Mature Gland. Mature glands occur in the skin of the various parts of the body. They reach their highest development and largest size in the central areas of the parotids of the adult toad (Fig. 14). The following de- scription of the mature type applies definitely to those occurring in the above region. A median longitudinal section of such a gland shows that it is relatively an enormous sac-like body with a short, thick outlet which consists of a neck and collar (Figs. 14, 25-a, 42, 58-a). The neck is in connection with the epidermis and the collar is a great accumulation of cells that marks the transition from the neck into the body of the gland (Figs. 78, 79). A duct leads from the cavity of the sac, the lumen, through the neck and collar to the surface. The most conspicuous feature of this type of gland is the great quantity of granular secretion, which completely fills its lumen. In the central areas of the parotids, the vertical diameter of these The Cutaneous Glands of Common Toads. oun gland sacs is always greater than the transverse and often reaches a depth of 1800 microns, and even as great as 1900 microns. If the sacs are not crowded, they are more or less circular in the transverse plane. If they are closely associated and are affected by pressure from neighboring glands, a variety of polygonal shapes arise (Fig. 16). The transverse diameter varies from 500 to 1200 microns. Wall of acinus. The gland sae consists of a homogeneous sub- stance or matrix. In the periphery are imbedded muscle fibers. To the lumen surface are attached epithelial cells or nuclei. In some eases the wall of the acinus is thin and the muscle fibers form a more or less continuous sheath, and the epithelial cells rest closely against the fibers (Figs. 44, 59, 60). In other instances the wall of the sae is thick, the individual fibers are less closely arranged in it and the fibers and epithelium are separated by a considerable amount of matrix (Figs. 45, 47, 56, 62, 65). The nature of the matrix of the acinus wall has been variously interpreted. Drasch is the only investigator who has made any reference to a substance which encloses the muscle fibers and on which rests the epithelium ; he says that the whole is surrounded by a membrana propria. Several others (Schultz, Weiss, Junius) state that the muscles rest upon a membrana propria or basement membrane. Schultz further states that each epidermal cell sends a foot in between two muscle fibers. Mme. Phisalix says that the membrana consists of smooth muscle fibers. Muscle fibers. The muscle fibers are of the involuntary kind, each elongated and spindle-shaped. An oval or elongated nucleus contains a single nucleolus (Figs. 43-46, 51, 54, 61). In many in- stances I have followed fibers through their whole extent and found them to be single spindles. Other fibers, with one end split in two, have been observed. Several writers (Drasch, Junius, Ancel) have stated that the muscle fibers are divided into fibrillee at one or both ends. I have never observed a similar condition in the toad. The fibers are meridionally arranged and adapted to the curvature of the gland body. The outer ends of a limited number of these fibers extend to the outer part of the gland collar and their opposite ends are overlapped by the tapering ends of other fibers. The number of 338 Etfa Funk Muhse. fibers gradually increases until the greatest circumference of the acinus is reached. From this region they decrease toward the lower pole over which they pass. The average fiber about a large gland has a length of from 130 to 150 microns and in the region of the nucleus a diameter of from 7 to 10 microns. Several fibers are re- quired to complete the cireuit of the gland. There is, therefore, no definite arrangement of the nuclei about any given part of the acinus. Seeck describes similar structures, 7. e., the spindle cells, and con- siders them replacement cells for the epithelium. He denies that they are muscle cells. The transverse sections of the fibers appear as a layer of cubical cells (Figs. 47, 51-53, 56, 61, 62, 65, 66, 75). Weiss evidently mistook this view of the fibers for epithelium. A discussion of the action of the muscle-fibers will follow later. Epithelium. The epithelium of this type of gland consists in large part of naked nuclei. They are attached to the matrix or are partly sunken in the same. They are not uniformly distributed. Over small areas they form a continuous pavement; over similar areas they may be entirely absent (Figs. 43-47, 56, 59-66, 75). The nuclei take in general one of two forms: (1) flattened, circular bodies, which appear quite uniformly dense when stained (Figs. 44, 59), and (2) those which arch out toward the secretion and show a clear area on the attached side (Figs. 45, 47, 60, 65). Mme. Phisalix studied the salamander with special reference to the part played by the nucleus in evolving the secretion. She describes nuclei, which take the form of parachutes and states that they are actively engaged in producing poison grains. The nuclei of the toad are much smaller than those of the salamander. An epithelial cell, that is, a nucleus situated in a small definitely limited cytoplasmic mass, in contact with the matrix, is seldom met with in the mature gland. The body of such a cell may be densely granular (Figs. 51, 52), or it may appear entirely homogeneous, or with but a few granules (Figs. 51, 61). One individual, collected with others at the breeding season, proved an exception in that a large number of cells were present. Even in this instance there was by no means a continuous cellular epithelium. The cells varied greatly in size. They were found in all parts of the acinus, and were here and there greatly The Cutaneous Glands of Common Toads. 339 crowded together. The nucleus was situated in that part of the cell which was attached to the matrix. The cytoplasm of the cell was very similar to the secretion in the gland lumen (Figs. 16, 52). Secretion. The secretion of the mature gland comes to the surface of the skin in drops. It is white or creamy in color, and has a strong disagreeable odor, very similar to that of the so-called Jimson weed (Datura). If the fresh secretion is placed on a slide and examined with the microscope, it is seen to be a liquid densely crowded with small globular bodies. The globules vary in size from 1 to 3 microns, but all are not perfect spheres. When in motion, the globules flow about in the liquid and the mount reminds one very much of a similar preparation of fresh blood, with the corpuscles in motion. The liquid in which the globules float is colorless. In consistency it probably is much like the plasma, for it permits of as free movement as in the case of the blood. ’ The secretion mass appears differently in different preparations (Figs. 25-a, 42, 59-68). It seems probable, however, that the al- veolar appearance in Fig. 64 may be due to the fixing agent. Judg- ing from the fresh secretion, the conditions represented in Figs. 59, 61, 63, 67 seem quite normal, except that it is hard to explain the separation of the more granular from the comparatively homogeneous or finely punctuated liquid. It is possible that in fully mature glands the granules may have drawn closer together in the center of the lumen, leaving near the outlet of the gland and about the periphery a stratum of the liquid secretion. As evidence of this there is often a change in the color and consistency of the secretion during the continuance of the flow, which will be spoken of later. Both the fresh and the stained globules may show one or more refractive bodies (Fig. 68). Large clusters of globules have been seen in certain preparations (Fig. 61). They are spheres from 15 to 25 microns in diameter, the periphery of which are made up of globules. The whole mass floats in the liquid and is filled with the same. We have no way of knowing if this is a normal condition, since such a cluster, if present in the secretion of the lumen, cannot pass in toto through the mouth of the gland, and appear in the freshly discharged secretion. 340 Effa Funk Muhse. I have not aimed in this investigation to make a cytological study of the parts of the skin, and the material has not been prepared with that in view. Consequently the question of the production of the secretion in relation to the parts of the epithelial cell or nucleus will not be treated from that standpoint. Further reference to the mature secretion will be made after we will have discussed the stages of gland development. Gland outlet. The gland outlet consists of a neck and collar, solidly built up from cells, except for a duct which passes through their vertical axis (Figs. 14, 25, 42, 67, 78, 79). In the mature gland there is a gradual transition of the neck and collar into each other. The latter has in general the form of a truncate cone, the base of which rests upon the wall of the acinus, squarely over the lumen of the same. From the top of the cone, the neck continues to the surface at a different angle, assuming more or less the form of a cylinder (Fig. 25-a). Concentric zones. The cells of the outlet are arranged in two concentric zones (Figs. 78, 79). The inner and outer zones are dis- tally continuous respectively with the melt stratum and with the outer layers of cells of the transitional stratum of the epidermis. Proximally their, position corresponds respectively te the epithelium and to the muscular part of the acinus wall. The molt cells continue down the duct to about the level of the lower surface of the epidermis. Their short diameter is in this position perpendicular to the duct. The whole layer is intimately bound together (Figs. 70, 71). At about the place where this lifeless layer ends, definite living cells are found; these continue to the lumen of the acinus, thus completing the inner zone. Their arrangement is radial (Figs. 71-74, 78). Those nearest the molt are more or less polygonal in form, and ar- ranged in a single layer (Figs. 71, 72). As the base of the collar is approached, these cells become more and more elongated and are so shifted in their position as to appear in several layers (Figs. 73, 74,78). A thick pad is thus formed in the collar to which reference will be made in connection with the emptying of the gland. The cells of the outer.zone, beginning at the distal end of the outlet, are but little modified from surrounding cells of the epidermis, except The Cutaneous Glands of Common Toads. 341 that they are somewhat flattened (Fig. 69). In the proximal part of the neck, the cells are polygonal in form. They are radially placed in at least two irregular layers (Figs. 70, 71, 78). In the collar the cells elongate, shift their position so as to appear as several layers, but maintain their radial arrangement (Figs. 73, 74, 78). Cog-wheel-like structure. In the outlet just below where it passes through the pigmented layer of the cutis, the cells form a cog-wheel- like structure (Fig. 72.) The cells of the inner zone, thirty or more in number, are radially arranged about the duct which at this point is more or less circular. The cells of the outer zone are in groups of ten or more, and the sixteen to twenty groups in turn radiate from the inner zone. This structure is a constant feature of the out- lets of the mature glands, and I have selected it to arbitrarily mark the outer limit of the gland collar. The distal ends of the first sixteen to twenty muscle fibers extend to this point. The spaces alternating with the groups of cells appear relatively clear when stained, for at this level only the cytoplasm of the muscle fibers is present; the nuclei are at a lower level. ‘Toward and passing into the acinus wall, the fibers constantly increase in number, overlapping in part those that have preceded them. At any given level in the outer zone between the cog-wheel and the acinus, the muscle fibers and the cells of the collar proper are intermingled with one another (Figs. 73, 74). This arrangement provides a firm attachment for the muscle fibers. Several authors (Schultz, Seeck, Phisalix) de- seribe the collar of the species dealt with as consisting of elongated eells, circularly arranged. Schultz even states that it approaches the nature of a sphincter muscle. As we have seen above, all the cells of the collar, have in the toad a radial arrangement. Esterly de- scribes dilator and constrictor muscles in cells of the neck, which lie against the duct. I have not been able to verify this in the toad. Duct. The ducts of the mature glands vary both in their shape and caliber. In the region of the epidermis, a duct has a slit-like form, simple or branched (Figs. 69, 70). Often the epidermis is depressed and appears in surface view as a pit or furrow at the opening of the duct (Figs. 25, 67). But occasionally there is no 342 Effa Funk Muhse. such modification in its surface (Fig. 42). A very large number of cells, forty or more in the layer surrounding the molt at the opening of the duct, may be counted in one section (Fig. 69). The slit in the region of the neck becomes more compressed as the base of the neck is approached (Fig. 70). For some distance its walls may even be pressed together and the duct may thus be closed (Fig. 71). At the beginning of the collar it becomes more or less circular (Figs. 72, 75, 76). Just before the gland lumen is reached, the inner, series of cells frequently entirely obliterate the duct (Figs. 74, 78). This is due in part to the action of the network of elastic fibers which surrounds the collar (Fig. 76). Relation of outlet to epidermis and cutis. Seeck, referring to the toad, states that the Malpighian layer of the epidermis passes directly into the neck of the gland. I find that the neck, except where it is continuous with the outer transitional layers of cells and with the molt, is in most cases easily distinguishable from sur- rounding cells. In the first place, the epidermis is usually somewhat increased in depth about the gland outlet. A limited amount of cutis often penetrates between the neck and the inner third or more of the epidermis (Fig. 78). We have above noted the radial ar- rangement of the cells in both zones, especially about the lower part of the neck. This likewise differentiates and separates the neck from the lower stratum of the epidermis. I do not believe that the germinating stratum of the epidermis, after it has given rise to the cell that becomes differentiated into a gland bud, has any further connection with that developing gland. T have not been able in the toad, say that development, it is simply a reservoir of poison.’ to learn if all or only a part of the mature glands are discharged as the result of a natural stimulus. The results of artificial stimuli differ. Electricity applied to a limited area of the skin causes a flow of secretion from that part alone. The same is true of mechanical stimulus. Pithing the animal in no case caused a discharge, but decapitation or killing with chloroform, in many instances, produced a general expulsion of secretion. Authors have quite generally agreed that the expulsion of the secre- tion is due to the contraction of the smooth muscles about the gland 344 Effa Funk Mubse. sacs. It is merely the nature of the cells that has, as a rule, given rise to this conclusion. Calmels and Seeck do not attribute the expulsion to smooth muscle fibers about the individual glands. Calmels does not describe or figure, as such, a layer of smooth muscle fibers in the acinus wall. His statements regarding muscles are not clear, and I shall not attempt to state his position. Seeck, on the other hand, describes the layer of spindle-shaped cells in the gland wall, but definitely states that they are not muscular in nature. According to him, the expulsion of the secretion is caused by the subeutaneous muscles. Also that the attachment of the skin to the underlying tissues differs according to the species, and thus the secretion flows differently from the toad than from the salamander. Further than this statement of Seeck’s, those who have held that the expulsion is due to the action of subcutaneous muscles have not stated in what way they act. The skin in the toad is very loosely attached over much of the body. While the glands are discharging, I have not been able to detect any change in the tension of the skin. I have experimented with several toads, the results of which prove beyond a doubt that the expulsion of secretion from the glands is not due to the action of subeutaneous muscles. Toads were killed by pithing, and no secretion was ex- pelled. At once a long piece of skin, containing a parotid, was cut on three sides, lifted and entirely freed, except at one end. Elec- trical stimulus was then applied to either the upper or under side of the parotid, and the secretion poured out in just the same way, and just as freely, as in those animals where the stimulus was ap- plied to the normal skin. Furthermore, there are present in the cutis of the skin of the toad no smooth muscle fibers, other than those definitely arranged in the acinus wall of the gland. It will be recalled that the collar of the outlet consists of two zones of cells radially arranged, and that the cells of the inner zone form a pad which rests upon the lumen, thus giving consider- able diameter to the acinus in this region. Also that the more or less continuous sheath of muscle fibers in the matrix of the acinus is firmly attached to that part of the collar which rests against and The Cutaneous Glands of Common Toads. 845 is in connection with the pad of cells. Furthermore, that the duct is usually closed at several points, especially in the base of the collar, against which the secretion presses. As the smooth muscle fibers contract, the cells of both zones of the outlet are drawn apart, and the duct is thus opened. Pressure is exerted on the periphery of the lumen, causing the secretion nearest the duct to escape first. As the effect of continued contraction, a large gland may be entirely emptied. Stages of the Gland other than the Mature. Developing stages. Glands younger than the mature cannot be classified into distinct types. It is safe to say that no two glands are alike in every way. ‘The degree of development is the prin- cipal reason for the great variation. But among other things that may modify the shape, and relative size and development of the glands, is the region in which a gland may occur, whether in the ventral skin, in the ordinary skin of the back, or in the parotids or other warts. I shall accompany the general description of the glands of the outer and transitional batteries with descriptions of specific glands, which represent certain stages in the development toward the mature glands. These have been chosen from the parotids, with the exception of the first stage (Fig. 26), which was in the ordinary skin near the parotid. The stages of the outer battery (1) may be divided into those that are not differentiated into neck, collar and acinus (a and b), and those that are (ce, d, e). The latter condition is also true of the glands constituting the transitional battery (11), of which I shall describe but three stages (f, g, h). I. (a) The youngest stage which I found in the adult toad is a bud, consisting of a group of cells contained in the epidermis, and producing a slight bulging toward the cutis (Fig. 26). (b) Slightly more advanced is a mass of cells which has partly pushed into the cutis. The cells forming the connection between the mass and the epidermis are slightly modified (Fig. 27). Later developmental stages show all the essential parts of the mature type. The acinus of the glands of the outer battery is spherical or somewhat compressed at the poles. The walls of the 346 Effa Funk Wahae acinus consist of a matrix in which are imbedded muscle fibers and on which rests the epithelium, whatever form it may assume. The number of muscle fibers increases with the size of the acinus. In the smaller glands, the fibers may be few and scattered. Compared with the depth of the acinus, the fibers are relatively very long, some- times reaching even through half its circumference (Fig. 34). The most apparent variation in the glands of the outer series is in the epithelium. Glands with a definitely branched acinus and a common collar and neck, others with both a branched acinus and collar, and the neck divided for part of the way, are found occasionally. Sacs more or less oval, or spherical, with a definite outpocketing or bud at one side, are common. This last has also been observed in the mature type. Two stages, a and b, have been described. (c) In the third stage a lumen is forming and is lined with a cubical epithelium (Fig. 28). (d) Fig. 29 shows that in this section of a given gland, the cells of the epithelium are uniformly cubical, each with a large centrally placed nucleus and homogeneous cytoplasm. In an adjoining sec- tion toward the center of the gland, four homogeneous cells have changed into reticular cells and have projected somewhat into the lumen. Part of the contents of these cells has streamed as an in- definite threadlike mass into the otherwise empty lumen. Fig. 30 shows a somewhat similar condition, except that relative to the other cells the reticular ones have enlarged more and projected further into the lumen. Fig. 31 shows a gland similar to the preceding ones, except that the cytoplasm of the enlarged cells has a granular appearance. The nucleus of the enlarged cells of the above glands has remained in contact with the base of the cell which rests upon the matrix. (ec) In a more advanced stage, the outer lower hemisphere of the gland is lined with enlarged cells. These are crowded together and all assume slightly polygonal forms (Figs. 32-35, 50). The cytoplasm of the enlarged cells may be homogeneous, reticular, or granular. All of these cells may be of one sort, or one cell may be The Cutaneous Glands of Common Toads. 347 homogeneous, another reticular and another granular. Even a cell may exhibit one kind of secretion in its upper region and another in its lower (Fig. 50). I have found many stages between what I have described under d ande. The glands referred to as d and e, and which make up the larger glands of the outer battery, measure on the average 96 microns in depth and 160 microns in transverse diameter. Il. The glands of the transitional battery are often not present in an area at a given time. But when these intermediate forms are present, there is no sharp dividing line between them and the outer and inner batteries. As said before, I have divided glands into these three batteries merely for convenience. In shape, the acinus of transitional glands tend to elongate in depth. The collar becomes somewhat conical in shape. ‘The neck becomes relatively shorter. But it is more particularly in the epithelium, secretion and lumen, that the transition from young to mature glands is apparent. (f{) A stage in advance of the last spoken of (e), is shown by a gland in which the acinus is somewhat elongated, and the epithelial cells in its lower half are greatly increased in height. The nucleus of each cell is crowded toward the edge and the cytoplasm of the cell is granular, presenting the same granular appearance as the secretion of adjacent mature glands. The cells about the upper part of the acinus remain cubical in form with homogeneous cyto- plasm. The lumen is still free from secretion (Figs. 36, 37). What appears as drops of secretion in the lumen, and probably what has been so described, are but the cut ends of cells, which have extended further into the lumen and because of the weight of the secretion are bent over at an angle. (g) Further advance is shown by a gland in which even the cells of the upper part of the acinus have elongated. The cytoplasm of the epithelial cells about the neck is reticular, and stains exactly as do part of the cells in neighboring glands of the outer battery ; that of the remaining cells is faintly granular. The lumen is almost filled with a secretion similar to the eyptoplasm in the last-mentioned cells (Figs. 38, 39). There is considerable difference in different individuals. 210. Fic. 11. Parotid of first summer toad (Fig. 8). Gradation. > 45. Fic. 12. Parotid of late fall toad (Fig. 9). Gradation. x 18. Fic. 18. Parotid of second year toad. Outer and inner batteries. > 10. Wic. 14. Parotid. Outer and inner batteries. x 10. Fic. 15. Ventral skin. m-mature gland. x 10. Fic. 16. Parotid. Transverse of glands. x 10. Fic. 17. Ordinary skin of the back; epidermis, cutis: a-outer loose, b- compact, c-inner loose strata, pigment, vertical strand; nerves and blood yessel. < 45. Fic. 19. Ordinary skin of back. Three strata of epidermis. Processes of germinating stratum. Blood loops. x 210. Fic. 20. Papilla, ordinary skin of the back. Capillary at base of end bulb. < 210. Fic. 21. Wart, and ordinary skin—o. s.—of the back. » 10. Fic. 22. Parotid of second year toad, showing growth and transitional glands. x 10. Fic. 28. Parotid showing replacement of degenerate glands. Transitional glands are present. x 10. Fic. 24. Parotid. Degeneration and replacement. >< 10. *The magnification of all figures takes into account the plate reduction. Plates II, III, V, and VII are the result of a one-fourth reduction, Plate IV of a one-third reduction, while for Plate VI there was no reduction. THE CUTANEOUS GLANDS OF COMMON TOADS. PLATE II. tte Fig.5 Fig.6 Fig.7 EFFA FUNK MUHSE. g Tor AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. PLATE III. Developmental stages of the cutaneous glands, found in the adult; batteries and gradation. Fic. 25. Parotid. Batteries: a-inner, b-transitional, ¢outer. Epidermis; cutis; outlet of glands. x 34. Iie. 26. Gland bud, ordinary skin of the back near parotid. > 210. Fie. 27. Undifferentiated gland, parotid. > 210. Fies. 28-52. Glands of outer battery, parotid. Gradation, « 210. Fies. 33-35. Gland of outer battery, parotid. First (34), third (35), fifth (83), sections of same gland. Fig. 34 shows the longitudinal view of smooth muscle fibers, and in the other two the muscle fibers are cut transversely, and appear in some cases as triangular cells at the base. 210. Fics. 36 and 387. Glands of transitional battery, parotid. >» 210. Fics. 38-41. Glands of transitional battery, with sections of large mature glands of inner battery, parotid. Figs. 88 and 39 are different sections of the same gland. x 45. Fic. 42. Mature gland of medium size, inner battery, parotid. > 45. THE CUTANEOUS GLANDS OF COMMON TOADS. PLATE III. EFFA FUNK MUHSE. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. ata > Pe a - peay Ply Cn ~ 7 fed + ry d- rr =e oa Sree PLATE IY. Detail of wall of acinus, mature glands of parotid. Detail of epidermis. Detail of gland of outer battery (Camera lucida). a. e. ¢—epithelial cell with cytoplasm alveolar in appearance. b. ¢—beaker cell, superimposed against an ordinary epithelial cell. c.—capillary and corpuscles. ce. t—connective tissue fibers. e. n.—epithelial nuclei, naked. g. e. c.—epithelial cell with granular cyptoplasm. g. S—germinating stratum. i. b.— intercellular bridges. m.—matrix. in. f.—smooth muscle fiber, and nucleus. m. s.—molt stratum. p.—processes. s.—secretion. t. s—transitional stratum. Fic. 43. Second year toad. Vertical section of gland. 413'/;. Fic. 44. Vertical section of adjacent glands, with intervening connective tissue. >< 4137/5. Fig. 45. Vertical section of gland. » 413'/;. Fic. 46. Second year toad. Vertical section of adjacent glands, with in- tervening connective tissue. >< 4137/s. Fic. 47. Vertical section of gland, near lower pole. 4137/3. Fic. 48. Vertical section of epidermis. 5537/5. Fic. 49. Second year toad. Vertical section of epidermis. Parotid. 5da'/s. Fic. 50. Vertical section near centre of gland of outer battery, parotid. SO AIS aN THE CUTANEOUS GLANDS OF COMMON TOADS. PLATE Iv. EFFA FUNK MUHSE. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. PLATE V. Structure of wall of acinus, mature glands of parotids: capillaries, muscle fibers, epithelial nuclei, and epithelial cells or poison sacs. Degenerating forms of glands. Figs. 51 and 54. Vertical, tangential sections of glands. Beginning at periphery of photograph,—connective tissue, network of capillaries, muscle fibers, naked epithelial nuclei. Fig. 51, in addition, shows secretion and three epithelial cells or poison sacs. The muscle fibers of the two glands appear in different states of contraction. \ 210. Fie. 52. Section horizontal to glands. Walls of two adjacent glands and intervening connective tissue. Upper gland shows a cluster of epithelial cells or poison sacs. Lower gland shows well the transverse view of muscle fibers. 210. Fic. 53. Vertical section of collapsed gland between two filled glands. Note the capillary network, and the similarity of the transverse view of the muscle fibers at the base, to a cubical epithelium, for which several writers have mistaken it. x 210. Fig. 55. Vertical section of glands and cutis, showing blood-vessels in lower Cutis layer, with branches leading to base of glands, and capillary net- work about same. 45. Fic. 56. Vertical section of gland at lower pole. From below upward, con- nective tissue, Capillary and corpuscles, matrix with imbedded muscle fibers and attached epithelial nuclei, and secretion and detached nuclei. 210. Fics. 57 and 58. Vertical sections. In the inner battery, appears part of a mature, completely filled gland, and degenerating forms, the lower one of which in 57 opens to the inner side of the cutis and is filled with corpuscles. In the region of the degenerating forms, occur a relatively greater number of small glands, which show considerable gradation. 45. THE CUTANEOUS GLANDS OF COMMON TOADS, PLATE V. EFFA FUNK MUHSE. SPs er eS < WAN ronare oy) tay Saas Fig. 57 Tur AMERICAN JOURNAL OF ANATOMY.—VOL. PLATE VI. Structure of wall of acinus and of secretion, mature glands of parotid. Vertical section of gland. Fic. 59. Walls of adjacent glands and intervening connective tissue. Longitudinal view of muscle fibers. Note stratum of finely granular secretion in right gland. >< 280. Fic. 60. Walls of adjacent glands and intervening connective tissue. Note the arched appearance of the epithelial nuclei with enclosed clear area. x 280. Fic. 61. Parts of three mature glands: granular secretion with globular bodies, peripheral stratum of finely granular secretion, and muscle fibers about central gland. Cutis. x 60. Fic. 62. Walls of adjacent glands and intervening connective tissue. Gland to left shows an oblique view of muscle fibers imbedded in matrix, that to right a longitudinal view. > 280. Fic. 63. Secretion—finely granular peripheral layer and the coarsely granular, together with a few detached nuclei. (Same as Fig. 67.) « 280. Fig. 64. Secretion. >» 280. Fic. 65. Wall of acinus and secretion. Imbedded in the matrix are several muscle fibers seen in transverse view, two of which show a nucleus. Attached to the inner side of the matrix are three arched, naked, epithelial nuclei. < 670. Fic. 66. Wall of acinus near lower pole of mature gland—muscle fibers, matrix, epithelial nuclei,—and secretion. > 280. | Fic. 67. Shows difference in appearance of secretion in a given gland. < 60. Fic. 68. Secretion. Black and white dots in the grains represent the refractive bodies. < 670. - THE CUTANEOUS GLANDS OF COMMON TOADS, PLATE EFFA FUNK MUHSE. aoe — th Fs te BigE Giz P 2B, eet Fig. 65 THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. PLATE VII. Structure of the neck and collar of the mature gland of the parotid. Fics. 69-75. Transverse section of the outlet and acinus of the same gland at different levels, the outlet similar in shape to that of the mature gland in Wig. 25, Pi. 1M: xX 210: Fic. 69. View of neck, third section from surface (sections 10 microns). a.—neck of small gland. Fic. 70. View of neck, seventh section from surface. Fic. 71. View of neck, ninth section from surface. Fic. 72. View of cog-wheel-like structure, twelfth section from surface. Fic. 73. View of collar, seventeenth section from surface. Fic. 74. View of collar, twenty-second section from surface. Fic. 75. View of acinus (upper gland). Fic. 76. Oblique section of collar, showing surrounding network of elastic fibers. xX 210. Fic. 77. Oblique section of collar of mature gland, showing two secreting cells. Note thin homogeneous stratum at lower border of epidermis. 49. Fic. 78. Outlet of mature gland (same as in Fig. 42). Note the separa- tion of the neck from the lower half of the epidermis. > 210. Fic. 79. Outlet of mature gland of late fall toad (Fig. 9), showing the two concentric zones of cells. > 210. THE CUTANEOUS GLANDS OF COMMON TOADS. PLATE VII. EFFA FUNK MUHSE. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 2. A CONTRIBUTION TO OUR KNOWLEDGE OF THE EARLIEST KNOWN STAGES OF PLACENTATION AND EMBRYONIC DEVELOPMENT IN MAN. BY MAXIMILIAN HERZOG. From the Laboratory of Pathology of the Michael Reese Hospital, Chicago, Illinois. WirH 30 ILLUSTRATIONS. CONTENTS. PAGE MIM GTO MITELOT Y; VOT ICS = 5 stata ty ent athens des eace ous te. «tstieplanejateV chet lopeehelo¥e) cla Me ctelisyenel skate 361 History of the Case from which the Ovum Came................s..00-- 367 General Mescriptionson they Oviuieys es ccs 3. ceeecensg aoe ot > eroded helen ole! ole elle el ellie 371 IDENOAYACI OIE WE: IDNA gd 6 soot cap aRucod 6 It olds Ouro pau cidawiartlc 310.6 373 Descriptionsof, Chorionvand Weciduans 20% cae erate teie ewes we wick + ls) ore etoeyade 376 INOMTETICIAEUIE Ces ety ain oe ces erate rented: chariate sie suaesli score cuatie MMoRoss lollsteldubncue pare cece 376 The General Position of the Ovum and its Mode of Entrance into LLG Ye D CCL sr cters, Scotts cuecsh owas roms Secor aa ake ic hiale ba eiereiers sua aiaye whales 379 LDpeoyersouen. wavele KChavorsvois) Ni leo oily es anon odeeso0 Oooo oue.o manic 385 THELETOPHOHlASE, AMG AIES eS WMG GLU =). cle cpl euers eee - nue tatmlel-iuieiol~ «rls «cuelss 387 NEE OLG Cle ZOMG trees ha aohes cen cl Ceatesthomer robe es tole chin gialemsie ens ehels, se) tee 391 A Min’sy3 DYEYGVUIUUs ies See ie eeoc & Bay OR ere na CRO Geice eotene 4 CARTIER REE Or COID Po cou iocs CaTThO: 394 SITUTANTAE TAC a yaclguaioncralo Obi okt Bin RDI aces RO eno OMRON ch omic o ccc LORE NO Da 398 PAS TRO Pel MTSE PLOTS sor tone cision oeatbnede a aierotes oteval a! sie caheliel stiel «10 ces lisive:te ape ceustal el eVox InrRopUCTORY REMARKS. The most important original contribution to our knowledge of the earliest stages of human placentation was made less than ten years ago by Peters, whose monograph, “Ueber die Einbettung des menschlichen Eies,’’ Wien, 1899, is well known to every student of the subject. The present author, a number of years ago, became interested in human placentation, primarily in connection with the morbid an- atomy and histopathology of ectopic gestation, and he has for years, as a matter of routine, searched for very young human ova in every uterus and Fallopian tube which fell into his hands, either Tub AMERICAN JOURNAL OF ANATOMY.—VOL, IX, No. 3. 362 Maximilian Herzog. as an operative or as a post-mortem specimen.’ He has finally been fortunate enough to obtain a uterus from an absolutely un- objectionable case, containing an ovum, approximately lke the Peters’ ovum, but including a well-preserved embryonic shield, younger, as it appears, than any human embryo heretofore satis- factorily described. The specimen was obtained in July, 1904, during his term of service as pathologist in the Government Bureau of Science, Manila, P. I. The number of very young human ova unobjectionably adapted for a reliable study of the earliest stages of placentation is still exceedingly limited, and it may be said that we have not up to date a single specimen which is ideal and which will compare favor- ably with what can be obtained from the lower animals by timing conception accurately, removing its product from the living, and fixing it at once by the best methods at our disposal. Jung,” recent monograph, to be mentioned more fully below, says in this connection: “The ideal specimen would be a uterus extirpated from a live woman, containing in situ’ an ovum of the first week after conception. This ovum in situ, after removal from the uterus, should then be treated by aproved technical methods. The uterus itself should be free from those morbid changes which, according to common experience, pathologically alter the implantation of the ovum (myomata, chronic metritis, ete.). We are yet far removed from this ideal. Not a single one of the specimens heretofore de- scribed satisfies even approximately these requirements, and it is at in a ‘Herzog: Description of an Early Placenta in situ, obtained from the living. The Am. Gyn. and Obst. Journal, April, 1898, and Transactions Chicago Pathol. Soc., Vol. III, p. 817. Herzog: Superfetation in the Human Race. Chicago Medical Recorder, 1893) Viole Sepals Herzog: The Pathology of Tubal Pregnancy. Am. Jour. of Obstet., 1900, Vol. 42, No. 2. Herzog: Placentation in a Uterus Duplex Bicornis Gravis—Menses 1-2. Trans. Chicago Pathol. Soc., 1904, Vol. 6, p. 51. ‘Jung: Beitrige zur frtihesten Hi-Einbettung beim menschlichen Weibe: Berlin, 1908. This author has very fully gone into the recent literature of the subject, and I have repeatedly quoted from his monograph. Embryonic Development in Man. 363 least doubtful whether we will ever have such a specimen at our disposal.” Jung then points out the great rarity of young human ova ob- tained in situ. He mentions those of Peters, von Spee, Leopold, van Heukelom and Frassi-Keibel, and he finds objectionable features in all of them. The youngest and undoubtedly most valuable of these specimens is that of Peters. Without trying to detract from the great value and fundamental importance of the Peters’ specimen, it is proper to point out that it came from a case which strongly suggests the probability of pathologic changes.* Peter’s ovum was obtained from a woman who committed suicide by swallowing a large quan- tity of caustic potash solution, and who died three hours later. Such a rapid death after the ingestion of the fixed mineral alkalies is extremely rare, and must be connected with marked changes of the blood itself and of its circulation. I can find only the very in- complete record of a single similar case, that of a boy, said to have died three hours after swallowing lye. . The embryo in Peters’ specimen is said to be less well preserved than the chorion. It was described by Count von Spee as show- ing two very small epithelial cavities (the amnion and yolk sac), embedded in the chorionic mesoderm. The connection between the embryo and the chorion was thus so extensive that it was impossible to speak of an isolated body-stalk. Count von Spee was uncertain whether “the first little anlage of an entodermal diverticulum (al- lantoic duct)” was present or not. The amniotic cavity was com- pletely closed. It was lined in part by the very thin amniotic epithelium and in part by the tall columnar cells of the embryonic plate. A thin layer of mesoderm extended between the plate and the yolk sae, crossing the median line except toward one end of the em- bryo (thought to be the cranial end). Nothing is said of a neuren- teric canal, or of blood vessels. Peters estimated the length of the embryo to be 190 microns. A reconstruction of Peters’ ovum was made by Keibel, from nine- *The conditions found, which are probably pathologic, will be pointed out below. 364 Maximilian Herzog. teen outline drawings on 2 mm. wax plates prepared by Selenka, and found after his death. Nothing is reported concerning the model except the following.* “Neither an allantoic duct nor an amniotic duct was found. The external surface of the yolk sac appeared uneven (héckerig), as if the ‘anlagen’ of vessels and blood had already been formed, but naturally this point could not be definitely decided from the wax plates and the model.” After Peters’ ovum, the next youngest embryo enumerated by Keibel and Elze in the ‘Normentafeln” is the one described by von Spee in 1896. It had a longest diameter of 1.84 mm., and a longest yolk sac diameter of 1.054 mm. Length of amnion and allantoic stalk, 0.76 mm.; greatest width of the two latter, 0.76 mm. ; greatest width of yolk sac, 1.083 mm.; length of embryonic shield, 0.87 mm.; width of ectoblast plate, 0.23 mm.; length of allantoic duct, 0.835 mm. No amniotic duct was present. Between mesoblast and entoblast there were blood islands which caused the mesoblast to project strongly. The authors of the “Normentafeln” believe that the embryo 1.74 mm. long described by Beneke, is older than von Spee’s embryo. They consider that the description of Beneke’s specimen is unsatis- factory. The Frassi embryo, the youngest examined in compiling the tables which accompany the ‘Normentafeln,” is described as follows: Length of embryonic shield, 1.17 mm.; width, 0.6 mm.; diameters of yolk sac, largest, 1.9 mm.; smallest, 0.9 mm. The embryonic shield is flat, with a long primitive streak (0.5 mm.) and a shallow medullary groove. The medullary folds are not yet sharply defined At the anterior end of the primitive streak the canalis neurentericus is indicated (whether it is pervious is questionable) ; at the posterior end is the “anlage” tomes. “Anlagen” of vessels and blood are found on the yolk sae, and there are vessels in the allantoic stalk and adjacent chorion. There are no vessels in the embryonic shield. The amnion is of the cloacal membrane. ‘There are no myo- ‘Keibel and Elze, Vol. 8 of Keibel’s Normentafeln zur Entwicklungsgesch. d. Wirbelth., 1908, p. 9. Embryonic Development in Man. 365 closed, and there is no amniotic duct. An allantoic duct is present. The embryonic clom is not yet indicated. Three embryos not considered in the ““Normentafeln” are worthy of note; these have been described by Leopold, Teacher and ie, and Jung, respectively. Leopold’s small ovum, which forms the basis of a Sie published in 1906, was obtained from a young woman who com- mitted suicide by taking phosphorus. After a prolonged search of the interior of the uterus, Leopold found at the posterior wall of the corpus a small point, somewhat lighter than the surrounding tissue. A cubical piece of tissue containing this lighter point was properly fixed and cut into serial sections, each five microns thick. One hundred and sixty sections showed a very small cavity, which presented the following measurements: Length, 1.4 mm.; height, 0.9 mm.; thickness, 0.8 mm. In no section could Leopold find any- thing like an embryonic shield or an amnion, and hence he himself raises the question whether this ovum may not be a pathologic specimen. We most decidedly believe that Leopold’s small ovum must be looked upon as pathologic. The absence of the embryonic shield must favor such a suspicion. We are either, it appears, dealing with some profound changes in an ovum, due to phosphorus poisoning, or possibly with the first stages of what would have been a hydatid mole. Since Leopold published this monograph, it has generally been the tendency of those writing on the first stages of human placentation to discard his specimen from the list of young human ova. This is done by Jung, and we have one year previous to this author placed ourselves on record in the same sense. Bryce and Teacher’ have recently published an account of an ovum which they believe to be between thirteen and fourteen days old, and one day younger than Peters’ specimen. They received their specimen in “a mixture of urine and blood clot,” in which ‘Bryce, Thomas H., and Teacher, John H.: Contributions to the Study of the Early Development and Embedding of the Human Ovum. Glasgow, 1908. 366 Maximilian Herzog. it had been immersed for twenty hours. It had been discharged by a healthy woman on November 5th, presumably as a result of the disturbance effected by coitus November 3d to 4th. The speci- men was hardened in absolute alcohol and sectioned in paraffin. Attached to the inner surface of the chorionic vesicle, two smaller vesicles were found. The larger, ‘torn and collapsed,’ was at- tached to the chorion “definitely only at one point,” but the smaller was bound to the chorion by mesoblast strands, with which it was closely surrounded. ‘The two vesicles were separate from one an- other. Bryce and Teacher consider that “after careful considera- tion of the sections and model the conclusion is inevitable that the larger vesicle represents the amnio-embryonic cavity and the smaller the entodermic vesicle or future yolk sac.” (Page 26.) However, the embryo is so badly preserved that even this conclusion may be questioned, and on page 34 Bryce and Teacher discuss a different interpretation. In the very. young embryo to be described in the present communication the yolk sac vesicle is larger than that of the amnion, and is attached to the chorion only at one point. Jung’s ovum, fully and lucidly described in his recent monograph already quoted, is one of the very youngest and best preserved now on record. It was, however, not obtained in situ, but as the result of a curettage of the uterus, and was preserved in 80 per cent. alcohol, which, according to Jung’s own comparative studies, is not very favorable for the fixation of young human ova. In spite of this, its fixation has been very satisfactory, and karyokinetic figures have been preserved in the trophoblast, the decidua, and the em- bryo itself. According to the description given of the latter (p. 102, Jung, l.c.), the “Embryonalgebilde” was found in nineteen sections each on an average of 13 microns thick, making a total length of 247 microns. However, as Jung states, some of the sections containing the embryo had been lost and an attempt at reconstruction had to be given up as futile. The whole “Embryonalgebilde” is‘said to be en- closed within a thickening of the mesoblast in the interior of the ovum, attached to its basal surface. ‘Within the mass of mesoblast we see the ectoderm shield (‘Keimscheibe’). In transverse section it is a crescentic formation, with its concavity towards the basal side Embryonic Development in Man. 367 of the ovum. It is composed of high cylindrical cells, some of which show mitoses. The extremities of the crescent are continuous with the closed amnion, which is composed of one layer of flat cells. The cavity formed by the embryonic shield and the amnion in trans- verse section has a lenticular shape, and it is filled with a finely granular coagulated mass. Externally the amnion and embryonic shield are surrounded by a thin layer of mesoblast, . . . which separates them from the yolk sac. The latter can only be seen dis- tinctly in twelve sections. The yolk sac shows a simple layer of flat entoderm cells. Toward the embryonic shield this ento- blast rests directly upon the mesoblast which separates the ecto- dermal shield from the yolk sac; toward the other side the ento- blast is strongly curved, and quite a distance removed from the mesoblast. The yolk sac, as a whole, has a somewhat hemispherical shape, with its base directed towards the embryonic shield. “The mesoblast, as already mentioned, extends from a broad base attached to the chorion towards the ‘Embryonalanlage,’ and forms at its basal side the allantoic stalk. The latter is composed of mesoblast cells only, and shows no trace of an epithelial-lined duct. . . There are no distinct vessels. However, one sees here and there at the periphery of the mesoblast accumulations of cells, occasionally arranged in a circular manner, with a lumen in the middle. Similar formations are seen on the lateral portions of the allantoic stalk.” I am unable to decide whether we are dealing with the first ‘Gefissanlagen.’? The circular rings do not show in their lumen anything like blood corpuscles. Numerous mitoses are seen in the embryonic shield and in the allantoic stalk.” History oF THE Case From Waicu THE Ovum Cameg.® Our own ovum, while not coming up fully to the most ideal re- quirements, approaches them so closely that it must indeed be a ‘The first more extensive communication concerning our specimen was made before the Section of Embryology of the Seventh International Con- gress of Zoology, held in Boston, in August, 1907. It was then the privilege of the author to demonstrate the sections to such well-known European and American embryologists as Professors A. A. W. Hubrecht, F. W. van Wijhe, 368 Maximilian Herzog. very uncommon combination of circumstances which will furnish an investigator with a still better, equally young, human specimen. The ovum was obtained im situ from a healthy woman, with absolutely normal and healthy genitalia, who was almost instantly killed by a most peculiar accident. The body of the woman was brought at once to the public morgue, and there placed in a large refrigerator. The post-mortem exam- ination was made by the author, and the findings, as usual were recorded in the shape of a post-mortem protocol, which here follows, verbatim, as written down at the time. Mrs. M. R., from Intramuros, Manilla; Filipina, aged 25, died July 17, 1904. The post-mortem examination was made July 18, twelve hours after death. Immediate cause of death not known. It was stated that she had been struck by a small carriage (called carromata in Manila) shortly before she died. Body of a well-developed young native woman, twenty-five to thirty years of age. Post-mortem rigidity strongly marked. Post- mortem lividity quite noticeable. Abdomen’ somewhat distended. A repeated careful inspection fails to show any signs of ex- ternal violence. No wounds, contusions or abrasions of any kind to be seen. On opening the thoracic cavity, the pericardium is found to be much distended, and shining through it there appears to be a firm, dark, blood coagulum. On opening the pericardium, it is found that it contains a large amount of dark, coagulated, gel- atinous blood, and blood-tinged serum, distending the pericardium ad maximum and compressing the heart. A careful examination fails to show any perforation in the pericardium. The heart, which weighs 226 grams, presents a perforation, which begins two centi- meters to the left of the anterior border of the interventricular sep- tum. The perforation extends almost horizontally toward the left, being a little downwardly inclined. It forms a slit 2.2 centi- Charles Sedgwick Minot, Franklin P. Mall, A. Playfair McMurrich, T. G. Lee, and F. T. Lewis. Previous to this communication, the specimens had been demonstrated in June, 1907, before the German Medical and the Gynecologic Societies of Chicago. Embryonic Development in Man. 369 meters below the sulcus of the heart. The edges of the perforation are almost clean-cut where they enter the myocardium, as if they had been produced by a dull, somewhat serrated knife. The cut takes a somewhat downward and inward course, traveling through the whole thickness of the myocardium. Where the cut enters the cavity of the heart, the margins are not very smooth, but rather irregular and ragged. The consistency of the myocardium is good. Its color is pinkish-brown, and all the serous surfaces are smooth. There are no atheromatous changes. The heart is covered with a very moderate amount of epicardial fat. In short, the whole organ, except for the wound, is absolutely normal. After the removal of the lungs (the apex of the right one showing a very few tubercles, and a little caseous nodule not larger than a lentil), it is seen that the second, fourth, and fifth ribs of the left side are fractured. The fracture of the second rib is found to be 7.5 centimeters pos- terior to the sternal articulation, that of the fourth one 9.0 ecm., and that of the fifth one 9.5 em. ‘The anterior fragments are di- rected inwards. The fragments of the fourth and fifth ribs are very sharp and are surrounded by an area of subpleural blood extravasa- tion. However, these fragments have not perforated the pleura cos- talis. The extravasated blood is strictly subpleural and no free blood is found on the surface of the pleura. The uterus appears somewhat enlarged, and the left ovary shows a fresh but already closed corpus luteum. On opening the uterine cavity a little hemorrhagic mass, about one-half centimeter or less in diameter, 1s found em- bedded in the mucosa of the posterior wall, near the entrance of the left tube. This mass is carefully cut out and placed in Zenker’s solution, as it may contain a very young ovum. All the organs of the body, with the exception of the apex of the right lung, and the perforated heart, are found to be absolutely normal. They are all more or less congested with dark fluid blood. It appears clear that the woman must have been struck at the side of her body, or in the back, by a swiftly moving force. ‘This force, however, did not produce any signs of external violence, particularly no con- tusions, abrasions, or wounds. ‘The force traveled through the soft parts, and, meeting the resistance of the ribs, fractured them. The 370 Maximilian Herzog. anterior sharp fragment of the fourth or of the fifth rib was evi- dently driven into the wall of the left ventricle, producing a com- plete perforation. A highly interesting point is that the sharp frag- ments neither perforated the pleura costalis nor the parietal layer of the pericardium. Only when the resistance of the firm wall of the ventricle was encountered did a rupture or perforation occur. A hemorrhage took place, and when the pericardium was completely filled, and the myocardium much compressed, the heart’s action came to a sudden standstill. Death occurred from syncope at once. ANATOMICAL DIAGNosIs. Fracture of the second, fourth, and fifth ribs of the left side. Complete perforation of the wall of the left ventricle. Hemorrhage into the pericardium. Compression of the myocardium. Begin- ning tuberculosis of the apex of the right lung. Microscopic examination of the myocardium showed it to be perfectly normal. To this history may be added the statement that it was later learned through the police reports that the woman had indeed been struck by the swiftly-moving shaft of a small carriage; that she fell forward, got up, staggered, fell again, and was dead within a very short time.’ The small piece of tissue removed from the uterus was placed in Zenker’s solution, later washed in running water, and was then embedded in paraffin. Since our facilities for cutting serial sections were not the very best at this time (July, 1904) in Manila, and since the writer was then engaged in the study of Bubonic plague, a few sections only were prepared, and the bulk of the block was preserved for future work. From the few sections examined, the firm impression was gained that the uterine mucosa presented the picture of a very early decidua, with cystic hemorrhagic gland spaces "The history of this case has previously been published by the author in a paper: “Peculiar Cases of Traumatism of Internal Organs, Some Due to Tropical Conditions and Practices.” Surgery, Gynecolocy and Obstetrics, Vol. IV, No. 6, p. 741, Chicago, 1907; and Philippine Journal of Science, Vol. 2, 1907. Embryonic Development in Man. 371 of the type of those of the Peters’ ovum. However, the first see- tions did not show either the cavity of the ovum or a trace of the trophoblast. GENERAL DescripTION OF THE OVUM. On June 15, 1907, the block of tissue was finally divided, and I am obliged to Dr. Day, Pathologist in the Chicago Laboratory of the Bureau of Animal Industry, for assistance in preparing a complete series of sections. The individual sections were all 7 microns thick; in all they numbered 303. About three sections were lost on the microtone; several subsequently floated off partly or en- tirely from the slides during the process of staining in hematoxylin and eosin, but fortunately none of the important sections containing the embryonic shield were lost.* The general outlines of the ovum are those of a bi-convex lens, a somewhat flattened elliptical body. The ovum was found in sec- tions Nos. 63 to 235; the embryonic shield in sections 142 to 164. The measurements obtained by micrometer are the following: Ovum (chorionic cavity-exoccelom): Greatest length (section 153), 2.326 mm. Greatest width (section 153), 0.804 mm. Great- est thickness (172 sections at 7 microns each), 1.204 mm. The trophoblast begins to show in section No. 47, and ends in section No. 264. Since the chorion mesoderm begins to show in section No. 63 and ends in section No. 236, the trophoblast on one pole is 16 sections or 112 microns thick, and on the other 29 sec- tions, or 203 microns. In section 150 the trophoblast, towards the muscularis, is a little over one millimeter thick; towards the upper surface, 0.9 mm. A list of the smallest human ova described, compiled from Peters’ and Jung’s collection, follows: ‘The author has in the case under discussion and in a few cases of early tubal pregnancies attempted to obtain complete and perfect series of ova in situ by the paraffin embedding method. However, experience has taught him that young human ova in sitw presenting very heterogeneous tissue ele- ments, including masses of maternal blood, are not best adapted for the paraffin method, but should preferably be embedded in celloidin. 372 Maximilian Herzog. Bryce and Teacher: 0.77, 0.63, 0.52 mm. Peters: 1.6, 0.9, 0.8 mm. Herzog: 2.326, 0.804, 1.204 mm. Graf von Spee: 2.5, 1.5 mm. Jung: 2.5, 2.2 mm. Mertens: 3.0, 2.0 mm. Beneke: 4.2, 2.2., 1.2 mm. Leopold: 4.0, 3.7 mm. Graf v. Spee: 4.0 mm. von Heukelom: 4.5, 5.5. Beigel-Loewe: 4.0, 5.0, 2.5-3.0 mm. It appears from the above table that the author’s ovum is some- what larger than Peters’ ovum. However, the real difference in the cubical contents of the two ova cannot be much, since the Ma- nila ovum, while markedly larger in its longest diameter, is indeed not strictly elliptical, like the Peters’ ovum, but has the shape of a biconvex lens. It is useless to speculate whether or not the Manila and the Peters’ ovum are of exactly the same age, or whether the former is a little older than the latter. We have in our case not made any efforts, at the time when the ovum was obtained, to get data as to menstruation and probable time of con- ception. Such an attempt would have been useless in the case of a full-blooded native from the lower walks of life. Even in those reported cases of very young ova, where these data were obtain- able, the estimates as to the age of the embryo are not very cor- rect. Peters estimated the age of his ovum as of three to four days; Leopold, an older ovum (4.0; 3.7 mm), as seven to eight days old. Jung thinks that the Peters’ ovum is considerably older than three to four days; Bryce and Teacher estimate its age at thirteen to fourteen days. Von Spee and Minot believe that seven to eight days intervene from the time of fertilization in the tube till implantation occurs in the uterus. The age of a human ovum, hke that of the lower mammalian animals, whose early embry- ology has been sufficiently studied, can probably be best estimated from the stage of development of the embryo proper, but the data in this respect as to very young human embryos are insufficient. Embryonic Development in Man. 373 Description OF THE EMBRYO. The sections of the embryo have been accurately drawn under my direction by Miss Grace Amadon, and are shown in Plates I to IV, Figs. 1 to 22. The embryo is first seen in section 164 (Fig. 1 of the embryo), and the ectoderm cells of the embryonic shield were last encountered in section 149. Hence the whole length of the embryonic shield proper is 16 by 7 microns equals 112 microns. However, the mesoderm extends as a thickened mass beyond the ecto- dermal limit of the embryonic shield from sections 150 to 1438, or over a distance of 8 sections, equal in all to 56 microns. We have here what appears to be an extension of the shield-mesoderm beyond a mesoderm “Vorhof.” Includ- ing the mesoderm “Vorhof,” the embryonic shield extends through twenty-two sections; its whole length therefore is 22 by 7 microns equals 154 microns. The first section (164) has hit the embryo in a tangential man- ner, and shows only a single layer of cells. The three following sections (Figs. 2 to 4) consist of an inner layer of ectoderm and an outer layer of mesoderm. In the next section (Fig. 5) a delicate strand of cells is clearly seen between the ectoderm and mesoderm. This must be interpreted as a subdivision of the meso- derm. It continues into the eighth section, in which the entoderm first appears. The entoderm is represented by a small group of the shield ectoderm and entoderm cells between the two layers of mesoderm, seen on the upper side of Fig. 8. In Fig. 9 the entoderm has expanded so that the cavity of the yolk sac now appears. It lies in the upper half of the figure. The embryo is anchored to the chorion by an allantoic stalk, com- posed externally of mesoderm and traversed by a rather slender, somewhat curved canal of entoderm cells (Figs. 9 to 14). The con- nection between the entoderm of the allantois and of the yolk sac presumably occurred in sections 156 and 155, but the continuity of the entoderm has been destroyed. Around the allantoic stalk (Figs. 10 to 13) where its mesoderm is continuous with the yolk sac mesoderm, there are found some solid and some open circular masses of mesoderm cells. The open 374 Maximilian Herzog. rings are composed of three to four to five mesoderm cells; the solid round or oval cords contain a larger number of cells. These formations undoubtedly represent the earliest “anlage” of the yolk sac blood vessels. The chorion mesoderm and the mesoderm where it extends somewhat into the trophoblast do not yet show any traces of blood vessels. The cavity of the yolk sac, which first appeared in section 156 (Fig. 9) remains small and slit-like through sections 157 and 158. It then gradually increases and attains a transverse lateral diameter of about 176 microns in sections 152 and 150. In section 143, where the last of the “Vorhof’ mesoderm is seen, the transverse diameter of the yolk sac is 192 microns. From here on it hangs down free for a considerable distance into the exocclom. It can last be seen in section 122. Its entoderm and mesoderm layers are there very distinct. The yolk sac must have extended somewhat beyond section 122, but from 121 on it has been lost in the sections. It extended through at least thirty-four, and probably through forty sections, hence its greatest sagittal diameter was between 250 and 500 microns. The cavity of the amnion, which has a diameter of from 100 to 160 microns in the first sections, becomes reduced to a canal in section 153, having a lateral diameter of 45 microns and a dorso-ventral diameter of 80 microns. This canal terminates in section 149 (Fig. 16). In the first sections of the embryo (Figs. 2 to 8) the amniotic cavity is almost circular. Internally, it is bounded below by the thick embryonic shield, which is curved so as to form a deep crescent. Its concavity is toward the chorion. The ends of the crescent are continued as the very thin amniotic epithelium. However, the amnion is not complete in this region because its two lateral wings stop short and do not meet in the median sagittal plane. This seems clearly due to the artificial rupture of the very thin membrane. With equal certainty it may be said that the great concavity of the ectoderm shield is not artificial, but was present ante-mortem. So thick a layer is not liable to distortion, and its cells show no evidence of disturbance. Some embryologists have expected to find an inversion of the germ Embryonic Development in Man. 375 layers in early human embryos. The “Blattumkehr” in monkeys has been defined by Selenka as follows (Biol. Centralbl., Vol. 2, p. 552) :— “Der Embryonalbezirk . . . ist gezwungen sich . . . im Innern der Keimblasse einzustiilpen, wobei das Entoderm zur kap- penartigen Hiille ausgeweitet wird, die Keimblitter sind daher an dieser Stelle umgelagert, umgekehrt, invertirt.” It is now known that no true inversion of the layers occurs in man, but, as stated by Bryce and Teacher, “there is no doubt that the plate of embryonic ectoderm is inturned, and there is strong prob- ability that the condition is a primary one and not due to a precocious formation of amnion folds.” (Page 34.) The primary infolding of the embryonic shield is strikingly shown in Figs. 2 to 8. Where the embryonic shield forms the floor of the amniotic cavity in sections 156 to 153 (Figs. 9 to 12), there is an opening through the ectoderm in the median sagittal plane of the shield. No other indication of a neurotic canal was found. The opening, however, is presumably an artefact. Similar ruptures through ectoderm are seen. in sections 13 and 15. Around the median aperture the ecto- derm is not continuous with the entoderm, as would occur if the structure were a true neurenteric canal. The cells of the three germ layers as seen in this embryo may be described as follows: The ectoderm of the shield is composed of more or less cuboidal or cylindrical cells, which are quite epithelial in character. The nuclei are round or oval, with a finely granular chromatin network, and with generally one, occasionally two, distinct, deeply-stained nucleoli. The cell protoplasm is generally very finely eranular and stains moderately deeply with eosin. The chromatin network can be particularly well seen in the first sections of the ectoderm shield where the cells have been cut very favorably for this observation and where they are not so densely crowded as elsewhere. The karyokinetic figures are generally in the monaster stage. No distinct diasters were found. One cell was seen with two small vesicular nuclei containing densely but finely granular chromatin, and one pair of cells with the same kind of nuclei and an incomplete division of the protoplasm. 376 Maximilian Herzog. The ectoderm cells of the amnion are in general of the same de- scription as the shield ectoderm cells; however, they have smaller protoplasmic bodies, which are not cuboidal, but rather elon- gated. The mesoderm cells of the embryonic shield have generally oval nuclei and exceedingly little protoplasm; they are connected with each other by very thin, filamentous, bipolar processes. The yolk sac and amnion mesoderm cells are elongated and connected with each other by bipolar processes. But where the mesoderm cells are most numerous, namely, at the allantoic stalk, their nuclei are larger and their protoplasm, which is fairly abundant, is generally irregularly polygonal. The entoderm cells of the shield have nuclei very much like the ectoderm cells, but the protoplasmic bodies are smaller and often somewhat elongated. These cells are likewise more or less con- nected with each other by bipolar though shorter and coarser pro- cesses. The entoderm cells of the yolk sac have rather small nuclei, rich in chromatin, and, where they can be seen at their best, are almost geometrically cuboidal. They are rather small in size in comparison with the shield ectoderm cells. The entoderm cells of the allantois are like those of the yolk sae. DeEscrRIPTION OF CHORION AND DercrIpua. Nomenclature. In order to facilitate the description of the chorion and of the decidua in which it was found embedded, as well as to avoid any misunderstanding on the part of the reader, it is well to outline shortly the nomenclature used. That part of the uterine decidua on which the inner pole of the ovum rests and which is characterized in our case by the presence of large cystic gland spaces and lacunz, densely crowded with blood, we will call the decidua basalis. That thin strip of decidual tissue which separates the ovum from the uterine cavity will be desig- nated as decidua capsularis, and that part of the decidua surrounding on all sides the equator of the ovum we will call the decidua vera. Embryonic Development in Man. 377 In the latter at some distance from the ovum an inner spongiosa and an outer compacta can generally be easily distinguished. The mesoderm lining the interior of the chorionic cavity or exoccelom will be designated as the chorion mesoderm, while the chorion ecto- derm will be called trophoblast. This term has been proposed by Hubrecht, and it is understood and used so generally that it is perhaps well to retain and not to replace it by other terms.° The trophoblast is composed of two kinds of tissue, the inner cell masses and an outer covering of syncytium. We want to state here that the examination of our young ovum has confirmed our opinion, expressed a number of times previously, that both the cells proper of the trophoblast (the future Langhans cell layer) and the syney- tium are derived from the fetal ectoderm. We have never pre- viously in hundreds of placentze in normal and in ectopic gestation, nor in our present case, found anything which would seriously sug- gest a derivation of the trophoblast syncytium from maternal cells. The ovum under discussion nowhere shows a possibility that the syncytium is derived either from surface or glandular uterine epithe- hum, from vascular endothelium, or from decidual cells. Hence the term ectoblast shell for the combined trophoblast cells and the syney- tium is correct. We retain the well-known term trophoblast in spite of the fact that we consider it as to its etymology a misnomer. We have previously expressed ourselves on this subject, as follows: “The term trophoblast has been given by Hubrecht to the extra- embryonic ectoblast shell under the impression that it had to do a good deal with the nutrition of the early embryo. We doubt, however, that *Minot, in an address on “The Implantation of the Human Ovum in thé Uterus,’ delivered in 1904 before the Gynecological Society and printed in its transactions, has proposed the term trophoderm, but he states himself: “In the address the term trophoblast was used in accordance with my under- standing of Professor Hubrecht’s views and consent; but, as Professor Hu- brecht has objected to this application of his term, it has been necessary to propose a new one. I regret that so good a name as trophoblast has to be dropped.” With this address the author only became familiar after the completion of the manuscript of this contribution. He now finds that his views of the physiology and the mechanism of the implantation of the human ovum given are in many respects identical with the previously pub- lished explanations of Minot. 378 Maximilian Herzog. this is the case. The mass of the trophoblast in our case is certainly many thousand times that of the embryo. It does not stand to reason to assume that nature in the phylogenetic development would pro- vide, so to speak, at an enormous expense, a very large apparatus for the nutrition of a very small embryo. It appears more reasonable to assume that the trophoblast with its great proliferative energy, which we have likened to the growth of a malignant tumor, has more exclusively the function to provide the means for the embryo to safely implant itself at the very earliest date into the maternal tissues. The reaction of the maternal tissues in contact with the proliferating trophoblast must not be leoked upon as due to mechan- ical causes only, but to fermentative action of enzymes secreted by the trophoblast cells and diffused into the neighboring maternal tissues.” The above statement we still hold to be correct on the whole. However, we agree with Bonnet*® that the syncytium presents feat- ures, namely its property to stain very deeply with eosin, which suggest the possibility that it takes up hemoglobin from the maternal blood for the benefit of the nutrition of the embryo. The term syneytium in connection with placentation has been used very promiscuously and has been inaccurately applied to de- generating confluent cell masses of maternal origin. According to Bonnet, the term syncytium has been introduced into histology by Haeckel, who designated by it a nuclei-containing plasma, formed by the confluence of previously separate and distinct cells. Taken in this sense, the term syncytium as appied to the human tropho- blast is probably a misnomer. It is very likely—though nothing about this is known from actual observation—that the syncytium of the human placenta is formed ‘‘ab origine,’’ as an outer strip or capsule of protoplasm which is provided with expelled nuclei from the cells of the inner cellular trophoblast. In a publication “On the Pathology of Tubal Pregnancy,” quoted in a footnote above, I have considered the syncytium of the human placenta as the homo- *Bonnet: Ueber Syncytium, Plasmodien und Symplasma in der Placenta der Siiugethiere und des Menschen. Monatschrift fiir Geb. u. Gyn., 1903, Vols als joy ae Embryonic Development in Man. 379 logon of the periblast of transparent pelagic fish eggs, such as those of Fundulus, which I had a chance to study in the Summer of 1899, in the Woods Hole Marine Biological Laboratory, under the direction of Professor C. O. Whitman. In these fish eggs the for- mation of the periblast—an outer ‘capsule of plasma without cell boundaries, but with numerous nuclei—can, of course, be studied from stage to stage under the microscope, and the expulsion of nuclei into the plasma can be seen. In spite of the fact that the syneytium of the human placenta does not deserve this name in the sense as originally applied, it is well to preserve its use, since it has been universally apphed to the outer covering of the trophoblast, and of the later chorionic villi. For degenerative confluent cell masses in the placenta, whether they be of maternal or fetal origin, Bonnet has proposed the term symplasma, and he distinguishes between symplasma maternum and symplasma fetale. These terms have been accepted by Jung in the descrip- tion of his ovum, and we will likewise introduce them into our considerations. Some of the German writers on placentation, fol- lowing Bonnet, have come to use the terms “Grundschicht,” for the cellular part of the “Trophoblast” (the later Langhans layer), and “Deckschicht,” for the syneytium. The General Position of the Ovum and its Mode of Entrance into the Decidua. At the time of the autopsy, as stated above, the ovum, or rather the small hemorrhagic spot, was found at the posterior wall of the corpus uteri, comparatively high up in the fundus, and near the entrance of the left Fallopian tube; that is, on the same side where the corpus luteum ovarii was noticed. The dark, hemorrhagic spot which contained, as was later on found, the ovum was only very slightly prominent over the remainder of the thick, velvety mucosa. The uterus, after the careful removal of the dark spot, which was excised as a cubical mass, was preserved in the patho- logic collection of the Government Laboratory, but I have not had a chance to re-examine it during my stay in Manila, and I do not know whether it has been preserved permanently or not. 380 Maximilian Herzog. The mass removed was sectioned from above downward, and the ovum, which can best be seen as a whole in photomicrograph (Fig. 24), was found situated with it long axis parallel to a line drawn from one ostium internum tubae to the other. A glance at the photomicrograph (Fig. 24) shows the follow- ing points as to the position and surroundings of the ovum in general: The ovum, including its trophoblast and the thin strip of decidua capsularis, protrudes very slightly above the surrounding surface. From the cavity of the uterus it 1s separated by a very thin decidua capsularis. This in the very center of the upper line of photomicrograph (Fig. 24, section 125) is slightly deficient, and here we see a teat-like process of the chorion mesoderm extend- ing almost to the surface. The inner pole of the ovum rests on a wedge-shaped mass of very large cystic gland spaces densely filled with blood. The decidua vera near the ovum shows densely crowded hypertrophied gland spaces. They are somewhat cystic towards the muscularis, and towards the free surface present enlarged tortuous tubes, separated by intervening septa. The differentiation into a decidua compacta and spongiosa, which is not well shown in Fig. 24, appears more clearly in the photomicrograph Fig. 26. Large cystic glandular blood-filled spaces are found not only at the base of the ovum, but also towards one side. Such spaces were found also in the first set of sections examined, 7. e., in sections which showed neither the trophoblast nor the chorionic eavity. If one studies sections 11 to 39 of the complete series, which hke- wise do not yet show any trophoblast, the following can be seen. Near the surface, under a thin strip of decidual tissue composed of large, partly-degenerated, decidual cells, there is an opening or hole surrounded on all sides by profoundly degenerated decidua. The decidua here is least degenerated at the upper stratum (capsu- laris), and most markedly degenerated at the inner (basal) aspect. The decidual cell masses are infiltrated with maternal blood, and are surrounded by large cystic gland spaces (Fig. 27) filled either with blood, with hyaline, eosin-staining balls or masses, with degen- erated, dropped-off glandular epithelium, or with networks of fibrin. The hole described is not empty, but more or less filled with blood Embryonic Development in Man. 381 and dropped-off degenerated decidual cells. In section 11 the canal or hole has no covering towards the uterine cavity, but the mass of blood and degenerated, chaotically distributed, decidual cells reaches to the very surface. It is clear that this canal, which is more or less circular in outline, with diameters varying from 1.5 to 1.0 mm., indicates the route over which the ovum traveled to the place where it was found in the decidua. It is probable that the ovum after having been fertilized in the tube was brought to a spot indicated by the surface opening in section 11, or thereabouts. The ovum when less than one millimeter in diameter (this measurement, including, of course, the chorionic cavity and the whole then existing tro- phoblast) began to make its way into the decidua. It did, however, not eat its way deeply in at all, but traveled under the surface almost parallel to it, being separated from the uterine cavity only by a very thin strip of decidual tissue, in some places so thin that it became slightly defective, or at least apparently so. After the ovum had traveled through the canal described, of which there are about 200 to 250 micra in length left, it must have become sta- tionary and must have begun to expand inwardly, towards the muscularis, and also laterally. It is clear that the ovum as found im situ in the decidua is much too large to have traveled through the canal, and it must have been much smaller, probably consider- ably smaller than one millimeter, at the time when this migration took place. The canal, as stated, was found moderately well filled with blood corpuscles and degenerated decidual cells. We have, therefore, in our case, unlike Peters, who had in his specimen a mushroom coagu- lum, a still patent, though partly filled, small canal, running almost parallel to the uterine surface, through which the ovum by its own inherent destructive tendencies made its way to the place of final implantation. While the outer end of the canal described com- municates with the surface and is practically open towards it, ex- cept as to the presence of maternal blood and degenerated decidual cells, the inner end is closed by the trophoblast of the ovum. On the opposite point of the ovum, which is found in section 264, where 382 | Maximilian Herzog. the trophoblast is last seen, we likewise have evidences of its great destructive tendencies. The next sections show numerous degenerating, dropped-off decidual cells, mixed with blood. Thus, there is formed here also a kind of cavity filled with blood and detritus. However, it does not reach the surface, but remains sep- arated from it by decidual tissue. We see the ovum then every- where more or less surrounded by cystic blood spaces or free blood. In this respect the early human ovum is very much like that of the hedgehog and mouse, as described by Hubrecht and Bonnet, surrounded on all sides by maternal blood, floating as it were in a lake of blood. The conditions in our ovum are more or less identical with what had been found by Peters in his case. It can be seen in Fig. 25 that the embryonic shield is anchored by its allantois to the chorion mesoderm near the inner pole of the ovum and farthest away from the outer pole and the thin capsu- laris. The position of the embryo with reference to its outer and inner poles is thus identical with that of Peters’ ovum. In the Jung ovum likewise the embryonic shield appears to hold the same relations to the maternal organism, through orientation in this last- mentioned specimen, which was not obtained in situ, but by curette- ment, is not absolutely certain. The apparently identical position of the embryonic shield in the three young human ova is very prob- ably not at all a matter of chance or accident, but is due to a very early automatic orientation of the ovum for the benefit of the developing embryonic shield—from a _ biogenetic standpoint the most important part of the ovum. Examining the sections which show the beginning and the course of the canal, the following conditions can be ascertained: The entrance of the canal is evidently not ina gland duct, but in an interglandular septum, through a decidua which here, in spite of profound degenerative processes, shows well the character of a compacta. On both sides of the entering canal we see the enlarged tortuous necks of uterine glands. Enormously enlarged capillaries reaching from the sides of the entering canal to the very surface can also be seen. . Both at its entrance and along its course the canal is sur- rounded by enlarged gland spaces. The glandular epithelium every- Embryonic Development in Man. 383 where shows profound degenerative changes, and the large eystic glands are partly or completely filled with blood and detritus, as already mentioned. There is no trace of glandular or surface epithe- lium left at the site where the canal takes its origin from the surface. Nor can one find any gland ducts opening into the canal, or into the cavity in which the ovum now is situated. All of the condi- tions point unmistakably to the fact that our own ovum, lke that of Peters, penetrated into the decidua not through a gland space, but by eating its way through interglandular tissue of the compacta. It does not appear necessary to us to assume that the ovum can make its way into the decidua only through a spot denuded of the surface epithelium. Since the enlarged gland spaces, even where not in direct contact with the trophoblast, exhibit most marked degener- ation of the lining epithelium, we may well assume that the tro- phoblast, in contact with the surface epithelium, can destroy it easily and make its way into the connective tissue of the decidua. In our preliminary communication; read in August, 1907, before the Zoological Congress of Boston, we said: “We cannot conclude this preliminary report without pointing out what we might call the pathologic aspect of the early stages of placentation in man. The proliferation of the trophoblast, the manner in which it invades the maternal organism, pushing aside, destroying and changing ma- ternal tissue elements, vascular and other structures, is the exact picture of malignant tumor proliferation, while the reaction of the maternal tissue, taken for itself alone, reminds one forcibly of a profound destructive hemorrhagic inflammation. It is very striking to the pathologist to behold in early placentation, in the apparatus and the phenomena which enable the young ovum to anchor and implant itself firmly into the maternal organism, the very para- digma of two such important pathologic processes as malignant tumor growth and hemorrhagic inflamation.” A further study of the sections has only strengthened the im- pression gained previously. The destructive tendencies of the early trophoblast of the ovum are certainly very marked. If some hypo- thetical speculations may be here permitted, we would like to ex- press our opinion that the trophoblast at a,certain stage of its 384 Maximilian Herzog. development secretes an enzyme which diffuses into the surround- ing maternal tissues and here causes coagulation necrosis and com- plete degeneration of cells. The trophoblast cells, as represented by our specimen, are certainly not phagocytic in the ordinary sense of the word. We have in vain examined and re-examined our sections to find included in the trophoblast cells or the syncytium maternal blood corpuscles, fixed tissue cells or fragments or remnants of the same. We must, therefore, conclude that the effect of the tropho- blast upon the maternal tissue is brought about not by true phago- cytosis, but through the action of an enzyme. If the latter destroys maternal cells to a large extent, and this destruction, of course, takes place, as can be seen, we have those conditions which under any circumstances would lead to violent inflammatory reac- tion, including enormous dilatation of veins and capillaries; free hemorrhages; and if the process take place in a glandular mucosa, with hypertrophy of the glandular apparatus. And, indeed, if we look upon the decidua in our specimen particularly, as seen in Fig. 24, to the left of the ovum, and in Fig. 26, in the whole section, the resemblance between them and a typical, well-marked case of endemetritis glandularis hypertrophica is very striking. In fact, when the set of sections represented by Fig. 26 was shown to a very competent pathologist with the statement that it was very probably a very early decidua and that an ovum would be found in the block of tissue, he ridiculed the idea and firmly held that the section simply showed a typical strongly-marked case of endometritis glandularis hypertrophica. If we behold the great destructive tendencies of the early tro- vhoblast, the question presents itself, Why does this process evidently come almost to a standstill somewhat later in the course of gesta- tion? Two possibilities present themselves. Either the secretion of the supposed destructive trophoblast-enzyme is limited as to time, or there is established a temporary immunity of the maternal tissues, These speculations, while at present entirely hypothetical, might perhaps be supported by experiments in which the effect of filtered extracts of animal placentee of various stages of development, and in repeated applications, upon the uterine mucosa, would have to be studied. Embryonie Development in Man. 385 Peters, in his case, has shown how the ovum did not make its way into a gland duct, but had eaten its path into the decidua and he opposed the old theory as to the formation of the decidua reflexa. He, however, conceded the desirability of demonstrating the mode of entrance of the ovum in more than one case examined in situ. Our own case, offering in some regards even better conditions, namely, almost absolute certainty of the absence of all pathologic deviations from the normal type, fully confirms the view of Peters of the mode of implantation of the ovum and of the erroneousness of the older theories. Both the Peters and our ovum show the cor- rectness of Count von Spee’s't hypothesis that the human ovum would be found to behave in its method of implantation into the uterine mucosa like the ovum of the guinea-pig. It was shown by von Spee that the dividing blastoderm of the guinea-pig eats its way through the uterine epithelium, into the connective tissue, causing here edema and hyperemia. Exocelom and Chorion Mesoderm. The chorionic cavity or the exoecelom, as to its size and shape, has been sufficiently described. The position of the embryonic shield, yolk sac and allantois have also been indicated. Aside from the ‘‘Keimanlage,” the exocelom shows in its interior a finely granular, irregularly lumped material, which has stained intensely with eosin. This material can be well seen in photomicrographs, Figs. 24 and 25. It is responsible for the fact that the sections of the embryo could not be photographed so that they appear on a clear homogeneous background. What is seen in the sections with reference to the eosin-staining granular material proves that the exocelom ‘inter vitam” was filled with a watery fluid rich in coagu- lable proteids. The strongly eosin-staining properties of the gran- ular material may perhaps be due to an absorption of hemoglobin indirectly derived from maternal sources. ‘Towards the periphery, that is, towards the lining chorion mesoderm, and running parallel “von Spee: Die Implantation des Meerschweincheneies. Zeitschr. f. Morph. u. Anthrop., 1901, Vol. 111, p. 180; and Ueber die menschl. Eikammer, ete., Verh. der Anat. Ges. zu Kiel, 1898, p. 196. 386 Maximilian Herzog. with it, are seen long slender fibers, more or less mixed with granular detritus. These fibers are evidently the remnants of de- generated, dropped-off lining mesoderm cells. The latter themselves are comparatively long and fusiform, with very gradually taper- ing long bipolar processes. Their protoplasm is finely and distinetly eranular, well eosin-staining. The nuclei are oval, sometimes al- most rod-like, with rounded ends like the nuclei of involuntary muscle cells. They have a fine but darkly-staining chromatin retic- ulum; often one or two nucleoli can be seen. The chorion mesoderm in most sections has slightly retracted from the trophoblast ectoderm and we here can see distinctly along the outer margin of the meso- dermal lining a fine, sharply-cut membrana limitans, as described by Bonnet, in a more advanced older ovum as separating the meso- derm of the villi from their ectoderm. The chorion mesoderm forms teat-like or finger-like processes arising from the periphery and extending outwards into the ectodermal trophoblast. These processes likewise show the fine limiting membrane. Sometimes these pro- cesses arise near each other, but they do not yet show any dichotomous division. Here and there are seen floating in the exoccelom bands or filaments of mesoderm cells. They are interesting from the stand- point of the pathologist, because their occasional growth and _ per- sistence may lead to the formation of those so-called amniotic bands responsible for disturbance in the normal development of the em- bryo. These mesodermal bands and strands, crossing the chorionic cavity, have also been described for their respective specimens by Bryce and Teacher, Peters and Jung. They are important because they have been interpreted as the remnants of a once solid mass of mesoderm, existing before the formation of the ccelom and exo- coelom. Keibel (Normentafeln, Vol. 8, p. 12) in discussing the early mesoderm and the formation of the exocelom says: “In man at a stage when a primitive streak cannot yet be demonstrated with certainty or even does not exist, we find the whole embryonic shield, yolk sac and amnion richly surrounded by mesoblast, as is also the internal surface of the chorion. Spee, in describing his embyro H, says: ‘It appears almost inconceivable that the region of the still Embryonic Development in Man. 387 smaller primitive streak of an earlier period has furnished these masses (of mesoblast). Probably a small mass of mesoderm 1s produced from a primitive streak at a very early stage, and later this mass proliferates independently.” “Although this view of Spee,” Keibel continues, “appears to be the most probable, it is well to point out that it is only an hypothesis, which encounters many difficulties. We cannot at present state anything more definite as to the origin of the mesoblast in man. Something more certain may be said of the origin of the ccelom, although its earliest stages have also never been observed in man. It is certain, that, as in mammals generally, the extraembryonic colom (the cavity of the ovum) is formed earlier than the embryonic caclom, and we are probably correct if we assume that the extraembryonic ccelom is formed by cleavage (Spaltbildung) in a mesoblast which has already developed. This would be in accordance with what is known of the other mammals. The Trophoblast and Its Syncytium. The early characteristic trophoblast of the human ovum, though first correctly described by Peters from his specimen, had pre- viously been hypothetically constructed from the observation of an older ovum in sifu, in which the peculiar ectoblast shell had been differentiated by the formation of the villi. We owe this description to van Heukelom’’, who gives it in the following words: “One can best get a conception of these conditions if one imagines all villi connected at their periphery by beams of ectoblast, so that they form an ectoblast shell full of small and large holes. This shell is unevenly thick and rests directly on the maternal compacta. ae The trophoblast as found in our specimen surrounds the chorionic cavity or exoceelom of the ovum lke a thick shell. It is, however, not equally powerful on all sides, as has been already indicated by the measurements of the trophoblast. Nor is it a solid mass of cells. It is, on the contrary, honeycombed by irregular communi- “Yvon Heukelom: Ueber die menschliche Placentation: Archiy f. Anat. u. Physiologie (Abth. Anatomie), 1898. 388 Maximilian Herzog. cating spaces contained in a network of irregular bands, strands and masses of trophoblast material. The cavities in the trophoblast are not empty, but well filled with maternal blood. There is a certain regularity of those trophoblast cavities which are situated next to the chorionic cavity. Here they are somewhat regularly cuboidal and they are placed around the chorion mesoderm like blocks of stone in a pavement. It is evident that this arrangement is brought about by two factors working in a certain sense against each other, namely, the pressure of the maternal blood and the growth energy of the chorion mesoderm. Towards the chorion meso- derm, these cavities are lined by a thin layer of trophoblast, com- posed of one cell layer of the “Grundschicht’’ and one layer of “Deckschicht” or syncytium. Towards the periphery these cavi- ties are lined by more powerful masses of trophoblast. In the middle stratum of the trophoblast there are irregular cavities of moderate size, and in the outer stratum the open, blood-containing spaces be- come very large and very irregular. In this outer zone we find the trophoblast material much diminished, and where it is in con- tact with the thin capsularis, it dwindles down to isolated thin threads or pillars of cells. The cells of the “Grundschicht,” or that part of the trophoblast which later becomes the Langhans layer of the villi, are most char- acteristic in the middle zone where they are present in the shape of bands and beams and irregular masses, and where they have not been exposed to considerable pressure, as in the zone next to the chorionic cavity. In the middle as well as in the outer zone the cells have the following characters: The protoplasm is generally almost spherical, or in consequence of mutual compression of the cells irregularly polygonal. The cell boundaries are so very distinct that it appears as if the cells had membranes. The protoplasm has stained very lightly with eosin and gives the impression that the cells “inter vitam” must have been very “saftreich.” The nuclei are large, round and vesicular, with a finely granular chromatin network. Frequently one or occasionally two nucleoli can be seen ; these likewise are more or less distinctly vesicular in appearance. The nuclei are about twice the diameter of the maternal red blood Embryonie Development in Man. 389 corpuscles, and the whole cells are three to four times as large in diameter as an erythrocyte. Karyokinetic figures are occasion- ally, though not very frequently, seen. (Fig. 29.) The trophoblast cells next to the chorionic cavity are cuboidal and compact. Their protoplasm is rather scanty, and it stains much deeper with eosin than that of the cells of the middle tropho- blast stratum; the nuclei are somewhat smaller, more oval and slightly richer in chromatin. Towards the uterine cavity the trophoblast cells form thin bands which in sections present them- selves as slender bridges connecting the trophoblast with the struct- ure mentioned before as “the thin strip of the decidua capsularis.” Where they lead up to the surface the trophoblast cells have often broken through the syneytium. Here the cells become fusiform, bipolar and while not showing any marked features of degeneration, it becomes difficult to distinguish them from what appear to be decidual cells. It has previously been stated that the decidua cap- sularis separating the outer pole of the ovum from the uterine cavity is deficient in some portions, so that the ovum, in fact, is not yet entirely separated from the uterine cavity. This impression is very strongly conveyed by one of the sections which for some reason was cut much thinner than the others (it is certainly less than 5 micra). Here one can see that the trophoblast cells have proliferated outwardly, have broken through the syncytium and have become fusiform. They reach to and form the very surface. In this section decidual cells appear to be absent from the strip which borders upon the cavity of the uterus. However, even in this strip some undoubtedly maternal cell elements can be recognized, namely, mononuclear lymphocytes and polynuclear leucocytes, and also, of course, infiltrating erythrocytes. The trophoblast cells, whether they be present in a single layer, as towards the chorionic cavity, or whether they form larger or smaller irregular masses honeycombed by blood spaces, are covered by the syncytium. This consists of a layer of protoplasm in which cell boundaries are not demonstrable. In sections the protoplasmic layer is generally rather narrow, but there and there it is thickened, form- ing projections. These are seen particularly in the most peripheral 390 Maximilian Herzog. parts. However, the numerous and large syncytial buds present in somewhat older placente are not seen. The protoplasmic strip is deeply eosin-stained, but it shows a tinge as if it had also taken up some of the nuclear stain (hematoxylin). The very margin, however, is purely eosin-stained. The protoplasm is finely vacuo- lated. The distinctly eosin-stained outer strip consists of a cuticle and cilia. The cuticle can only be distinguished here and there in favorable places, but the cilia are almost everywhere easily seen. (Fig. 30.) The syneytium in our specimen fully conforms to the description given by Bonnet as found in an early human ovum, fixed like ours in Zenker’s solution. This author says: ‘Towards the periphery the plasma of the syncytium is condensed into a stratum frequently staining very intensely with eosin, rubin or Heidenhain’s iron hematoxylin. This outer strip, variable in thick- ness and distinctness, in fact appears like a cuticle. Its free surface in all sufficiently thin sections (3 to 5 micra) shows a very dis- tinct and beautiful lining with cilia (‘‘Biirstenbesatz”’). This lining has also been described by Marchand’*® and Lenliossek. Ac- cording to the latter, these cilia or rods are not motile (they were studied by Lenhossek in a fresh preparation), and in specimens stained with iron hematoxylin they exhibit basal granules in the cuticle.” Bonnet has not been able to see such basal granules, nor are they shown in our specimen, which, however, has not been stained with iron hematoxylin. The cilla in our sections present themselves as stiff, fairly slender, moderately high rods, which form very regu- larly parallel rows. They are deeply eosin-stained and of the same color as the cuticle. As stated, they can be seen without the least difficulty almost everywhere in the sections where syncytium is found. These rods are unlike the cilia of the glandular epithelium, which can still be well seen in the innermost portion of the decidua spongiosa towards the muscularis. Since cells provided with a cuticle and cilia or rods generally have “Marchand: Beobachtungen an jungen menschlichen Hiern, Anat. Hefte IES AVE PA ey Pale Embryonic Development in Man. 391 a secretory function, it is possible that this apparatus of the syney- tium has something to do with the secretion of the hypothetical enzyme of the trophoblast mentioned above. The nuclei of the syncytium are generally oval, elongated and rather densely pro- vided with chromatin. I have, like Bonnet, not been able to find any cuticle or basement membrane between the syncytium and the cells of the trophoblast. In speaking about the outermost processes of the trophoblast, we described above how the trophoblast cells have invaded the narrow strip of outer polar tissue designated as decidua ‘apsularis. Such processes of proliferating trophoblast elements, in- cluding both cells and syneytial masses, are found extending into the maternal tissues around the whole circumference of the ovum. This zone directly surrounding the ovum, forming the soil into which such invading processes extend, has been called the “Umlagerungs- zone,’ by Peters, a term which perhaps may be best translated by the ‘‘Border Zone.” The Border Zone. The tissue which forms the bed of the ovum (Kilager) surround- ing it more or less from all sides may be divided into three parts, the decidua basalis, the decidua capsularis, and the equatorial zone or decidua vera. The border zone at the base and around the equa- torial planes of the ovum is characterized by the presence of large blood sinuses, originally formed from the capillaries and small veins of the uterine mucosa. Some of these blood lacunze have retained the outlines of vessels; others have become irregular spaces which have no resemblance to ordinary vessels. The largest of these blood sinuses in our specimen are found in the decidua basalis. near the inner pole of the ovum. But very large thin-walled blood spaces surround the ovum on all sides. They proceed from the basal decidua into the equatorial border zone, bend around the upper or outer hemisphere of the ovum, and very nearly reach the thin polar cap of tissue, the decidua capsularis. No real blood spaces are, however, found in this thin cap of tissue, but only free blood corpuscles mixed with cells either of maternal origin (decidual cells) or derived from the ectoblast shell. Very much enlarged capil- 392 Maximilian Herzog. laries and veins can also be seen at a distance from the border zone in the spongiosa and compacta of the decidua extending nearly to the surface. In the border zone of our specimen near the inner pole of the ovum there is a large irregular blood sinus about 1 to 1.5 mm. in diameter, which has been completely opened by the trophoblast and is in free communication with the blood-filled cavities of the trophoblast. On its inner side (towards the mus- cularis) this sinus is still lmed by vascular endothelium. The wall towards the trophoblast has been extensively destroyed, so that the Jarge blood space is lined on one side by much stretched but still fairly well preserved endothelium, and on the other by the irregular, ragged trophoblast. The border zone at the base of the ovum also shows many large cystic gland spaces filled with blood. Most of them can be recognized as derived from glands by remnants of dropped-off, degenerating epithelium, while the densely filled blood spaces, on the other hand, can be identified by their endothelal ning and the remnants or dropped-off floating portions of the same. But there are some cystic blood-filled spaces which cannot be readily identified as being originally glands or blood vessels. Nowhere in the border zone does the glandular epithelium or vascular endothe- lium show any proliferative processes; degenerative processes only are seen. At the base of the ovum in the layer of the border zone nearest to the trophoblast a small, rather delicate strip of canalized fibrin is found. This strip consists of a network of fibrin in which are em- bedded decidual cells, red blood corpuscles and polynuclear leucocytes. There is a more powerful mass of fibrin found in the equatorial tract of the border zone. This mass (Fig. 28) contains rather coarse threads of fibrin and great numbers of red blood corpuscles. It appears that this mass has formed in and is filling out the lumen of an enlarged blood space. We have described how the trophoblast in approaching the cap- sularis sends out. bands and filaments of cells which become fused with and are lost in the thin capsular strip. The same process can be seen around the whole periphery of the trophoblast. Par- ticularly around the equatorial plane the syncytium can be seen to Embryonic Development in Man. 393 take part extensively in this process of fusion. It appears that the syneytial masses after penetrating into the border zone have a tendency to break up into cells. Individual detached pieces of syneytium can often be recognized as such by the deep eosin stain of the protoplasm and by the rods lining the external surface. How- ever, other portions of what appears to be detached syncytium in the border zone have lost their characteristics. It is in the border zone and only in it, in our sections, that marked degenerative pro- cesses are seen, and these appear to be mostly confined to cells and tissues of maternal origin. In the border zone are also seen larger protoplasmic masses containing several generally pyknotic nuclei. We think that these are detached degenerating portions of the tro- phoblast; whether they are portions of the “Grundschicht” or the “Deckschicht,”’ we are not able to decide. We believe that the large, irregularly round cells with vesicular nuclei are derived from the syneytium, since their protoplasm stains very deeply with eosin. Peters describes numerous and profound changes in the tropho- blast. These changes Marchand has already considered to be patho- logic and probably due to the fact that the woman from whom this ovum came died from the effects of a rapidly fatal dose of caustic potash. However, Marchand also believes that the extensive pres- ence of blood in the trophoblast of Peters’ ovum is abnormal. In this respect he is mistaken, since our own ovum shows the identical condition. It appears from Peters’ monograph (p. 50 and p. 51) that he found in plasmodial masses and in the syncytium more or less normal and also much changed fragmentary red and white blood corpus- cles. He describes this quite fully, and he draws from this observa- tion the remarkable conclusion that the maternal blood with its own corpuscular elements contributes to the formation of the syneytium. Neither Bonnet nor Jung have found anything like this. Not a trace of any such process or condition can be found in our own ovum. Nowhere were red blood corpuscles in toto or in fragments seen included in the trophoblast elements. Jt is quite probable that in Peters’ case the profound intoxication with fixed alkali had so changed the red blood corpuscles, had, as we would express it to-day, 394 Maximilian Herzog. so “opsonized” them, that they became liable to be taken up by the phagocytie action of other cells. We know that red blood cor- puscles, in consequence of the action of certain bacterial toxins, are so changed that this phagocytosis occurs. We want to mention in this respect what occurs in typhoid fever when numerous red blood corpuscles are taken up by the pulp endothelial cells of the spleen. That the elements of the trophoblast of the human ovum under absolutely normal conditions do exhibit towards the maternal blood corpuscles truly phagocytic properties is certainly not proven. Our own specimen, which we consider perfectly normal, shows abso- lutely nothing which would justify such a conclusion. We see in the border zone, particularly around the equatorial planes, cells which show already almost all of the characteristics of the later decidual cells. These cells exhibit a large vesicular nucleus, with rather scanty, finely granular chromatin and obtusely fusiform or irregularly polygonal protoplasm. Between them are found small mononuclear cells and polynuclear leucocytes. This is the picture seen in places a little distant from the ovum. Towards the very interior of the “Umlagerungszone,” the outlines of almost completely destroyed gland spaces with dropped-off degenerating epithelia, red blood corpuscles and fibrin are seen abundantly. Here also are seen these cells or cell remnants with pyknotic, irregular, shrunken nuclei, and a very dense, deeply cosin-staining protoplasm. The latter we consider as detached portions of the ectodermal tro- phoblast. Protoplasmic masses containing several nuclei, which we also take to come from the trophoblast, have already been men- tioned. We have, however, not found larger masses of fused de- generating cells, either of maternal or fetal origin, hence we have no occasion in our ease to make use of either one of the terms, symplasma maternum or feetale. The Decidua. The character of the decidua as it exists in our ovum is well illus- trated in Figs. 24 and 26. In the “Umlagerungszone” and right next to its periphery the degenerative processes and the hemorrhages predominate. At some distance, however, we find a decidua well Embryonie Development in Man. 395 differentiated into a compacta and a spongiosa. How far distant from the ovum these characters have already been established we cannot say, since only the ovum and its next neighborhood have been sectioned and examined. Jung, who, in his ovum, likwise found a distinct differentiation into a compacta and spongiosa quoted Hitchman and Adler’s observa- tion of the uterine mucosa before and during menstruation. These authors, even in the absence of gestation, found in the premenstrual period a temporary formation of a compacta and spongiosa. We have ourselves studied the menstrual changes on several specimens obtained per operationem and at once properly fixed, and we have previously (The Pathology of Tubal Pregnancy), summarily de- scribed them as follows: “The capillaries of the inter-tubular con- nective tissue are enormously dilated and densely filled with red blood corpuscles. Many of the latter are also found free, outside of the capillaries, between the connective tissue cells of the inter- glandular spaces. The whole mucosa is edematous and the connec- tive tissue cells are pushed apart by the edematous and hemorrhagic infiltration. Some of the connective tissue cells, which in the inter- menstrual periods are normally all of the type of small lymphoid cells, are enlarged, oval or fusiform. ‘They assume a type found in certain forms of endometritis interstitialis and approach the type of decidual cells. It may really be said that the uterine mucosa in menstruation shows to a very slight extent the beginning stage of a decidua. Most of the surface epithelia of the mucosa are pre- served; only a few are missing here and there. Changes similar to those described as characteristic for the menstruating uterine mucosa I have twice observed in the tubal mucosa during menstrua- tion.” The decidua spongiosa is considerably thicker than the compacta (Fig. 26). In the former we find irregular gland spaces, much crowded, and separated from each other by small bridges of tissue. The proliferative energy of the glandular epithelium is shown by the fact that it is found inside of the gland space proper, in the shape of projecting papillary ridges, septa and digit-like processes. All of these masses of epithelia are lined up on a slender basis of con- 396 Maximilian Herzog. nective tissue. The interglandular connective is composed of distinctly fusiform cells with elongated deeply-staining nuclei. In some places the gland spaces project considerably into the muscularis. Towards the latter there are occasionally found between the glands groups of small round lymphoid cells of the type of the cells seen in the intersti- tial tissue of the non-pregnant uterine mucosa. In the spongiosa at some distance from the ovum there are no very markedly enlarged veins or capillaries seen. But they appear towards the zone where the spongiosa goes over into the compacta. In the latter we see the more or less straight or decidedly tortuous ducts of the glands lead- ing to the surface. Here also the epithelial lining projects in ridges and bands. Between the gland ducts solid septa are present. In the direct neighborhood of the ovum these septa contain enormously en- larged blood spaces (capillaries or veins); at some distance they show the tortuous cork-screw arteries, characteristic for the decidua compacta. The edema existing in the decidua is demonstrated in the spongiosa even at a distance from the ovum by a coagulated granular material found in the gland spaces. In the compacta the edema can be recognized in the solid septa. The cells here are distinctly pushed apart, they are embedded in a granular coagulated material. In the compacta are found cells which exhibit already quite well the characters of decidual cells. They possess a large oval nueleus, with distinct nuclear membrane, scanty chromatin and one or two nucleoli. They have a large protoplasmic body, oval, fusiform or irregularly polygonal in outlines, finely granular and well eosin-staining. Among these larger cells, small mononu- clears of the type of lymphocytes or young connective tissue cells are quite numerous. The epithelium lining the glands is best preserved in the deeper layers of the more distant compacta. It is high columnar with nuclei near the basement membrane. The cilia in favorable locations are still preserved. The profound degenerative processes seen in the glandular and surface epithelium towards the ovum have several times been referred to. No karyokinetic figures were seen anywhere in the glandular epithelium. However, a few cells with two small densely stained round nuclei were found. I am, of course, well Embryonic Development in Man. 397 aware that the specimen described is one obtained post-mortem. However, the rapid cooling on ice and the subsequent proper fixa- tion had well preserved it. This, among other things, is attested to by the fact that mitotic figures were found in the embryonic shield and in the trophoblast. None of the decidual cells, either large or small, show any karyo- kinetic figures. Jung, who describes mitoses in the decidua of his specimen on page 29 of his monograph, says: “Marchand and Bonnet are the only authors who have heretofore described mitoses in the decidua. . The present author in a paper published in July, 1898 (Super- fetation in the Human Race), described among others an aborted speciinen of superfetation. Both embryos were contained in the intact fetal membranes. One embryo was 8.6 em. long; the other 16.5 mm. The superfetation was proven by a microscopic examina- tion of the embryos and of their placentee, showing in both the different stages of development. In the description of the placenta- tion of the small ovum the following passage occurs: “Large ap- parent islands of decidual cells: In some places the decidual cells present a very beautiful feature which I had not observed before in the decidue of many other placentzee examined. As is well- known, the large vesicular, round or oval nuclei of decidual cells are, as a rule, quite poor in chromatin, which is distributed in the form of small granules in a peripheral manner near the nuclear membrane. In some places of the young placenta of this case the nuclei are rich in chromatin, consistine of coarse granules and masses, arranged in an aster-shaped manner, occasionally a disaster is fairly well recognizable. We have to deal with karyokinetic figures. That this is really the case, and that we are not dealing with a degenerative process of the nuclear chromatin is proven by the fact that the aster stage can be well recognized, and, secondly, by the observation that leucocytes are absent at the place where the karyokinetic figures in decidual cells are found. I have pre- viously pointed out that where a degenerative process—coagulation necrosis—is going on in the decidua we find great numbers of poly- nuclear leucocytes, many of which show nuclear fragmentation.” 398 Maximilian Herzog. The paper in which this statement was made shows a photomicro- graph of these cells with karyokinetic figures. In re-examining the photograph now, I find that these cells are comparatively small cells. This would agree with the description of dividing decidual cell as given by Marchand. The muscularis uteri, as far as it is included in the sections, appears to consist of muscle fibers already somewhat hypertrophied. Measurements by micrometer were not made. SUMMARY. From the object described in the preceding pages, an ovum al- most identical in size and type with the Peters’ ovum, one may draw the conclusion that these two ova represent the normal type of the earliest known stage of human placentation. The mode of placentation and implantation in both are practically alike. Cer- tain retrograde changes described in the cells and in the syncytium of the Peters ovum must be looked upon as histopathologic changes, probably due to the poisoning of the mother. This conclusion is justified not only from our own case, but alike from the studies of Marchand, Bonnet, Jung and others. If we now attempt a summary description of the ovum and its manner of implantation and placentation, we have to make a few hypothetical statements, but, on the whole, we can give a resumé based upon actual facts, as they are clearly represented by the speci- men studied. A human ovum at the earliest stage of normal development hitherto known, a stage which perhaps represents one to two weeks after fertilization, is found interstitially embedded in the decidua. It is incompletely separated from the cavity of the uterus, because it is very superficially embedded and its outer pole is protected by a thin, incomplete decidua capsularis or a coagulum only. The ovum, after having been fertilized, has been transported to or near to the place where it is found embedded. By the aid of an ecto- dermal trophoblast shell, which probably secretes an enzyme de- structive to the epithelial cells and the connective tissue of the uterine mucosa, which is then in a premenstrual or menstrual Embryonic Development in Man. 399 condition of congestion and glandular hypertrophy, the ovum pro- duces necrobiosis or coagulation necrosis in the structures of the mucosa. At the same time the trophoblast exhibiting great pro- liferative energy now penetrates through the necrotic tissue, into the connective tissue of the mucosa. Here the phenomena of edema and of a violent hemorrhagic inflammation are now established. Veins and capillaries become enormously dilated, the blood current becomes sluggish, edematous infiltration becomes pronounced. The ovum automatically orients itself, so that the embryo comes to be situated towards the muscularis. The proliferating trophoblast with its syneytium, provided with cilia or rods, at this time begins to break into and to open up dilated maternal capillaries. Maternal blood now makes its way into the trophoblast, whether it here finds preformed cavities or whether it forms these cavities in a loose protoplasm in consequence of hydrostatic pressure, we do not know. While the trophoblast opens up the enlarged maternal blood lacune, the hypertrophy of the mucosa, as a whole, goes on. ‘The gland spaces become large and cystic, their ducts lead to the surface in a tortuous manner. A separation into a spongiosa and compacta becomes early established, and in the latter some cells early assume a marked decidual character. The ovum is now interstitially embedded in the mucosa and sur- rounded by a border zone which is composed of an admixture of still attached or detached trophoblast elements, degenerating fixed maternal cells, both connective tissue decidual cells and glandular epithelia. In this border zone (“Umlagerungszone’’) are also con- tained enormously enlarged maternal blood vessels, cystic blood-filled gland spaces and free blood. The ovum almost floats as it were in a lake of blood partly contained in the trophoblast cavities, partly in the cystic maternal gland spaces, partly freely infiltrating more or less all of the tissue in the direct neighborhood of the growing germ. When the preliminary paper was read in Boston, Mass., Au- gust 20, 1907, before the Section on Embryology of the Seventh International Zoological Congress, Professor A. A. W. Hubrecht, the Chairman of the Section, in the discussion of the paper, called 400 Maximilian Herzog. attention to a misconception of the writer as to certain relations of the amnion and yolk sac. It is needless to say that the con- ception of Professor Hubrecht proved to be the correct one. This necessitated on the part of the author a careful re-examination of the sections and some changes in the manuscript for the Transac- tions of the Congress. Professor C. S. Minot then kindly placed at my disposal his laboratory and his library to enable me to make the necessary re- examinations and changes. To him, as also to Professor F. T. Lewis, I am under obligations for the great kindness shown on this occasion, and in the final revision of the manuscript. The literature on human placentation is extensively given in Keibel’s Normentafeln ; in Peters’ monograph; in Webster, ‘Human Placentation” ; in Pfannenstiel’s article in Winkel’s “Handbuch der Geburtshilfe.” The most recent contributions are quoted in Jung’s monograph and Bryce and Teacher (1. ¢.). Stahl, in his article on “Die Embryonalhiillen der Siuger und die Placenta,” in Vol. I, Part 2, Hertwig’s “Handbuch der Entwickelungslekre der Wirbel- thiere,” gives the literature on placentation among mammals in general. Fic. 28.—Colored plate drawn by Miss Katharina Hill, artist of the De- partment of Anatomy of the University of Chicago, from an enlargement of Fig. 25, photomicrograph from section 153. The embryo shown in this plate is drawn after section No. 155, because in it the yolk sac, embryonic shield proper, allantois and allantoic duct are best seen. All. S.—Allantoiec stalk. C. A.—Cavity of amnion. Can. F.—Fine fibrin threads and leucocytes (early canalized fibrin). Cho. M.—Chorion mesoderm. D. C.—Decidua capsularis. Exoc.—Exocelom. Gl.—Gland space (epithelium partly preserved). Mat. Bl. 8.—Large cystic gland spaces filled with blood. S. V.—Yolk sac. Syn.—Syncytium. Tr.—Trophoblast. aise PLACENTATION AND EMBRYONIC DEVELOPMENT IN MAN.—M. HERZOG. PLATE I, DEG: ChoM DiS: Mat.Bl.L. Tit AMERICAN JOURNAL oF ANATOMY., Vou. IX. Fics. 1 to 22.—Drawings from twenty-two serial sections of the embryo (Sections Nos. 164-148 of the entire ovum). C. A.—Cavity of amnion. C. M.—Chorion mesoderm. D. A.—Duct of allantois. Em. S.—Embryonic shield. EHxo.—Exocelom. Mes.—Mesoderim. P. A.—Allantoic stalk. S. V.—Yolk sac. V.—Anlage of blood vessels. EMBRYONIC DEVELOPMENT IN MAN. MAXIMILIAN HERZOG. CM. ‘ ‘ere ve) pas e<0 ® oy ‘ o «ec ) \ C3) < ? eer S59 Mees Rei ® gy? a a © @ by Ke e °, i Serie eed Exo wu ’ Fig. 1, C. Mm. wi S hie a . a ean o> oo : Ney fs? i! t 2 re er CA $ Ry BS ¥ y ie “o = Vga >, Pad \s . Exo Fic, 4, THE AMERICAN JOURNAL OF ANATOMY.—VoL, IX, No. 3. Fie. 5 ie) = eee im Se - . =! ‘TP 1? mer Pie ae ? b - am) ed 2. d ' 29 Ni5ass opine Fae yk : : *) be r, : : Hut i; ar Oat 1 = 7 - md . EMBRYONIC DEVELOPMENT IN MAN. ‘ MAXIMILIAN HERZOG. Fie. 12. CM. ss va PA ‘ ¢@ on fy ‘ Ty (Ds i Bese} tf en Ya aT i gaze” & \ f . 4 be. hehe aye i & NS Race ve e e * So (Sol ry “ite & ‘Fr, ¢ a) wh a é ‘ at Le ¢ q : SV SS PS be ol i? , veins Sates * cue a * 8 ¢ 4 ene | @ ae Pha) Y 0. i ee Ne, 4 oe & © e a | ; : wy, ae ‘ Exo ™> Fie. 14. THE AMERICAN JOURNAL OF ANATOMY.—Vot. IX, No. 3. PLATE IV fo. e. oe ee? Pt Sea Ce tay ae Tete eS wit Set SV 2 ¢ a to es wv “g e ot ee, i,” baer, 4 ra é ¥ @ ne ne ve f é §& #0,6° aA 8 be ea § ‘ ‘@ ‘ Lyaeeree © 5 ° 4b Se ba as) ee eeeeére ¢ i, Te we @' Mrs e. LF - of Geceres . ‘ tc ee” 56 6,8 00.7 a p fe *eagee!, rd Be 2, 4h \! Exo ev a Ps | as ‘ Fie. 13. cM 4, 0f PA DNs SRE di Ps 5 f ' ee! eget ty ¥ ~ 4 Pa) % ,) re 7 Ba a ~S 2 i : feo oe %% Fan Si Sv 0: ates, Ss x ’ a6 . OV nag ' rH P , (0 Wz Pe ey (e -pat at SV o, t ; * é fhnr V a @, 7 te, aa 6 Mi ° ¢ Padi oe 4? 7) ar te ; 4 é (pp teat ene %, : Se f% , ae } - Exe = wf Fie. 15 ny 7 nan ‘ ait an . = te EMBRYONIC DEVELOPMENT IN MAN. 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PLATE V. ae ce @ A, x aS ; ge” & Page \ @ Fa) ad 2” ¢ & G &) ‘ ‘ way )) a4 ® use? ‘ \ S ‘ ‘> aH Len ® - Seng SoSD wy Ee ee a SBC orwdowe DQG OD Exo Fig. 17.) & @® & ® ® - & & “= ® : & os ee ? fe e g oo, Ss & w € Be, & 8 o Cs « se @ 3 2 POE © ®@ ) ) “ip “2S @ — . ors iv iv) & ® sae e = fo 3 ® a) % © “QL & e e&sgsea \, & bey es 3 wip - , Be SSS é ® ga (ip) easter se Ves n @ & ew aSee Ge Q & ® & > a we on @ ee “eae? ef > @ So x ly Aj & pee se ~ . a3 E r . “ =k ~ >» ye a . ” » = & “~ oe * ~~ =” “ = = bal - nae Hie. 19. ; ¢ el \ ae ee ila ay i 0 Sc Fie. 20. Fig, 24.—Photomicrograph from section No. 125. Zeiss, planar, X 15 diam. (approximately). This photomicrograph shows the relation of the ovum to the parts where it has implanted itself. It is separated from the surface by a very thin capsularis and in the middle of the upper margin of the photograph may be seen a place where the chorion mesoderm is adherent in the form of a teat-like projection to the capsularis, which is defective at this place. The open ring seen a little to the right of the center of the chorion cavity (exocelom) is a transverse section of the yolk sac which hangs down beyond the end of the embryonic shield. The chorionic mesoderm is surrounded by the trophoblast. The ovum rides as it were on a wedge-shaped group of enormously, cystically, enlarged gland spaces, and lacune, filled densely with maternal blood. To the right and to the left are seen the enlarged glands of the uterine mucosa. Differentiation into a decidua spongiosa and compacta is not well seen in this section. At the left lower corner there is a strip of the muscularis. Fig. 25.—Photomicrograph from section No. 153. Zeiss, planar, xX 29 diam. Showing the chorionic cavity filled with a coagulated granular mate- rial. To the right is seen the embryo anchored by the allantoid stalk to the chorion mesoderm. In the upper corner a thin strip of degenerated decidua capsularis. Fic. 26—Photomicrograph from a section outside of the implanted ovum (from first series of sections, unnumbered), Zeiss, planar, xX 15 diam. Showing large cystic gland spaces partly filled with blood. To the left a differentiation into a decidua compacta and spongiosa is well marked. The lower margin of the. photomicrograph shows the muscularis uteri. Fie. 27.—Photomicrograph from section No. 83. Zeiss Apochromat. 8 mm., proj. oce. No. 4, X 210 diam. Gland spaces near the ovum filled with spherical hyaline masses. EMBRYONIC DEVELOPMENT IN MAN. MAXIMILIAN HERZOG. THE AMERICAN JOURNAL OF ANATOMY.—VOL, IX, No. 3. PLATE VII. Rs ce 7 SER Ab gf Pinas id * s Sas ASP : : Pur) ‘ es oa ; Fs “ = ’ a i eee ¢ Y ae ’ 7 ae of ‘ : =m x] ® U 7 » m * £3539. Pal ote Serpe ibe i : Pie 7 ; ‘ J 3 %, , . i rk ites rat iV’ t Pn. ‘<< £ be ’ a P ” >= 7 » 7 : * Ite. 28.—Photomicrograph from section No. 101. Zeiss Apochromat. 16 mm., proj. oce., No. 4, XK 100 diam. A coagulated crescentic mass containing a coarse network of parallel fibrin threads stretches across the field. Fic. 29.—Photomicrograph from section No. 210. Zeiss Apochromat. hom. oil. imm., 2 mm., proj. occ., No. 4, x 1000 diam. 900. the blood vessels to this tissue during development. The relation of the blood vessels to the preadipose tissue of the renal region does not impress me as being especially close ; but no one can fail to notice such a close relation as shown in Text Figs. 1 and 4, where the true adipose tissue is beginning& to form. Equally impressive are sections of the liver in many specimens. In several fcetal livers the cells adjacent to the veins were full of fat when little or none was found elsewhere (Fig. 1, Plate 1). In two young steers fat droplets were found only in the cells adjacent to some of the medium sized veins. When we compare the deposit of fat in the liver with its first appearance in connective tissue there is seen to be a striking 426 EB. -E. Bell. resemblance. The fat passes out of the blood stream and is taken up by the adjacent cells. Possibly the fat itself (in some soluble form’) acts directly upon the relatively undifferentiated connective tissue cells causing them to pass into the preadipose and later into the adipose condition.'® Detailed changes in the fat cell during development. As was said above, the preadipose cell is a branched cell, in sections appearing usually bipolar and frequently spindle-shaped. lis long coarse processes le among the collagenous fibrilie (Figs. 6 and 9, Plate IL; and Fig. 8, Plate I). It may accumulate small fat droplets at this stage long before any further changes occur. A 24.7 em. foetus showed whole lobules of renal preadipose tissue in which nearly every cell contains fat droplets (Fig. 7, Plate IT). Usually it seems that no fat is deposited in the cell until about the time its transformation into a true rounded fat cell begins. In the first formation of a fat lobule the deposition of fat begins in the pre- adipose cells adjacent to a blood vessel and extends outwards. The blood vessel is the center of the lobule. As the lobule grows the pre- adipose cells around its periphery are gradually converted into fat cells. Some fat cells are formed inside the lobule. Most of the drawings of individual fat cells were made from cells on the periphery of the lobule, since the cells are not crowded there and can be more easily studied. Text Fig. 7 shows some early stages in the formation of the fat cells. The cells were drawn from the edge of a renal fat lobule of a 42cm. foetus. Cell A shows the first appearance of Altmann gran- ules (2), but has not yet developed a cell membrane or fat droplets. It is spindle-shaped and has long processes. Cell B is not ali included in the section. It has fat droplets and Altmann granules. A thin cell membrane (m) is present. In cell C a number of fat droplets “The physiologists have shown that fat is moved through the tissues in some soluble form and not as small droplets. “This assumption does not, however, explain why the fat passes out of the vessels at any particular place. Histogenesis of the Adipose Tissue. 497 are present separated by thin layers of protoplasm. The cell mem- brane (m) is now clearly shown. The cell is still pointed at the ends. Text Fig. 6 shows two cells from the subcutaneous tissue of the brisket of a 24 em. feetus. They lie near a blood vessel around which a few fat cells have appeared. The processes of the preadipose cells of this region are somewhat thinner than those of the renal tissue. Both cells, especially cell A, show fat droplets in their processes. I have not determined the exact way the cell process disappears, but several cells like cell A have been observed. It seems that in these Text Ficure 7. Three cells from the edge of a renal fat lobule of a 42 cm. foetus. A, before formation of cell membrane. B, shows first appearance of cell membrane. (, later stage. f, spaces occupied by fat droplets; g, Altmann granules; m, cell membrane; n, nucleus. Fixation, Zenker’s fluid. Stained with iron-hem. and eosin. 1200. cells the cell process is transformed into fat droplets which are moved up into the body of the cell. Text Fig. 8 represents two cells well into the edge of a fat lobule, They were drawn from the same section as those shown in Text Fig. 7. The cells have increased greatly in size, though the processes have not yet disappeared. The protoplasm is thickly studded with Altmann granules. The cell membrane is sharply marked except on the processes. Text Fig. 9 was drawn from the same specimen from which Text Figs. 7 and 8 were taken. The cells le well into the edge 428 K.-T Bell: of a fat lobule. These cells have lost their processes and become rounded. Cell B has a large amount of protoplasm, but only a little fat. This multiglobular cell is somewhat similar to the cells of brown adipose tissue. In both cells the cell membrane is readily seen. Figure 10, Plate 11, was drawn from the inguinal adipose tissue of a 32 cm. foetus. Fat cells of different stages of development are shown. Most of the smaller cells are of the type shown in Text figure 9—rounded cells with considerable granular protoplasm and TEXT FicurE 8. Two young fat cells from the renal adipose tissue of a 42 cm. foetus. The cells lie well into the edge of the lobule. f, spaces occu- pied by fat droplets; g, Altmann granules; m, cell membranes; n, nucleus; pr, processes of cell. Fixation, Zenker’s fluid. Stained with iron-hzem, and eosin. < 1200. Text Figure 9. Two young fat cells from the renal adipose tissue of a 42 cm. foetus. The cells lie well into the edge of the lobule. f, spaces occu- pied by fat droples; g, Altmann granules; m, cell membrane; n, nucleus. Fixation, Zenker’s fluid. Stained with iron-hem. and eosin. 1200, one or more fat droplets. Most of these celis were not derived directly from the original branched cells of the preadipose tissue, but from the division of small cells in the interior of the lobule. As will be described later, the fat lobule grows, in its early stages at least, by division of small fat-free cells inside the lobule as well as by the addition of cells on its periphery. The cells formed inside the lobule are not branched; they become rounded directly by increasing their protoplasm and accumulating fat. The largereells (about 25 microns Hlistogenesis of the Adipose Tissue. 429 in diameter) show a well developed peripheral zone of protoplasm containing Altmann granules. It will be noted that many colla- genous fibers (ef. stained blue) occur crowded between the fat cells. These fibers become included in the adipose tissue during its forma- tion. The preadipose tissue often contains a number of coarse collagenous fibers (note lower right-hand corner of figure) and these are crowded together in thin layers as the fat cells increase in size. These fibers do not occur in renal adipose tissue. The fibers of the renal preadipose seem to be completely absorbed. Text Figure 10. Cell from the renal adipose tissue of a 105 em. foetus (about full term). ff, spaces occupied by fat; g, Altmann granules; m, cell membrane; 7”, nucleus. Fixation, Zenker’s fluid. Stained with iron-hem. < 1200. Text Fig. 10 represents a fat cell from the renal adipose tissue of a foetus about at full term (105 cm.). Nearly all the cells of the tissue at this stage are similar to this cell. The border of the cell is wavy because of shrinkage from fixation and embedding. The cell membrane is readily seen. In some parts of the circumference, particularly around the nucleus, a thick zone of granular protoplasm is present. The zone of protoplasm contains Altmann granules and often small fat droplets. ; Text Fig. 11 is from the scrotal adipose tissue of a steer about 314 years old. The animal was fairly well fattened and weighed 1,260 pounds. The drawing shows only a part of the wall of a fairly large 450 E. T. Bell. fat cell. A mass of protoplasm containing small fat druplets is shown. No other thickening is present in the wall of this cell. A great many cells of this type are present in this animal in the scrotal fat and in various portions of the subcutaneous fat. Many of the cells show more than one thickening in the wall caused by small accumulations of protoplasm. Similar small masses of protoplasm containing small fat droplets were found in cells of all sizes up to 125 microns, but the protoplasm is present less frequently and in smaller amount in the larger cells. The same kind of fat cells were found in large numbers in corresponding portions of the adipose tissues of several other fat steers. These cells are clearly similar to the cells of the full-term foetus (Text Fig. 10). They are probably merely young fat cells. High magnification reveals the presence of some very small TEXT FicureE 11. Thickened part of wall of fat cell from a fat steer. A great many cells in the animal are of this type. /, spaces occupied by small fat droplets; /’, position of main mass of fat; g, Altmann granules; m, cell membrane; 2, nucleus. Fixation, 20 per cent formalin. Stained with iron- hem. < 900. masses of protoplasm immediately under the cell membrane in many large fat cells, but the protoplasm does not form a continuous layer. The cell membrane is differentiated from the peripheral proto- plasmie layer of the cell. It begins to develop when the cell is yet branched (Text Fig. 7, B and C; Text Fig. 8, A and B). It develops only a short distance on the cell processes. The membrane increases in thickness as the cell grows. - It has long been known that the cell membrane is different chemically from the protoplasm and con- nective tissue fibers. It is not stained by Mallory’s anilin blue™ “Tf the tissue be fixed in formalin, anilin blue will stain the membrane of the fat cell. Histogenesis of the Adipose Tissue. 431 nor by Weigert’s elastic tissue stain. It may be stained by fuchsin, eosin, Congo red, ete. Altmann’s granules are present in the protoplasm of the fat cell from the time it begins to assume the rounded form. There is no conclusive evidence, however, that they stand in any intimate rela- tion to the formation of fat, since protoplasmic granules which stain similarly are of stich widespread occurrence. Metzner (23) has attempted to show that these granules are directly transformed into fat droplets, but his observations are far from convincing. Text Ficure 12. Fat lobule from renal adipose tissue of a 40 em. foetus. The dark nuclei are in some stage of division. f, spaces occupied by fat; mv, mitotic figures. Fixation, Zenker’s fluid. Stained heavily with iron- hrm. >< 400. Growth of adipose tissue. It has been pointed out that the blood vessel is the center around which the fat lobule develops. Whether in a mass of preadipose tissue, or in ordinary connective tissue, the first fat cells appear immediately around the blood vessels. The lobules thus established increase in size to a large extent by the addition of cells adjacent to the periphery. The increase in the number of fat cells is, how- ever, to a considerable extent due to the division of fat-free cells 432 Hei Bellk: inside the lobule. Text Fig. 12 represents a lobule of renal adipose tissue from a 40 cm. foetus, under a low magnification. A number of nuclei in the lobule stain intensely with hematoxylin and are prob- ably in some stage of division. ‘Two mitotic figures (m) are shown. Fig. 10, Plate II, shows clearly these young fat cells among the older cells. Probably only a few of these young cells were developed directly from the original branched cells of the preadipose tissue. They arise by the division of fat-free cells inside the lobule. In almost any section of adipose tissue a few nuclei may be seen crowded in the angles between the fat cells. The protoplasm around these nuclei is so small in amount that it can hardly be demonstrated. From a study of fattening animals I am convinced that these in- terstitial cells may form many new fat cells when the animal fat- tens. I have not,. however, found mitoses in the interstitial nuclei. It is generally believed that a cell does not divide after any consid- erable amount of fat has been deposited in it. A great part of the growth of the fat lobule is certainly due to the increase in size of the individual fat cells. A cell increasing its diameter from 15 to 150 microns (the latter being a common size in fat cattle) increases its cross-sectional area 100 times and its volume 1,000 times. The subcutaneous fat cells of thin cattle are markedly smaller than those of fat cattle. The increase in the volume of the fat cells already present is sufficient to account for a large part of the increase in the mass of the adipose tissue during fatten- ing, but a numerical increase of the fat-holding cells also occurs. As pointed out above, fat cells with a peripheral protoplasmic margin containing small fat droplets (Text Fig. 11) were found in large numbers in the subcutaneous adipose tissue of several fat- tening steers. This type of cell is usually comparatively small, and is essentially identical with the fat cells of the fetus. (Text Fig. 10 and Fig. 10, Plate II.) They are very probably newly-formed fat cells developed from the interstitial cells referred to above. During the fattening of the animal it is evident that some new fat lobules are formed around old or newly-formed blood vessels. It can no longer be maintained that the fat cell is a special kind of cell in the sense used by Toldt, distinctly different from the ordi- Histogenesis of the Adipose Tissue. 433 nary connective tissue cells. In the ox it seems that in general the blood vessels are important factors determining the development of adipose tissue. The connective tissue cells which happen to lie ad- jacent to a blood vessel at the time a fat lobule is forming are the ones which become fat cells. In certain situations, however, as on the kidney and in certain parts of the omentum, so-called primitive organs are developed which bear no close relation to the blood vessels. The blood vessels cannot be regarded as the main determining factors here, though they may take part. There is not, however, a random formation of fat cells in the con- nective tissues. The lobules of adipose tissue, in which nearly all new fat cells appear, are well-defined structures. It is only in naan TEXT FIGuRE 15. Two leucocytes trom the omentum of a 16 cm. feetus. n, nucleus; v, vacuole. Stained with iron-hem. Fixation, Gilson fluid. x 1200. the early stages of their formation that cells are added to the lobule from the connective tissue adjacent to the periphery. I intend to treat this phase of the subject more fully in a later paper. Text Fig. 18 shows two leucocytes from the omentum of a 16 em. foetus. A great many such cells are present at this stage. They usually contain several vacuoles. By staining with Scarlet red it is seen that these are not fat droplets. It is possible that such cells have lent support to the erroneous conception that leucocytes may form fat cells. In conclusion I wish to thank Dr. R. R. Bensley for sending me some necessary material. Most of the drawings accompanying this paper are the work of Mr. G. T. Kline; a few were made by Miss MeGill. 434 EK. T. Bell. SUMMARY OF PART II. A peculiar open-meshed tissue (preadipose) precedes the formation of true adipose tissue. The renal preadipose tissue is formed into well-defined lobules a long time before the true fat cells appear. The masses of preadipose tissue in the omentum are much less sharply marked off than those of the kidney and persist only a short time before the formation of the fat cells begins. The subcutaneous preadipose tissue is found as masses around the blood vessels (Text Fig. 1) and persists only a short time in the preadipose condition. The preadipose tissue consists of loosely-arranged cells with two or more long coarse processes. A great many fine connective tissue fibers occupy the ground substance of the tissue (Fig. 8, Plate I; Fig. 6, Plate IL). The branched cells may contain fat droplets a long time before they begin to assume the rounded form. The preadipose tissue is clearly a fibrillar connective tissue. My results support Flemming’s view that adipose tissue is a modified fibrillar connective tissue. In the formation of a fat lobule the cells adjacent to the blood vessel are filled with fat first. The filling of the cells with fat ex- tends from the blood vessel outwards in all directions. This process is closely similar to the deposition of fat in the liver where it is deposited first in the cells immediately adjacent to a vein and later into those lying farther out. The branched preadipose cell becomes rounded by the accumula- tion of fat in its interior. Its processes are absorbed. The cell membrane is differentiated from the peripheral proto- plasmic layer of the cell. It begins to form when the cell is yet branched. The Altmann granules are found in the protoplasm of all fat cells. They are first observed when the cell is yet branched and before the first fat droplets are formed. The mass of adipose tissue increases in amount in fattening (a) by the increase in the size of its cells, (b) by the formation and filling of new cells in the interior of the lobule, (c) by the formation of new lobules. Histogenesis of the Adipose Tissue. 435 APPENDIX. Since receiving the page proof of this article I have discovered that after formalin fixation, usually only a part and often none at all of the fat-content of muscle fibers and epithelium can be stained with Searlet red, Sudan, or osmic acid. If the tissue be stained fresh in the stains mentioned above, a considerable amount of fat can be demonstrated in the muscle fibers of a number of animals including the ox; but if the tissue be fixed in formalin a few days it 1s often impossible to demonstrate any fat in this situation. I have found that after a few days’ fixation in formalin little or no fat can be stained in the muscle fibers of the frog or chicken, and with the exception of a few samples the same is true of the ox muscle fibers. Mr. H. H. Bullard has recently prepared special concentrated solutions of Scarlet red and Sudan which stain a great many more droplets in muscle fibers and epithelium than are brought out by the ordinary stains. We have not yet decided whether all the droplets brought out by Bullard’s stain are neutral fat, but it seems probable that they are all of a fatty nature. The use of Bullard’s stains on fresh material gives such splendid pictures that it seems certain there must be a revision of all the work that has been done on the fat-content of tissues both physiological and pathological, In a short time papers will appear setting forth the results men- tioned above. 436 Hd el LITERATURE LIST. 1. ALTMANN. R. Ueber die Fettumsetzungen im Organismus. Archiv f. Anat. u. Phys., Anat. Abth., 1889, Suppl. Bd. 2. AscHorr, L. Ueber den Fettgehalt fotaler Gewebe. Centralblatt f. allg. Path. u. path. Anat., Bd. 8, 1897, Autoreferat. 3. AUERBACH, M. Das braune Fettgewebe bei schweizerischen und deutschen Nagern und Insektivoren. Archiv f. mikr. Anat., Bd. 60, 1902. 3a. Bosprirzky, C. Zur WKenntniss des Baues, der Entwickelung und der regressiven Metamorphose der Fettzellen. Centralblatt f. die medicin- ischen Wissenchaften, Jahrg. 23, 1885. 4. Bourn, M. Origine des corps adipeux chez rana temporaria. Bibliog. Anat, I VI, 1899: 5. Corrry, D. J. The development of the fat cell. Trans. Royal Acad. of Med., Ireland, Vol. 24, 1906, pg. 468-469. 6. CZAJEWICZ. Untersuchungen tber die Textur, Entwickelung, Riickbil- dung und Lebensfiihigkeit des Fettgewebes. Reichert und Du Bois’ Archiv, 1866. 7. ErpuHEemM, J. Zur norm. u. path. Histologie der Glandula thyreoidea, parathyreoidea, u. Hypophysis. Ziegler’s Beitriige, 1903. 8. FLEMMING, W. Ueber Bildung und Riickbildung der Fettzelle im Binde- gewebe. Archiv f. mikr. Anat., Bd. 7, 1871. 9. FLEMMING, W. Weitere Mittheilung zur Physiologie der Fettzelle. Archiv f. mikr. Anat., Bd. 7, 1871. 10. FLEMMING, W. Ueber das subcutane Bindegewebe und sein Verhalten an Entztindungsheerden. Virchow’s Archiv, 1872. 11. FLEMMING, W. Beobachtungen tiber Fettgewebe. Archiv f. mikr. Anat., Bd. XII, 1876. 12. Fremminc, W. Ueber die Entwicklung der Fettzellen und des Fett- gewebes. Archiv f. Anat. und Physiol., Anat. Abth., 1879. 13. FROMMANN, C. Structur der Fettzellen und ihrer Membran. Jenaische Zeitschrift f. Naturwissenschaften, Bd. XVII, 1884, S. 217-220. 13a. Gace, S. H. Observations on the fat cells and connective tissue corpus- cles of Necturus. Proceedings of the American Society of Micro- scopists, Vol. IV, 1882. 14. Hammar, J. A. Zur Kenntniss des Fettgewebes. Archiv f. mikr. Anat., Bd. XLV, 1895. 15. HANSEMANN, D. Ueber Fettinfiltration der Nierenepithelien. Virchow’s Archiv, Bd. 148, 1897. 16. Hoaean, G., and Fr. E. On the development and retrogression of the fat cell. Journal of the Royal mikr. Society, Vol. II, 1879. (Quoted from Flemming.) 17. 29. 33. Histogenesis of the Adipose Tissue. 43,7 JAKOWSKI, M. Hin Beitrag zur Lehre von der Entwicklung des Fettge- webes. Sep. Abdr. aus der Festschrift Feier des 25-jihrigen Jubiliiums von Prof. Hoyer, Warschau, 1884; (Hofmann u. Schwalbe’s Jahresbericht). KeriInatH. Ueber den mikroscopischen Nachweis von Fett in normalen Muskeln. Inaug. Dissert., Freiburg, 1904. Kemp and Hatt. The formation of fat in animals fattened for slaughter. The American Journal of Physiology, Vol. 18, 1907. (Proceedings of the Physiol. Soc.). KLEIN, E. The anatomy of the lymphatic system, I, London, 1873. (Quoted from Shaw.) KXOLLIKER, A. VON. Zur Entwicklung des Fettgewebes. Anat. Anz., Bd. I, 1886, S. 206-208. Lowe, L. Zur Kenntniss des Bindegewebes. Archiv f. Anat. u. Physiol., Anat. Abth., 1878. Mati, F. P. On the Development of the connective tissues from the connective-tissue syncytium. American Journal of Anatomy, Vol. 1, 1901-02. METZNER, R. Ueber die Beziehung der Granula zum Fettansatz. Archiv f. Anat. u. Physiol., Anat. Abth., 1890. Morpureo, B. Sur la nature des atrophies par inanition. Arechiy, ital. de biologie, T. XII, 1899. Nemivorr, A. Zur Frage tiber den Bau der Fettzellen bei Acipenser ruthenus. Anat. Anz., Bd. 28, 1906, S. 513-522. PFEIFFER, W. Ueber den Fettgehalt foetaler Organe. Inaug.-Dissert., Freiburg, 1899. POLJAKOFF, P. Ueber eine neue Art von fettbildenden Organen im lockeru Bindegewebe. Archiy f. mikr. Anat., Bd. 32, 1888. RABL-RUCKARD, H. Fettzellen von eigentiimlicher Form. Archiv f. mikr. Anat., Bd., 32, 1888. RanvigErR, L. Hléments cellulaires du tissu conjonctif. Arch. de physiol., 1869, p. 471. (Quoted from Shaw.) Sara, A. Ueber das Vorkommen von Fett in der Haut und in einigen Driisen, den sogenannten Eiweissdrtisen. Ziegler’s Beitrige, Bd. 27, 1900. ScHos., J. Ueber Wundernetzbildungen im Fettgewebe. Archiv f. mikr. Anat., Bd. 24, 1885. SHaw, H. B. A contribution to the study of the morphology of adipose tissue. Journal of Anatomy and Physiology, Vol. 36, 1901-2. Smiru, J. L. On the simultaneous staining of neutral fat and fatty acid by oxazine dyes. Journal of Path. and Bacteriol., Cambridge, 1907, p. 1-4. 458 od. 3D. 36. Br Sake lt Sotcer, B. Zur Kenntniss osmirten Fettes. Anat. Anz., Bd. 8, 1893. Totpt, C. Lehrbuch der Gewebelehre. 3 Aufl., Stuttgart, 1888. TRAINA, R. Ueber das Verhalten des Fettes und der Zellgranula bei chronischem Marasmus u. Hungerzustiinden. Ziegler’s Beitriige, Bd. 35, 1904. WALBAUM, O. Untersuchungen iiber die quergestreifte Muskulatur mit besonderer Beriticksichtigung der Fettinfiltration. Virchow’s Archiv, Bd. 158, 1899. WALDEYER, W. Ueber Bindegewebszellen. Archiy f. mikr. Anat., Ed. 11, 1875. EXPLANATION OF PLATES. PEATE Ae ad Fic. 1. From a section of the liver of a 7 cm. fetus. The fat droplets in this and the succeeding figures are colored red. an, annular fat droplet; h, hepatic cell—those near the vein (V) are filled with fat droplets. Fixa- tion, 20 per cent formalin. Frozen section stained with Scarlet red and hem. > 800. Fic. 2. Cartilage cells from the centrum of a vertebra of a 10.1 cm. foetus. Fixation, 20 per cent formalin. Frozen section stained with Scarlet red. < 13800. ics. 8 and 4. Cross-section of muscle fibers of psoas muscle of a 17 cm. foetus, showing fat droplets in the interior. Fixation, 20 per cent formalin. Frozen section stained with Scarlet red. x 1300. Fic. 5. Cartilage cell from the innominate cartilage of a 17 cm. fetus, showing many fat droplets. Fixation, 20 per cent formalin. Stained with Scarlet red and hem. >» 13800. Fic. 8. Renal preadipose tissue from an 18.5 cm. fetus. f, fine fibrille; n. nucleus: pr, process of cell; s, space. Fixation, Zenker’s fluid. Stained heavily with iron-hzem. and not decolorized. x 800. HISTOGENESIS OF THE ADIPOSE TISSUES.—E. T. BELL. PLATE I. Gre BI THH AMERICAN JOURNAL OF ANATOMY.—VOL. IX. PATH ell: Fic. 6. Renal preadipose tissue from an 18.5 cm. foetus. f, fine fibrille; n, nucleus; pr, cell process; s, space. Fixation, Zenker’s fluid. Mallory’s anilin blue. > 1200. Fic. 7. a, b, ec, d. Cells of the renal preadipose tissue of a 24.7 cm. foetus, showing fat droplets. Fixation, 20 per cent formalin. Frozen section stained with scarlet red. 1700. Fic. 9. Preadipose tissue of the omentum of a 40 cm. foetus. f, fine fibrillze; be, blood capillary; cf, coarse collagenous fiber; , nucleus; pr, process of cell; s, space. Fixation, Zenker’s fluid. Mallory’s anilin blue. » 13800. Fic. 10. Inguinal adipose tissue from a 32 cm. feetus. cf. collagenous fibrille; f, spaces occupied by fat; mn, nucleus. Fixation, Gilson’s fluid. Stained with Mallory’s anilin blue. >» 1200. acta i a » ih Pee HISTOGENESIS OF THE ADIPOSE TISSUE.—E, T. BELL. Puare Il, THE AMERICAN JOURNAL OF ANATOMY. VOL. =, IX. ON THE DEVELOPMENT OF THE SUPERFICIAL VEINS OF THE BODY WALL IN THE PIG. BY HELEN WILLISTON SMITH. From the Anatomical Laboratory of the Johns Hopkins University. WitTH 11 FIGURES. Satisfactory studies of the vascular system of young embryos have been impossible until quite recently, for the embryologist has been unable to see much more than mere fragments of the growing ends of this system in embryos treated by the usual methods of prep- aration. During the past few years the method of injection of the vascular system of young embryos has been perfected more and more in this laboratory, so that now we are able to procure complete injections in the very youngest stages. In order to make proper headway in the study of the morphology of the vascular system it is necessary to study the development of the primitive vessels in the embryos. For instance, more fundamental conditions can be ob- tained for the study of development in the umbilical vein, than in the vessels of an organ; in the former case there is but one vessel to be followed, while in the latter there are millions, and it is practically impossible to find the same terminal twig from stage to stage. The key to the situation is obtained when the main trunks and all their branches are brought out sharply by means of injection in their very earliest and in subsequent stages. This has now been accomplished by a number of investigators in this laboratory, most successfully, probably, by Dr. Evans, who injected many of the embryos I have studied. In order to obtain perfect injections it 1s necessary to inject live embryos, and, in addition to tadpoles and chicks, an abundance of THE AMERICAN JOURNAL OF ANATOMY.—VOL, IX, No. 3. 440 Helen Williston Smith. pigs of various stages of development are available every day. In the latter case the injection of India ink is made into the um- bilical artery towards the heart which, while beating, enables one to obtain perfect injections. Studies in this laboratory have shown conclusively that the entire vascular system is developed from a common plexus of capillaries which gradually extends over the whole body, a part of which is transformed into arteries and a part into veins. Throughout de- velopment the vessels are functioning, and the formation of arte- ries and veins is only an expression of the law of functional adapta- tion of the extensive capillary plexus peripheral to a beating heart. How could it be otherwise, for the arteries and veins are not formed step by step by sprouts, or by the union of independent anlages, but they are functioning from the time of their simplest beginning until the animal dies. The present study was undertaken, at the request of Dr. Mall, in order to follow, in a relatively simple field, the gradual evolution of the vascular system from its first appearance until its adult form is reached. This study is one of a series, two of which by Dr. Evans are now in press, and others are in preparation. If cireum- stance will permit I hope to follow this with an account of the development of the deeper vessels of the body wall.’ We find in the earliest stages considered in this paper a relatively simple circulation in the body wall, one in which the posterior cardinal and the umbilical veins are both formed, but have com- paratively few ramifications. As the embryo grows the limb buds appear and the membrana reuniens closes in around the umbilical ‘Yhe literature on the standpoint taken in this study is as follows: Aeby, Der Bau des mensch]. K6rpers, 1871; Baader, Inaug. Diss., Bern, 1866; Thoma, Untersuch. tiber die Histogenese und Histomechanik des Gefiiss- systems, 1893; Flint, Johns Hopkins Hospital Reports, IX, 1900, Amer. Jour. Anat., II, 1903, and VI, 1907; Mall, Amer. Jour. Anat., IV., 1905, and V, 1906; Rabl, Arch. f. m. Anat., LXIX, 1907; Evans, Anatomical Record, II, 1908, and Amer. Jour. Anat., IX, 1909. The specimens on which the present article is based were demonstrated at the last meeting of the Association of American Anatomists, Baltimore, Decem- ber, 1908. Superficial Veins in the Pig. 441 cord. The posterior cardinal and the umbilical veins share in drain- ing the limb buds, one dorsally, the other ventrally, and the umbilical drains as well a plexus which forms in the membrana reuniens. As the posterior cardinal vein sinks into the depth, though it con- tinues to partly drain the limb buds and to receive its segmentals from the myotomes, the relatively vast area of the membrana re- uniens drains entirely into the umbilical vein. Then, as the plexus in the membrana increases in complexity and the muscle layer shifts into it ventrally, larger longitudinal anastomoses form along the body wall draining up under the anterior limb bud to the ves- sels there, which connect in turn with the cardinal veins. Soon a definite vessel is formed, the thoraco-epigastric, which increases rap- idly in size until it collects to itself almost all the tributaries of the umbilical vein. The plexus in the membrana reuniens, in conse- quence, gradually dies out until only a few vessels in the median line and in the lower ventral region remain. Meanwhile on the mesial side of the muscle layer and ribs, other vessels, namely, the in- ternal mammary and the deep epigastric veins and arteries have been formed from a longitudinal plexus, and the intercostal vessels have been spun out like strands of cobweb in the intercostal spaces. All these vessels not only anastomose among themselves, but also have very numerous communications with the superficial vessels. Finally, it comes about, by changes given in detail later, that the thoraco epigastric loses its axillary connections, and drains into the internal mammary. After this the vessels of the body wall are not changed in kind, but in degree only, and the condition of the adult is practically achieved. Turning to the literature concerning the development of the super- ficial blood vessels of the body wall, we find a number of observa- tions upon the subject, but these are given, in great part, either as isolated or unexplained steps, or in an attempt, more or less satisfactory, to explain the condition in the adult. These latter observations, for the most part, are made upon feetuses, and so only a clue can be gleaned here and there concerning the early deyvel- opment. Among observations upon very young embryos are those of Coste, Kolliker, His, and Mall. 449 Helen Williston Smith. His describes a human embryo in which the membrana reuniens contains vessels emptying into the sinus reuniens above, and into the umbilical vein below. This is a condition found in pig embryos of about 7 mm. and one that is easily interpreted by a study of younger and older stages. This observation is confirmed and ex- tended by Mall, who says, “It appears, then, that during the third week of development, while the umbilical veins still empty into the sinus reuniens, an extensive plexus is formed throughout the greater extent of the membrana reuniens, which receives blood from the aorta on its dorsal side, and empties into the umbilical vein on its ventral side. As the umbilical vein changes its position to enter the liver, this circulation through the membrana reuniens is broken up as a much earlier circulation through the umbilical vesicle was broken up.” K6lliker and Coste give a description of a somewhat older type. Kolliker pictures a cow’s embryo in which the membrana reuniens is filled with a minute plexus radiating from the myotomes to the umbilical vein, and Wertheimer quotes Coste as follows, speaking of the vessels in the abdominal wall of the adult: “Ces vaisseaux sont les restes du riche appareil veineux transitoire, qui des parois abdominales sur lesquelles ils étaient repandus, se portent vers la veine ombilicale ou allantoidienne droit dans laquelle ils pénetrent par une foule de trones, placé les uns a cdté des autres. [ls suivent la destinée de la veine qui les recoit et s’éteignent complétement avec elle.” That I am correct in naming the vessel described above as the thoraco epigastric is evident upon comparison with the thoraco epigastric in the human adult. This is a vein subject to consider- able variation, and described somewhat differently in the text-books. Toldt and Spalteholz and Piersol describe it as extending subcu- taneously from the superficial epigastric vein, on the anterior and lateral surface of the trunk, to enter the long thoracic. Sabotta says it may either enter the long thoracic or the axillary vessel directly. In this laboratory I have examined a number of cadavers which show variations, agreeing for the most part with the two types given by Sabotta. The thoraco epigastric vein is unques- 9 Superficial Veins in the Pig. 445 tionably the same as that described by F. T. Lewis in rabbit em- bryos, and called by him the external mammary vein. (Amer. Jour. Amat.,1906.)* In general we may say that the thoraco epigastric is a vessel con- necting the veins of the epigastric plexus with the axillary veins. This is exactly the condition of the vein in the pig embryo of about 20 mm., and though in the adult we find it has undergone further change, embryologically it must be regarded as the same vessel. Joris, after speaking of this same stage, says, “Il est done un moment oti les veins umbilicales représentent les seul trones collecteurs des vaisseaux pariétaux, enfin, les veines pari¢tales perdent leur trones collecteurs par l’atrophie de la partie supérieure des veins ombilicales, et finissent par se rattacher au systéme veineux cave.” There is never a time in the embryo pig in which the umbilical vein drains the whole body wall, taken in a broad sense to include the limb buds, but it is certainly the chief collecting vessel for a considerable period. Finally the vessels emptying into it do atrophy, as the blood flowing from the body wall is directed into the cardinal veins. The method by which this is effected is, as I have said, chiefly through the development of the thoraco epigastric vein, the description of the origin, growth and permanent condition of which gives the connecting links between stages in the embryo and those of the adult. Concerning the adult condition in man, especially in the ventral body wall, much has been written, and though an account of that literature does not come strictly within the bounds of this paper, it may be of interest to give a brief résumé of it here. It deals chiefly with the question whether or not the umbilical vein remains patent, and the various points of communication between the portal and systemic circulation. There is considerable difference of opinion, due perhaps to the different methods of attack upon the problem, and to the great amount of anatomical variation which unquestionably must exist. *Since this article went to press Dr. Lewis has also adopted the term thoraco epigastric. Amer. Jour. Anat., Vol. IX, No. 1, Feb., 1909. 444 Helen Williston Smith. The names chiefly associated with this work are those of Robin, Baumgarten, Wertheimer, Sappey, Burow, Pfeifer, His, and recently Joris. I have not seen Robin’s papers, but he is quoted by most of the other authors, generally with more or less indignant sorrow because of the incorrectness of his views, namely, that the umbilical vein never receives vessels from the abdominal wall in the feetus, and is completely obliterated after birth. Baumgarten, on the other hand, according to Pfeifer and Joris, admits the existence of collateral veins in the adult, describes their arrangement as constant and normal and says that the part of the umbilical vein remaining patent is connected with the deep epigastric veins. Wertheimer comes to the conclusion, from injections of thirteen foetuses and young infants in the hepatie extremity of the um- bilical vein, that ordinarily the umbilical vein becomes absolutely occluded. However, there is a venule of later formation running in the obliterated cord in the adult, and he admits that cases may exist in which the umbilical vein remains open permanently. This possibility he explains by reference to comparative anatomy, for in amphibians the umbilical vein persists as the anterior abdominal vein. Sappey is somewhat more positive than Wertheimer in stating that the umbilical vein is absolutely occluded in the adult, but he says accessory veins exist, which he divides into a superior and an inferior group. “Le groupe supérieur est constitué par des veinules que descendent de la partie médiane du diaphragme vers la face convexe du foie et qui viennent se distribuer sur les lobules auxquels adhére le liga- ment suspenseur. Par une de leurs extrémités, ces venules com- muniquent avec les veines diaphragmatiques, et par l’autre avec les divisions sus-lobulaires de la veine porte. “Le groupe inférieur comprend toute une série de veinules qui se portent de la partie sus-ombilicale de la paroi abdominale anté- rieure vers le sillon longitudinal du foie. Ces derniéres, comprises dans la partie du ligament suspenseur qui renferme le cordon de la Superficial Veins in the Pig. 445 veine ombilicale, se trouvent en communication, 4 leur origine, avec les veines €pigastriques et les veines tégumenteuses de l’abdomen.” Burow describes a vein formed from fhe union of branches from the right and left epigastric. This vein passes up towards the liver and enters the upper part of the umbilical vein. Joris maintains that this vein is the same one as that described by Sappey, with the difference that in the one case the vein enters the liver, and in the other enters the unobliterated portion of the umbilical vein just before it reaches the liver. He says, moreover, that the right as well as the left umbilical vein may persist in the adult as a com- munication between the liver and the right epigastric vein. His work was done by injecting the portal vein after ligature of the vena cava above and below the liver. These injections were made on foetuses two months old and older, and on a few infants. His has made a classification of these veins as follows: “(1) VV parumbilicales (Sappeyi) welche von der Nabelgegend aus zur Leber emporsteigen und in deren Substanz sich einsenken. “(2) V supra umbilicalis (Baumgarten’s Burowsche Vene) welche in das obere, offen gebliebene Ende der V. umbilicalis einmiindet. “(3) VV umbilicovesicales. (Braunes’ Burowsche Venen. ) “(4,) VV umbilicoepigastricae, welche beiderseits in die VV epi- gastricae inferiores profundae einmiinden.” Summing up these observations it seems evident that ordinarily there are some small venules running from the region of the dia- phragm to the umbilicus, and that these connect the portal system with small branches of the deep epigastrics. In pigs about three centimeters, in which all the vessels of the membrana reuniens appear to have atrophied, sections show that small vessels pass from the liver in the median line to the umbilical vein, and that there are also some twigs connecting the umbilical vein with the plexus in the region of the epigastric veins. However, from a study of the vessels of the membrana reuniens in younger embryos it is evident that the probability of variation is great which naturally complicates studies of this kind very much. The smallest injected embryo considered here is one about 6 mm. (Fig. 1). It may be noted that this specimen is twisted upon 446 Helen Williston Smith. itself through a considerable angle. This twisting is probably not normal, but it permits of a very good view of the blood vessels, and this embryo has therefore been selected for illustration. The strik- Fic. 1.—Embryo 6 mm. long. Enlarged 17 times. Cor, heart; V.o, omphalomesenteric vein; Ao, aorta; V.cps,. left posterior cardinal vein; V.ws, left umbilical vein; V.wd, right umbilical vein; V.cas, left anterior cardinal vein; V.cpd, right posterior cardinal vein; A.om, omphalomesenteric artery; A.ws, left umbilical artery. ing thing about it is the extreme simplicity of its vascular system. The aorta is large. It gives off a number of spicule-like vessels, many of which on the ventral side unite to form the omphalo- Superficial Veins in the Pig. 447 mesenteric artery, a conspicuous, bulky vessel that increases in size as it passes anteriorly. Just below the omphalomesenteric artery the aorta divides into two vessels which in turn break up into two groups of capillaries, each of which reunites to form an umbilical artery. In the head region there are only two branchial arches formed. From the anterior of these a branch is given off which passes into the capillaries that unite to form the anterior cardinal vein (V. cas.). The anterior cardinal vein runs back to meet the posterior cardinal (V. eps.) and the two form a plexiform union before they enter the heart together. The posterior cardinal cannot be traced below the mesonephros, but it is possible that the injec- tion is incomplete as in embryos a very little older than this it extends dorsally to the posterior limb bud. The umbilical veins are plainly visible, running from the allantois along the edge of the membrana reuniens to the sinus reuniens which they enter together with the omphalomesenterie veins. (Vv. om.) These vessels are not clean cut, but show clearly their plexiform origin. Along the left umbilical particularly, we see many loops and open- ings. There are also some spicule-like projections here and there along the course of the umbilical veins that form the anlage of the future plexus of the membrana reuniens. The second embryo, 7 mm., pictured here in Fig. 2, corresponds roughly, as I have said, with that described by His. Probably the most striking thing about it is the size of the umbilical veins (Vvu). In the figure the right vein is seen to run from the cord in a long curve for the whole length of the mesonephros to the liver. At a point very little below the liver, a relatively small vein is seen looping up superficially and emptying at the anterior portion of the liver into the sinus reuniens (SR) above. This vessel is a part of the umbilical that does not sink into the depth, and receives numerous tributaries from the arm bud. The arm bud, however, drains also into the posterior cardinal vein (Vep) above, and below by five good connections, directly into the large umbilical vein (Vud). Below these connections, until the posterior limb bud is reached, there is, as yet, only a very narrow strip of body wall to drain. Such few vessels as there are here run into the 448 Helen Williston Smith. posterior cardinal (Vep), but lower down there is a very rich plexus, lying ventral to the anlage of the posterior limb bud, which com- municates freely with a similar plexus on the opposite side of the 4 Fic. 2.—Hmbryo 7 mim. long, in which the injection is complete. The ves- sels in the head are semi-diagrammatic. Enlarged 21 times. V.lf, linguo facial vein; SR, sinus reuniens; V.ud, right umbilical vein ; vein; V.us, left umbilical vein; A.om, omphalomesenteric artery; Ao, aorta; V.ca, anterior cardinal vein; V.cp, posterior cardinal vein; Pars sup. V.u, superficial part of umbilical vein. body, and as the figure shows, with its respective umbilical vein. Dorsal to the posterior limb bud the posterior cardinal (Vep) arises from numerous capillaries which unite to form a vein running along Superficial Veins in the Pig. 449 the body wall. This receives some small veins and finally sinks into the depth under the anterior limb bud, receives two or three twigs from it, and uniting with the anterior cardinal (Vea) enters the sinus reuniens in a graceful, sweeping curve. The sinus re- uniens also receives a vein, the inferior jugular, formed by the union of a number of capillaries which rise in gill arches, where they anastomose with twigs to the anterior cardinal. The veins that form the anterior cardinal (Vea) are very large and striking, par- ticularly the one which curves in a half cirele above the anastomosing tips of the cervical segmental arteries to enter the anterior cardinal. A chain of anastomoses along the spinal cord, formed from the tips of the segmental arteries passes from the region of the head to a point below the anterior limb bud. This plexus drains back into the posterior cardinal vein through its segmentals. The aorta is enormous. Three aortic arches are shown. The most anterior of which is not well injected. The omphalomesenteric artery (Aom) is represented by four vessels which unite at some little distance from their origin. In the tail the aorta divides into two vessels which anastomose at the tip. The next embryo, Fig. 8, is a very little larger than that just described, the chief point of difference being the presence of a thick capillary mesh in the membrana reuniens. As before, there remains “a superficial part of the umbilical vein (Vud) draining the posterior limb bud, which also drains largely into the posterior cardinal vein. The limb bud contains a fine plexus of veins which tend to form a border vein, while dorsal to it the well- developed mesh runs out upon the body wall to unite with the gen- eral plexus of the membrana reuniens. The plexus of the mem- brana reuniens is evidently the same as that described by Coste. Tt is very characteristic of the membrana, being made up of com- paratively large vessels anastomosing among themselves, but for the most part passing very directly to the umbilical’ vein which they enter by parallel veins. The next embryo in the series, Fig. 4, shows these vessels even better developed, since the membrana has progressed farther. It rep- resents most completely the phase in the body wall when it drains 450 Helen Williston Smith. \ chiefly into the umbilical vein. The lower part of the membrana re- uniens (MR) as high as the point where the umbilical vein enters the liver, is seen to be full of veins. These rise from a fine network of capillaries which is fed by the segmental arteries and extends is te oh a pene ze i SS SNR A) Fie. 3.—Embryo 8 mm. long. Enlarged 14 times. Only the superficial vessels are shown. Vif. linguo-facial vein; SR, sinus reuniens; Pars sup. V.u, superficial portion of umbilical vein; MR, membrana reuniens; V.ud, right umbilical vein; V.ca, anterior cardinal vein; V.cp, posterior cardinal Vein. in a crescent shape, with the concavity directed ventrally between the two limb buds. In the lower portion, this network is entirely irregular, but higher up it forms a more or less connected plexus which passes partly into another plexus extending ventrally to the Superficial Veins in the Pig. 451 anterior limb bud out towards the region over the liver, and partly, into two distinct, though very fine ropes of capillaries running up under the anterior limb bud, the more dorsal of which is the anlage of the thoraco epigastric vein (Vte). Over most of the ar SN at RUA ( on DTGan SS ¢ Oa: &y “pa ao SS QE Mitr 4 Ali OX i Jar | : yh wu x ! i ‘ Fic. 4.—Embryo 10 mm. long. Enlarged 13 times. | V.te, thoraco epigastric vein; J/R, membrana reuniens; Vu, umbilical vein. upper part of the membrana there are no blood vessels visible and the superficial part of the umbilical vein, shown in Figs. 2 and 3, appears to have atrophied. When compared with Fig. 5 it is evident that the system draining into the umbilical vein is receding and giving place to one draining longitudinally between the limb buds. 1, SSS > x SAWN ST. AN x \ ws CA e. SZ rit ss gd Plexus ves aed Fie. 5.—Embryo 151% mm. long. Enlarged 10 times. A few vessels in the depth are dotted. V.m, border vein; MR, membrana reuniens; V.te, thoraco epigastric vein; Ves, superficial epigastric vein: V.u, umbilical vein. Superficial Veins in the Pig. 453 There are still many blood vessels in the membrana (MR), but they show a tendency towards atrophy. The thoraco epigastric (Vte), on the other hand, which in the previous figure (Fig. 4) was nothing but a thread of capillaries, in Fig. 5 shows as a very distinct vessel passing up under the limb bud, receiving the prim- itive ulna, and running on up to enter the posterior cardinal at its junction with the anterior cardinal. The central connections of the thoraco epigastric, in this figure, show very well, but the picture is not always so uncomplicated. It will be noticed that the vein runs dorsal to the artery while in the adult it is normally ventral. The change is effected as follows. There is at the root of the arm bud a capillary mesh surrounding the artery connected above with the car- dinals at their junction and below with the primitive ulnar. In young stages the dorsal portion of this capillary mesh or loop is often the more prominent, but, later, the blood tends to take the ventral short cut and the dorsal part astrophies. The thoraco epigastric may be looked upon as a continuation of this mesh extending out upon the body wall. It drains a small area, below and dorsal to the limb bud and-has numerous connections with the capillaries supply- ing the lower part of the membrana reuniens. These latter capil- laries, it will be noticed, show a tendency to form into a long rope- like plexus, running from the region ventral to the posterior limb bud up towards the anterior limb bud. This rope of capillaries later forms the superficial epigastric (Ves) and may therefore be consid- ered as part of the permanent type. That the injection over the heart of this embryo is not complete is very probable because sections of this same stage show injection all over the upper heart region. This is shown in Fig. 6. Here, on either side of the U-shaped aortic arches, we find good-sized veins that empty into the internal jugulars (Vji). These veins drain the upper part of the membrana over a bib-shaped area, somewhat greater than is shown in the section, and anastomose on the sides with veins that enter the posterior cardinal at its junction with the anterior cardinal. Fig. 7 is from a section of this same embryo at the Jevel of the omphalomesenteric artery and shows the way in which the blood is supplied to the muscle layer and to the body wall. The 454 Helen Williston Smith. paired segmental arteries are seen passing out at a wide angle from each other, and each breaking up into a tuft of vessels, twigs of which run into the inner and outer side of the muscle layer and to the an- terior spinal artery and cord. These twigs pass into fine capillaries which may then be traced into the veins. The venous blood is ear- Fic. 6.—Section one-half millimeter thick of an embryo 151% millimeters long. MS, spinal cord; V.ji, internal jugular vein; 7, trachea; Aa, fourth aortic arches; MR, membrana reuniens. ried off in one of three ways. Either it may run through the thoraco epigastric to the posterior cardinal, or it may run into the membrana to the umbilical vein, or it may run back through the dorsal seg- mental veins into the mesonephros to the posterior cardinal veins (Vep). The sections of this embryo also show that at this stage the internal mammary vein and artery are not present, as such, but that Superficial Veins in the Pig. 455 the capillaries on the mesial side of the muscle layer have a tendency to unite and knot together to form a chain, the upper end of which runs into the posterior cardinal. The artery is represented by a few fine capillaries which run from the subclavian artery out under the arch of the thoraco epigastric. Fie. 7.—Section one-fourth of a millimeter thick of an embryo 154% mm. long; taken just below omphalomesenteric artery. MS, spinal cord; Vte, thoraco epigastric vein; Ao, aorta; Vv.cp, posterior cardinal veins; V. plerus MR, veins of the plexus of the membrana reuniens ; A.om, omphalomesenteric artery; V.uws, left umbilical vein; V.ud, right umbilical vein; 1M, mesonephros. From this stage on, however, the internal mammary vein and ar- tery as well as the thoraco epigastric and the plexus of the superficial epigastric developed rapidly. This further development is seen in Fig. 8. The thoraco epigastric (Vte) and superficial epigastric plexus (Ves) are now draining practically all of the dorsal side of the muscle layer of the body wall which has grown considerably farther Enlarged 12 times. $.—Embryo 18 mm. long. Fic. shown. Only most superficial vessels are al mammary vein; “MR, membrana V.te, thoraco epigastric vein; V.mi, intern reuniens ; vein. plexus of superficial epigastric vein; V.wv, umbilical V.es, Plezus Superficial Veins in the Pig. 457 forward carrying the blood vessels with it. The internal mam- mary (Vmi) is represented in the figure by a straight line ventral to the thoraco epigastric (Vte). It les on the mesial side of the muscle layer and has very numerous capillary connections with the thoraco epigastric. Posteriorly it anastomoses with the plexus of the deep epigastric, which runs beneath the superficial epigastric and is not shown in the drawing. The membrana reuniens is still large and well supplied with blood vessels, though it is evident that less blood runs from the body wall to the unbilical vein than formerly. As the internal mammary and thoraco epigastric veins now lie, their paths to the heart are about equal in length, and it is natural there- fore that blood supphed to the outer side of the muscle layer should pass back through the thoraco epigastric and that to the inner, through the internal mammary. It is evident, however, that, as the muscle layer grows forward, it will carry with it the internal mammary. The thoraco epigastric being an axillary vessel must still continue to empty into the axilla and therefore were the lower part of the vessel carried forward, the course of the blood through it, on the outer side of the muscle layer, would become more round about than that through the internal mammary. It is therefore to be expected that the blood, following the path of least resistance, will tend to flow from the lower part of the thoraco epigastric through the numerous con- nections into the internal mammary and that, these vessels enlarg- ing in consequence, the path to the internal mammary will become so easy that practically all the blood from the lower outer body wall will pass that way. This is what proves to be the case. In embryos about 18 or 19 mm. long the thoraco epigastric, as such, reaches its maximal development. Then it drains practically all the outer body wall between the limb buds, back as far as the circulation connected with the spinal cord, while ventrally it receives vessels from the mem- brana and anastomoses very frequently with the supers.cial epigastric and with the internal mammary. These anastomoses grow larger so that while the more posterior part of the thoraco epigastric becomes practically continuous with the superficial epigastric, anteriorly it begins to drain largely into the internal mammary. At this latter point a characteristic elbow is usually formed from which, as is RR, 4 ese ee: —, oO as JA = Lia Fic. 9.—Embryo about 8 em. long. Enlarged 6 times. All vessels except those immediately connected with the thoraco epigastric vein are omitted. Pars acillaris V.te, axillary portion of thoraco epigastric vein; Vv. et a.mi, internal mammary veins and artery; V.te, thoraco epigastric vein; MR, membrana reuniens; A.ep, deep epigastric artery. Superficial Veins in the Pig. 459 shown in Fig. 9, vessels pass to the internal mammary. Only one of these persists in older stages. There are all manner of variations as to the proportion of the thoraco epigastric that is left draining into the axilla. Sometimes almost the whole vessel is taken over com- pletely and sometimes, as in Figure 9, a considerable portion is left. This figure shows very well the transition stage of the thoraco epi- gastric. The vessel is seen passing on along the edge of the muscle layer to a point where a fan-shaped plexus of vessels conceals its anastomoses with the internal mammary. Here it swings in a long loop back to its axillary connection. This loop is, however, noticably smaller than the vessel below the turn, and it is evident that a good deal of the blood has already been deflected into the internal mam- mary. This connection with the internal mammary is plainly shown in the next figure. (Fig. 10). It also shows how far the internal mammary has developed (AmiVvmi), that it is now a double vein with the artery running between, receiving anterior intercostal veins, which anastomose with the intercostals proper. It also receives a great many fine, anastomosing vessels, which have been dissected away from the upper part of the membrana reuniens. The mem- brana is still of considerable size but the blood vessels are of a very feeble type compared with the earlier ones, and drain almost entirely back to the body wall through the superficial and deep vessels. As the membrana grows smaller and smaller, the blood vessels on the surface atrophy until only those along the edge of the advancing muscle layer are left visible. This is shown in figure 11, which is from an embryo 3 em. long. This embryo illustrates chiefly, how- ever, a case where almost all the thoraco epigastric along with the superficial epigastric with which it forms a continuous vessel, drains into the internal mammary. The internal mammary has been carried forward, while the stump of the thoraco epigastric has been left well back in the axilla. This is a very typical case. The chief variation being, as I said before, in the proportion of the thoraco epigastric taken over to the internal mammary. As the membrana continues to recede, and the muscle layer advance, the internal mammary ves- sels are carried nearer together and along with them the superficial veins, so that ultimately we find the transferred portion of the Fie. 10.—Embryo 3 cm. long. Enlarged about 5 times. The anterior limb is removed. Pars axillaris V.te, axillary portion of thoraco epigastric vein; Vv. et a.mi. internal mammary artery; Vv.mi, internal mammary veins; MR, membrana reuniens; V.te, thoraco epigastric vein; A.ep, deep epigastric artery. os 7-7---Pars axillaris Vte Fig. 11.—EHmbryo 3 cm. long. Enlarged 6 times. Pars avillaris V.te, axillary portion of thoraco epigastric vein; Vv. et a.mi, internal mammary artery and veins; V.te, thoraco epigastric vein; MR, membrana reuniens; Aep, deep epigastric artery. 462 : Helen Williston Smith. -thoraco epigastric as a very superficial vessel that may be seen plainly in uninjected specimens, running from its forked origin near the hind leg, along the milk ridge up to the manubrium at which point it dips sharply down to enter the internal mammary vein. This completes the history of the changes in the superficial blood vessels of the body wall. During this development, four circulations obtained in the super- ficial layers. First, the circulation in which the posterior cardinal plays a part. Second, the circulation across the membrana re- uniens to the umbilical vein; then, thirdly, the formation and growth of the thoraco epigastric and superficial epigastric on the outer side of the muscle layer; and somewhat later, that of the internal mam- mary veins and artery on the mesial side. The thoraco epigastric erows with the superficial epigastric until they drain the whole superficial body wall dorsalwards as far as the spinal cord. Mean- while the internal mammary vessels have grown. The intercostal veins and arteries also have been spun’ out of the plexuses of the segmental vessels, as the muscles and ribs invade the membrana reuniens, and they anastomose with the anterior intercostals from the internal mammary vessels. The internal mammary vein increases in size. Its connections with the thoraco epigastric increase also, and gradually the end of the thoraco epigastric is switched off, and we reach the fourth and final stage in which the superficial body wall drains largely into the internal mammary vein. After this con- dition is reached further change is not so much in kind as in degree. REFERENCES. Ba.rour, F. M. Comparative Embryology, II, 658. Burow. Archiv f. Anat. u. Phys., 1838, S. 44. ECKER U. WIEDERSHEIN. Anatomie des Frosches, 8. 418. His. Anat. mensch. Embryonen. III, 8. 206, also Fig. 130. His. Die anat. Nomenclatur. Leipzig, 1895. Joris. Recherches sur les Veines Ombilicales et Para Ombilicales. Bull. de ’Acad. Royale de Médecine de Belgique, 1905. KOLLIKER. Grundriss d. Entwickl. d. Menschen, S. 103, Fig. 58, 1884. Matt. Journal of Morphology, XIV, p. 362, 1898. PFEIFER. Zur Kenntnis des histolog. Baues und der Riickbildung der Nabelgefiisse und des Duct. Bot. Arch. f. Path. Anat., CLXVII. SAPppEy. Veines portes accessoires. Jour. de ]’Anatomie, XIX, 1883. WERTHEIMER. Recherches sur la veine ombilicale. Jour. de l’Anat. et de la Phys., XXII, 1886. THE VASCULARIZATION OF THE HUMAN TESTIS. BY EBEN CLAYTON HILL, From the Anatomical Laboratory of the Johns Hopkins University. WitnH 9 FIGURES. The literature of investigations on the vascularization of the testis is very meagre, the absence of recent studies of the blood supply of the testis being evident in studying text books on anatomy. The descriptions in these books are taken from Arnold,’ who in 1847 published the first serious attempt at a solution of the blood supply to this gland. Shortly after Arnold’s publication Huschke and Kdélliker added to a small degree to his description, but practically all anatomical text books and atlases reproduce the original Arnold illustrations and present his original description. The cause of this is manifest and can be readily assigned to the necessity of awaiting the invention of the newer, simpler and more perfect methods of injection which have recently been furnished. Arnold had at his disposal only the most difficult means for injections and it is surprising that he was able to carry his studies as far as he did. He was aided only slightly by these injections, and like von Baer before the days of the microtome, had to depend prin- cipally upon careful dissections. He succeeded, however, in trac- ing the course of the spermatic artery and its branches through the cord to the testis. In 1904, under the guidance and at the suggestion of Professor Mall, I undertook an investigation of the vascularization of the testis of the pig. This study was begun with the idea that a knowledge of the blood supply of the male sex gland of this mammal ‘Arnold. Handbuch der Anatomie des Menschen, 1847. Surgeon-General’s Library, Washington, D. C. The original article could not be located. THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. / 464 Eben Clayton Hill. would greatly simplify the later research on this gland in man. Such, however, was not the case and results obtained in late years by various investigators tend to disprove the theory that the general structure and blood supply of the organs of lower mammals is al- ways similar in detail to that in man. Text books have too frequently adopted the results obtained from investigations on dogs, pigs or other mammals and have incorporated the illustrations and descriptions without specifying that such re- sults have not as yet been verified for man. So very marked are the differences of the gross structure and blood supply of the human testis as compared with that of the pig that the study of the human testis was practically an entirely new research. The methods used were similar and will be briefly de- tailed, but in no other way did a study of the testis of the pig aid in solving the problem of the blood supply of the male sex gland of man. Professor MacCallum, of the pathological department, very kindly placed at my disposal the testes from autopsy subjects, and for. this courtesy I desire to express my thanks. These specimens were usually brought to the anatomical laboratory within twenty-four hours after death and were in excellent condition. Professor Mall and Professor Brédel also aided me in obtaining several valuable specimens of human embryonic testes, and to them I wish to ex- press my appreciation. Embryonic material in fit condition for injection is most difficult to obtain, and I regret that a series of human embryonic testes could not be injected similar to that of the pig.” My injections were made with either India ink or celloidin. The detailed technique of these injections is given in my separate publication® on clearing methods, but the following improvements should also be noted: The gland or organ under investigation must be fresh and must contain no clotted blood. Warm, normal saline "Hill, E. C. On the Gross Development and Vascularization of the Testis, Am. Jour. Anat., Vol. 6, No. 4. *Hill, E. C. On the Schultze Clearing Method as Used in the Anat. Lab. of the Johns Hopkins University. J. H. H. Bull., Vol. 17, No. 181. The Vascularization of the Human Testis. 465 solution is forced into the artery under a pressure of 60-80 mg. of mercury, and is allowed to pass through the vascular tree and issue from the returning veins. After a short interval, depending upon the size of the organ, the fluid pouring from the vein will contain little blood and later will be clear. The gland, however, has become firm from the passage of fluid from between the endo- thelial cells of the capillaries. It seems odd that a pressure below the normal systolic blood pressure should cause this extravasation, but this may perhaps be due to the rapid death of the endothelial cells. Air is then forced into the artery and in an incredibly short time bubbles of air are seen at the openings of the veins, the organ becomes soft and no sign of tension is manifest in the vessels, which are now thoroughly emptied. Then follows a similar injection of equal parts of absolute alcohol and ether, and after the appearance of this mixture at the openings of the veins a second “blowing out” is resorted to. In this instance, however, the cells lining the vessel walls are more or less fixed with absolute alcohol and ether and their lumen is open and ready for the celloidin injection mass. The celloidin, usually 7 per cent., colored with vermillion or finely powdered lamp black, readily enters the vessels against no backward pressure and flows into and completely fills the smallest vessels. By using a thin celloidin for the arterial and capillary vessels, and a thicker celloidin for the veins, a very instructive and beau- tiful double injection is obtained with comparative ease. Such specimens are valuable for microscopic work, for clearing in 1 per cent. potassium hydroxide and glycerine and for corrosion work. The vermillion and lamp black withstand the ordinary laboratory reagents even in concentrated forms. The method of corrosion used is simply that of peptic digestion as devised for such studies by Dr. Mall.* In digesting the testis it was found advisable to place the gland in concentrated hydrochloric acid for 6 or 8 hours in order to soften the tough fibrous albuginea. This was followed by a digestion at 38 degrees C. in the thermostat with pepsin and ‘Mall, F. P. A Study of the Structural Unit of the Liver. Am. Jour. Anat., Vol. 5, No. 3. 466 Eben Clayton Hill. .38 per cent. hydrochloric acid. These specimens in pure glycerine can be permanently kept cleansed of any small particles of un- digested gland. They are not at all friable, and one perfect double injection clearly defines the entire blood supply of the gland. Other thick blocks of the testis were cleared without digestion in 1 per cent. potassium hydroxide after hardening in 95 per cent. alcohol. These specimens showed the capillary network around the lobules and careful dissection revealed the lobular arrangement with its blood supply. Water macerations similar to those used by Dr. Mall’ in studying the structure of the spleen were attempted without success. This was rather puzzling until investigations of the reticulum surround- ing the lobules was begun. So dense and firm are the bands of reticulum encasing these tubules that the entire gland will macerate in water before any of the cells can be shaken out. Also the trabeculae, which figure so conspicuously in all illustrations of the human testis, disappeared under maceration. ‘The causes of this will be taken up in discussing the blood supply. In studying the reticular structure entering into the gland, Mall’s method® used so successfully by Flint’ in his work on the adrenal was followed. Arnold, in 1847, described the course of the spermatic artery and its branches. His beautiful illustrations show quite accurately and clearly the anterior and posterior scrotal arteries and the ex- ternal and internal spermatic arteries. He traced the latter in the cord where it gives off two or three branches to the epididymis, one of which anastomoses with another small branch of the internal spermatic artery which follows down the vas deferens. His dia- gram and account show these branches to the gland proper passing under the albuginea, but not penetrating the testis at the medias- tinum. Arnold noted in his article the observations made by "Mall, F. P. The Structure of the Spleen. Johns Hop. Hosp. Rept. *Mall, F. P. Reticulated Tissue and Its Relation to the Connective Tissue Fibres. Johns Hop. Hosp. Rept., Vol. 1. "Flint, J. M. The Blood Vessels, Angiogenesis, Organogenesis, Reticulum and Histology of the Adrenal. Johns Hop. Hosp. Rept., Vol. 9.- The Vaseularization of the Human Testis. 467 Huschke as to the comparative size of the renal artery and sper- matic vessels. Huschke states that the calibre of the renal artery is 15 times that of the internal spermatic artery, although the weight of the testis is only one-eighth as great as that of the kidney. This is a most interesting observation and explains the difficulty met with in attempting injections of the testis. Not only is the spermatic artery very small, but it is long and near the human sex gland becomes tortuous, although this is hardly appre- ciable as compared to the great tortuosity met with in the spermatic artery of the pig. Arnold in describing the veins states that these veins “entsprechen in ihrer Anordnung im Allgemeinen den Ar- terien.” He traces the blood from the testis through the pampino- form plexus and its subsequent branches back into the renal vein or aorta. Thus it is seen that he does not trace the blood farther than to the gland itself, and makes no attempt at interpreting its course after reaching the mediastinum or albuginea. That he should have been in doubt about the distribution of the arterial supply near the mediastinum is easily understood because only the most perfect double injection made by methods but re- cently developed, could aid in unravelling the profusion of veins and arteries at this portion of the gland. Astley Cooper has in- vestigated a capillary plexus covering the inner surface of the tunica albuginea, which he has termed the tunica vasculosa. Aside from these valuable contributions, practically no work has been done on the blood supply of the testis; hence the following results seem worthy of record. The spermatic artery arises from the abnormal aorta as a long slender branch passing into the abdominal rings, and from here follows in the spermatic cord to the testis. The vessel which directly supplies the testis proper and which is a direct continua- tion of the spermatic artery has been named by Arnold the in- ternal spermatic artery. The spermatic artery, before giving off its terminal branch or branches to the testis, gives off ordinarily a branch, the external spermatic artery, high in the cord, just below the external abdominal ring which in time divides in two or more branches to supply the membranes of the testis. Until the sper- 468 Eben Clayton Hill. matic artery ends in the one or more terminal branches to the testis, Arnold’s original description is excellent. These terminal branches (A and B, Fig. 1) become tortuous just before reach- ing the mediastinum of the testis and near the globus major of the epididymis send one rather large vessel to supply the tunica albuginea. This branch, one of the capsular branches, encircles the gland on the inner side of the albuginea and sends deep branches into the glandular substance which anastomose with the ascending arteries given off at the mediastinum. The terminal arteries, after giving off the capsular branch, break up near the mediastinum and send a great number of small arteries into the gland. A small branch from the terminal arteries descends to the globus minor and _pass- ing under the tunica albuginea (C A, Fig. 1) runs under this capsule and anastomoses with the capsular branches given off at the level of the globus major. These capsular branches send out many small arteries, most of which are rather tortuous and encircle the gland on the inner side of the albuginea. These vessels and their branches supply a capillary plexus on the inner side of the tunica albuginea which has been called the tunica vasculose by Astley Cooper. Branches from these penetrate the glandular substance and anastomose with the arteries given off at the mediastinum from the spermatic artery. Also the capsular artery sends small branches to the tunica par- ietalis visceralis. In most of the specimens examined a large branch (F, Fig 1) passes from the capsular artery to the medias- tinum and anastomoses with the arteries in this portion of the gland. Generally at the level of the globus minor of the epididymis a Fig. 1.—Arterial supply of human adult testis. A portion of the gland has been removed so as to show the penetration of the arteries through the medi- astinum into the glandular tissue. A. B., main terminal branches to testicle; C., branch following spermatic cord and encircling and supplying vas deferens; C. A., capsular artery—a branch from B.; C. E., caput epididymis—shown in outline; D., branch of capsular artery lying on innermost side of albuginea; E., outline of epididy- mis; F., central artery connecting vessels of mediastinum with capsular branches; M., mediastinum. x 3%. THE VASCULARIZATION OF THE HUMAN TESTIS. EBEN CLAYTON HILL. THE AMERICAN JOURNAL OF ANATOMY.—VoOL. IX, No. 4. THE VASCULARIZATION OF THE HUMAN TESTIS. EBEN CLAYTON HILL. THD AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. 71 7 oe q , ay Yr Ss) va ¥ ‘+500 i i). 2 ar ' oar : ; eS te ve er n me er cut boa OWS he a C es Ae a Li's Pts | en en ee THE VASCULARIZATION OF THE HUMAN TESTIS. EBEN CLAYTON HILL. Fig. 3. THD AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. The Vaseularization of the Human Testis. 469 branch is given off from the spermatic artery which anastomoses with the artery supplying the vas deferens (C, Fig. 1). The blood supply of the tunica vaginalis parietalis (Fig. 2) is uninteresting. The arteries pass to this tunic near the epididymis, are slightly tortuous, give out small branches, which ending in capil- laries, form a plexus. The blood from this plexus is collected into veins which in turn empty near the epididymis. The blood supply of the tunica albuginea and the branches given off from the capsular arteries is shown.in Fig. 3. A thick section, including the albuginea and a portion of the glandular substance, taken from an injected gland and cleared, shows three general group- ings of vessels :— First (A), those dipping perpendicularly into the glandular sub- stance and following the trabeculae to supply the lobules. Second (B), capsular arteries and their branches on the inner side of the tunica albuginea, and Third (C), the small branches given off from the capsular ar- teries which pass outward and penetrate the albuginea supplying it and the tunica vaginalis visceralis. Corrosion specimens of the human testis show very clearly the arrangement of the vessels, and these, when placed in glycerine, may be isolated in order to study their characteristics. (Fig. 4.) In this connection it may be of interest from the standpoint of comparative anatomy to note the marked differences- between the blood supply of the testis of the pig and that of man. This is especially well shown by isolating arteries from corrosion specimens of each. In man the distribution of these vessels and their course Fic. 2—Blood supply of tunica parietalis of the human testis. Injected with India ink under 20 mm. Hg. pressure. Drawn with camera lucida. < 26%. A., artery; V., vein. Fic. 3.—Celloidin arterial injection of capsule and outermost glandular tissue of human adult testis. Specimen cleared in 1 per cent KOH and glycerin. Three arrangements of vessels are shown. ist. Surface vessels on outer side of tunica albuginea. (c) These arise from branches of capsular arteries (b) which lie on inner side of albuginea. From these latter vessels branches (a) penetrate the glandular tissue and anastomose with the as- cending arteries coming from the mediastinum. X 10. 470 Eben Clayton Hill. is “rational.” If a student knew that the vessels entered the mediastinum and also penetrated the substance of the gland through branches from the capsular artery, he could readily picture the distribution of these vessels. In the pig testis the vessels enter from the capsule, but form most unusual loops. Reference to my former article on the pig’s testis’ will serve to bring out the contrast with the arrangement in man; both in the penetrating vessels and their branches and in the supply to the albuginea, ete. Practically the whole blood supply of the testis of the pig comes from one large capsular artery which encircles the gland sending tortuous rib-like branches around it. These branches send other branches deep into the gland to the mediastinum without giv- Fic. 4.—Isolated arteries of human adult testis. These vessels were in- jected with celloidin and the testis was then digested in HCl and pepsin. x 2%. ing off any branches, then loop back and supply the tubules through the return loops. This unique arrangement may be occasioned by the central location of the mediastinum (Fig. 5) which is so essen- tially different from the lateral location of this collecting portion of the human testis. A cross section of the fresh testis gives an impression that very thick, strong trabeculae pass from the mediastinum to the albu- Fic. 5.—Sagittal section through testis of adult pig to show central location of mediastinum. E., epididymis; M., mediastinum; S. C., spermatic cord. THE VASCULARIZATION OF THE HUMAN TESTIS. EBEN CLAYTON HILL. Geo: THD AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. tubules. The Vaseularization of the Human Testis. 471 is true, but their apparent thickness is due to the fact that arte- ries and veins follow in radiating lines in the very fine connective tissue bands, and it is principally the presence of these vessels that has given rise to the erroneous idea of heavy trabeculae, (Dt Bs. an \ Ss ——— > a af iV KS Fic. 6.—Reticulum of human testis, showing reticular formation around Digested in HCl and pepsin, stained with picric acid and acid fuchsin. > 10. Drawn with camera lucida. Whitehead*® has published some very instructive results accom- panied by illustrations of the reticulum of the testes of various ®Whitehead, R. H. Studies of the Interstitial Cells of Leydig, No. 6. Ana- tomical Record, No. 8, 1908. 472 Eben Clayton Hill. mammals including man. On one point he has not laid especial emphasis, and that is the great strength of the reticulum of the testes of man and its resistant powers. Testes treated with weak KOH do not macerate readily, and even after the capsule has become disintegrated the tubules are firm and resistant. They are elastic and can be teased out of their full length without breaking. This is due to the great amount of reti- culum surrounding them (Fig. 6). A similar reticular structure is found in the tubules of the kidney (Mall), only the reticulum of the testis is denser. It is the presence of this large amount of reticulum that prevents the preparation of specimens of the gross structure of the lobular arrangement of the spleen which Professor Mall found cf such value in studying that organ. Turning again to the description of vessels we find that the com- paratively large branches given off at the mediastinum divide into many small branches which radiate toward the albuginea like spokes in a wheel. These ascending arteries (A A, Fig. 7) pass between the lobules and give off capillary branches to the tubules. An anastomosis may be noted between the ascending arteries and the descending arteries (D. A., Fig. 7) given off from the capsular arteries and their branches. The veins follow the general course’ of the arteries. There are several large capsular veins which en- circle the gland, lying on the inner side of the capsule and empty- ing into the pampiniform plexus. These capsular veins ‘receive blood from the capillaries and veins on the inner surface of the tunica albuginea, from the tunica vaginalis visceralis and from anastomoses with the ascending veins which enclose the lobule (A. V., Fig. 7). The blood is also returned to the pampiniform plexus by descending veins which follow the course of the arteries and empty into the venous plexus at the mediastinum (D. V., Fig. 7). od Fic. 7.—Sagittal section of human testis; to show blood supply. Injected with red and blue celloidin, cleared in 1 per cent KOH and 20 per cent glycerine. x 4. A. A., ascending artery; A. V., ascending vein; D. A., descending artery; D. V., descending vein; M., mediastinum; V. D., vas deferens; T. A., tunica albuginea; T. P., tunica parietalis. THE VASCULARIZATION OF THE HUMAN TESTIS. EBEN CLAYTON HILL. THn AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. Sine 2) od ease ee = » * , = 7 THE VASCULARIZATION OF THE HUMAN TESTIS. EBEN CLAYTON HILL. Fic. 9. HIG Se THE AMERICAN JOURNAL OF ANATOMY.—VOL IX, No. 4. The Vaseularization of the Human Testis. 473 Mall several years ago advanced a theory as to the presence of certain units of the blood system which may or may not be peculiar to the organ in which they are found and which correspond to the histological or structural unit of the organ. These units are composed of small branching blood vessels which pass into capil- laries, the blood from which is collected into small veins. This theory of vascular units may be briefly summed up in the statement “sim- ilar blood supply to similar histological units.” These vascular units have since been proved to be present in the liver, spleen and adrenal, but in the testis of the pig I could make out no definite units. In man, however, the lobular arrangement is less complex, and I was enabled to make out vascular units which correspond to units of structure and which repeat themselves similarly throughout the organ, the one bearing a definite and constant anatomical relation to the other. To determine the units, a testis, after receiving an arterial and venous injection, was placed in 95 per cent alcohol until well hardened, then cut into blocks measuring about .5 em. in thick-- ness and later cleared in 1 per cent. potassium hydroxide. In this way the tubules remained firm and the lobules could be teased out with their vascular supply. When placed in 20 per cent glycer- ine the vascular units were clearly defined. Fig. 8 indicates the arterial supply to a lobule. There is a very profuse anastomosis between the arteries enclosing the lobule and each lobule receives blood from two or more ascending arteries and a like number of descending arteries which are branches of the capsular arteries. From the vessels enclosing the lobules small arterioles are given off which in turn encircle the tubules ending in a plexus around them. Thick microscopic sections of the injected Fic. 8.—Arterial supply of lobule of human adult testis. Injected with celloidin, cleared in 1 per cent KOH and glycerine. The testis was then cut into large pieces and the lobules teased out. C., capsule showing capsular artery on inner side of albuginea; M., mediastinum. Fic. 9.—Microscopic section of adult human testis. Injected with 4 per cent celloidin to show arterial and capillary blood supply to tubules. Sec- tions cut to 200 microns in thickness, stained in H. and EH, and cleared in creosote. >< 50. 474 Eben Clayton Hull. testis lightly stained show very beautifully these small capillaries surrounding the tubules (Fig. 9). The arteries and veins supplying each lobule run in the trabecule, and it is principally to their presence that the testis has the appear- ance of such definite lobular divisions. STUDIES ON THE VASCULAR SYSTEM OF THE TEY RORD GiAsNy. | BY RALPH H. MAJOR. From the Anatomical Laboratory, Johns Hopkins University. WitH 10 FIGURES. It has long been known that the thyroid gland is a very vascular organ. This we would expect both from anatomical and physiologi- eal reasons, having such a rich gross blood-supply and exerting such a profound influence through its secretion upon the physical and nervous development of the body. Tschuewsky (1), by a series of carefully performed experiments, has supplied us with data upon the subject. He found that the amount of blood flowing through the thy- roid per 100 gram weight of the organ to be 560 ccm. a minute. This same observer, using a like standard of calculation, found the amount of blood flowing through the head to be 20 cem. per minute, and through the kidney 100 ccm. per minute. Thus the thyroid, according to him and using the blood-flow per gram weight as the standard of comparison, is twenty-eight times as vascular as the head and five and one-half times as vascular as the kidney. Tschuewsky also estimates by a series of calculations that in the dog the entire amount of blood in the body flows through these small glands sixteen times in one day. This enormous blood-supply has led to a great deal of physiological speculation and it has even been suggested that the main function of the tyroid gland consists in acting as a vascular shunt to protect the circulation in the brain (2). In these studies, begun at the suggestion of Dr. Mall and finished through his constant advice and encouragment, an attempt is made to study a few of the main points of the microscopic blood-supply of this gland. The thyroid glands of the cat, dog and man have been studied principally and the glands were those of the adult ani- THE AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. 476 Ralph H. Major. mal. I wish here also to express my thanks to Dr. H. M. Evans for the use of several injected specimens of human thyroids which he had injected in connection with his work upon the parathyroids (3). The histological structure of the thyroid has been the subject of considerable literature in the past. Baber’s (4) classical studies of the minute anatomy of the structure of the thyroid gland was one of the most important of the early contributions to this subject. He described particularly the histology of the follicles and the lymphatics. Langendorff (6), Wéolfler (7), Lustig (8), Biondi (9), Hiirtle (10), Andersson (11), Kohn (12); Streif (13), 0 Bia (14), Coco (15), and others have given us important contri- butions upon the subject. These studies show that consid- erable differences of opinion have existed as regards its histological structure. W6lfler found that the thyroid of the child as well as of the adult contains solid rows of cells at the periphery while the center of the gland is composed mostly of vesicles filled with colloid material. He does not seem, however, to have attempted to divide it definitely into a cortical and medullary portion. Flint does not believe that thyroid gland, of the dog and man at least, can be di- vided into either lobes or lobules, but that the septa from the capsule penetrate the parenchyma of the gland in an irregular fashion. He has, however, observed pictures that suggest definite lobulation, but thinks that if the original lobulation is present in the embryo it is later lost. Regaut and Pettijean (16) studying the dog, cat, pig and other animals also do not believe that the thyroid gland can be divided into lobules. They maintain that there is no such architecture present, that there is no distribution of blood vessels or lymphatics to war- rant the conception of a lobule. Coco (15) gives a description of the structure of the dog’s thyroid that summarizes and agrees in general with the conclusions of most observers, and with the descriptions in most text-books. According to him, the thyroid gland is surrounded completely by a thick capsule which gives off numerous prolonga- tions of connective tissue. These septa penetrate the parenchyma of the gland, dividing and sub-dividing it into lobes and lobules. The septa which also support blood-vessels and nerves, become thin- ner and thinner and finally end by surrounding each follicle in the Vascular System of the Thyroid Gland. 477 form of a delicate membrane which supports the epithelial cells. The sections examined in the course of my own studies indicate a division of the gland by septa into lobes and lobules, and as will be shown in this paper, definite vascular units can be dissected out, which correspond to these structural units. Streif (13), by use of the Born wax model method, has shown that the glandular structure of the thyroid consists of closed follicles which are separated from each other by fine connective tissue. He also showed that a system of canals is not present and that the follicles do not communicate with each other. There seems to be no extensive literature upon the microscopic blood-supply of the thyroid. Kohlrausch (17) in 1853 drew atten- tion to the fact that the follicles are surrounded by a rich capillary net- work. This fact, as well as the presence of many lymphatic spaces surrounding the follicles, is mentioned in the textbooks of anatomy and histology. Wé6lfler devotes some space to the consideration of the blood-vessels. He studied them principally from the embryological standpoint, in their relation to the developing gland. He observed a network of capillaries surrounding each follicle and twigs from the blood-vessels to the follicles, but does not seem to have studied them further and no drawings illustrating these points are found in his monograph. Streckeisen (18) has made a very careful study of the gross distribution of the arteries supplying the thyroid gland. Landstrém (19) studied the gross distribution of the arteries and paid especial attention to the subject of the arterial anastomoses. His / article gives a résumé of the work done upon this subject with a com- | plete bibliography. In my own studies the injection method was used practically altogether. Specimens were injected with various injection masses— India ink and carmine, ultramarine blue, vermillion (mercuric sul- phide) granules in various per cent. gelatine solutions. In some cases single injections were made either into the arteries or veins, in other cases double injections were made, filling both arteries and veins. India ink and carmine both give very good injections of the follicular blood-supply, as the masses on account of the small size of their granules easily penetrate the capillary bed. Some very good 478 Ralph H. Major. double injections were made by using carmine and ultramarine blue injection masses. The carmine was first injected into the arter- ies and continued until the capillary bed was filled; then ultramarine blue was injected into the arteries, forcing the carmine out through the capillary bed and over into the veins. The ultramarine blue granules fill the arteries, but fail on account of their large size to pass over into the capillaries, and if we stop at the proper moment we have a double injection, in which the arteries are injected blue, the capillaries and veins red. Partial injections also gave some very in- structive specimens. After injection the specimens were hardened in alcohol or formalin, imbedded in paraftine or celloidin, cut and cleared in creosote. It was also possible, by taking a small piece of an injected gland, to dissect out under the binocular microscope, lobules and even single follicles. This, in the case of the human thy- roid, gives perhaps the most instructive specimens, as a mounted specimen thick enough to contain a whole lobule, is usually too thick to be studied succesfully under the microscope. Also a lobule or fol- licle when dissected out, can be turned about and studied from va- rious sides. The general form and shape of the human thyroid as well as its gross blood-supply is described in almost any text-book of anatomy. Also the variations in shape and position of the thyroid gland in various animals is described in the text-books of comparative an- atomy. It will be remembered that the thyroid of the cat and the dog differs from that of the human in consisting of two bean-shaped lobes, one on either side of the trachea, which are connected in the cat by a very thin strip of an isthmus and more completely separated in the dog. In these animals, too, the inferior thyroid artery is of very small size and by far the greater part of the blood reaches the gland through the superior thyroid artery. There seems to be considerable discussion as to the existence of anastomoses in the human thyroid, between the superior and the inferior thyroid arteries. Landstrém (19) and also Streckeisen (18) | show clearly by their injections that such anastomoses are present | upon the surface of the gland, not only between the arteries of the same | side, but also between the two sides. In my own injections such sur- Vascular System of the Thyroid Gland. 479 face anastomoses were easily demonstrated. Landstrém also expresses the conviction that anastomoses also occur within the gland, but did not succeeded in demonstrating them. The method that Landstrom employed was that of injecting the arteries with Woods’ metal, and then taking a Roentgen-ray picture of the gland. In my own studies several glands were injected with celloidin, and then by di- gesting with artificial gastric juice, corrosion specimens were ob- tained in which the arterial tree could be followed from its trunk to its termination in the individual follicles. In no case was it pos- sible to find definite arterial anastomoses beneath the surface of the gland. The arterial tree is so exceedingly complex that it is difficult to decide this point absolutely. It is also easy to imagine what a com- plex picture would be obtained by Landstrém’s method. In many cases when from study under the microscope a definite anastomosis appeared to exist, yet after carefully moving the blood-vessels with a pair of fine needles it was seen that the two arteries did not really anastomose, but ran out eack to its termination, entirely independent of the other. In many eases, large branches of the thyroid arteries would turn and twist about in great confusion, without, however, anastomosing. In the cat’s thyroid, anastomoses occur as are seen in Fig. 1. Gelatin injections of the dog’s thyroid, show a few anasto- moses, but they occur between branches of the same artery, and not between branches of the superior and inferior thyroid arteries. At any rate, if anastomoses are present in my specimens of human thyroids they are of small size. The capsule of the thyroid gland, like similar tissues, has a very seant blood supply. The arteries upon entering the gland give off small branches at various places which join each other to form a net- work throughout the substance of the capsule dividing it into large diamond-shaped areas. Each artery is accompanied, as a rule, by two veins, which are connected at various places by bar-like veins which run across the artery. The veins anastomose in the same manner as the arteries, and empty at various places into the large veins that are emerging from the interior of the gland. Occasionally a capsule vein empties into a vein within the gland, but such anatomoses are infrequent. The same general scheme of this circulation is ob- 480 ; Ralph H. Major. served in the cat, dog and in man. The capsule of the thyroid gland has been divided by some into an internal capsule which can be stripped off only with some difficulty, and an external capsule which strips off readily. The above description refers to the blood-supply of this outer capsule. Fic. 1.—Corrosion specimen of arterial injection of cat’s thyroid, showing numerous anastomoses. X 5. The manner in which the arteries approach the thyroid gland show some variations in the cat, dog and man. In the dog the superior thyroid artery gives off two main branches, one anterior branch and a posterior branch. Each of these in turn give off four or five smaller Vascular System of the Thyroid Gland. 481 branches which penetrate the gland. In a certain specimen the total number of these branches from both anterior and posterior branches was nine. These branches plunge into the gland and immediately give off branches which run in various directions, some attempting to gain the periphery of the organ, others running still deeper towards Fie. 2.—Gross blood supply of cat’s thyroid. x 5. A.—Artery. V.—Vein. P.—Parathyroid gland. the center. These arteries of the second order surround definite di- visions of the gland and give off no branches, as a rule, to the follicles. The veins follow more or less the same course, but show frequent anastomoses. The course of the arteries to the cat’s thyroid is somewhat similar to that of the dog. The superior thyroid artery, however, gives off a 482 Ralph H. Major. much larger number of branches before penetrating the gland. This is illustrated in Fig. 2. The superior thyroid divides into two main branches which course down the sides of the gland, each giving off a large number of branches, some of which are branches of the second order, others of which give off branches of the second order. The total number of the branches given off by the two main divisions of the superior thyroid artery in the specimen drawn is forty-one. The arteries of the second order are distributed as in the dog and pass between lobes of the gland. The course of the arteries in the human thyroid resembles some- what that of the dog, but presents differences which are apparent at lig. 3.—Gross arterial blood supply of human thyroid. RS T—Right superior thyroid artery. RIT—Right inferior thyroid artery. LS T—Left superior thyroid artery. L1IT—Left inferior thyroid artery. first sight. The human thyroid gland differs from that of the dog in shape, in the presence of a well-defined isthmus and in the fact that in man the inferior thyroid artery is as large or larger than the superior thyroid. Variations in the gross blood-supply of the human thyroid are common, but a general scheme seems to be present, with differences in method of anastomoses. Such a general scheme is shown in Fig. 3. Here we see the superior thyroid artery approach- to Vascular System of the Thyroid Gland. 483 ing the upper poles of the gland, and the inferior thyroid arteries ap- proaching from beneath. Each artery gives off four or five branches, some of which supply the anterior, some the posterior surface of the gland. The main continuations of these arteries are prone to run along and upon the margins of the gland, and the superior and in- Fig. 4. —Drawing illustrating the venous network upon the surface of human thyroid with accompanying arteries. A.—Arteries. V.—vVeins. ferior thyroid arteries upon the same sides anastomose, two anas- tomoses occurring between each artery in the specimen from which 484 Ralph H. Major. Fig. 3 was drawn. The general scheme of these anatomoses varies considerably in the different human thyroids. Landstrém, in his article to which reference has previously been made, gives excellent drawings of some of these variations. In the human thyroid, few large arteries are present in the depths of the gland, and in this respect it differs from the dog. In other words, in the human the branching of the large arteries takes place mostly upon the surface of the gland, and having by their branching obtained their approximate distribution, the smaller branches are sent in. Fic. 5.—Drawing illustrating arteries of third order passing between lobules and arteries of fourth order supplying the lobules in the human thyroid (partly diagrammatic). The further distribution of the arteries is essentially the same in both the dog and man. The arteries of the third order, as is shown in Fig. 5, pass between the lobules and give off arteries of the fourth order which supply the lobule. Each lobule is composed of a num- ber of follicles and is supplied usually by from two to five arteries, the number of arteries depending upon the size of the lobule. Fig. 6 and Fig. 7 show two lobules which have been dissected out ii a (aca CCC Fic. 6. Lobule of human thyroid dissected out. Fic. 7.—Lobule of human thyroid dissected out. 486 Ralph H. Major. with their blood-supply intact. They resemble, to use the classi- cal Malpighian expression, a cluster of grapes, over which arte- ries and veins can be seen twining about. These arteries of the fourth order run over the surface of the lobules and give off fol- Fic. 8. lig. 8.—Single follicle of human thyroid dissected out, showing its follicular artery A, and follicular vein V. x SD. I'tc. 9.—Single follicle of human thyroid dissected out, showing a follicle receiving its blood-supply from its own follicular artery A, and also from the follicular artery supplying an adjacent follicle A’. V.— Follicular vein. Sb. licular arteries to each follicle. The follicilar arteries end in a rich capillary network which surrounds each follicle. The vein which arises from this capillary network upon the far side to the artery, follows fairly closely the course of the artery. Figs. 8 and 9 show Vascular System of the Thyroid Gland. 487 the termination of the follicular artery at the follicle, the capillary network and the vein arising from the far side of the follicle. The relations between the capillaries and the cells of the fol- licle are seen in Fig. 10. The capillaries lie just outside the cells in the connective tissue that forms a sort of capsule for each fol- Fic. 10.—Drawing illustrating the relation between the follicular network of capillaries and the cells of the follicle in the human thyroid. Capillaries in solid black. X 200. licle. It is also noted that* compared with the size of the individual cells, the capillaries are very gross structures. This is interesting from the standpoint of the secretory changes in the thyroid. Ac- cording to Andersson, Hiirtle, Biondi and others, the colloid mate- 488 Ralph H. Major. rial passes from a follicle into the lymph spaces by a gradual oblit- eration, “melting” or bursting, of some portion of the wall of the follicle. As the meshes in the capillary net are large as compared with the size of the cells, it can be conceived that quite a number of cells can be destroyed without affecting the integrity of the capillary network. Thus an opening sufficient to permit the escape of the colloid material can take place without rupturing a capillary and causing hemorrhage, or at most only a few capillaries need be ruptured. That some capillaries are often ruptured, is shown by the frequent finding of red blood cells in the lymph spaces and in the cavity of the follicle. The veins that return the blood from the follicles follow closely the path of the arteries, show frequent anastomoses and finally reach the surface of the gland where they anastomose freely. The average size of the arteries of the first order is .15 mm.; those of the second order .1 mm.; those of the third order .03 mm.; and lastly the follicular arteries are .0125 mm. in size. The eapil- laries of the follicular network average .008 mm. in size. These measurements are those of the normal human thyroid. The finer distribution of the blood vessels in the cat’s thyroid differs somewhat from that of the dog and man. In the cat the arte- ries of the second order pass between lobes and the arteries of the third order pass between lobules just as in man. Arteries of the fourth order passing to the lobules are also present, but not so constant. In the cat, however, no follicular arteries are present. Each follicle is not surrounded by a rich capillary network and supplied by its own follicular artery, but the follicles are placed in a loose, wide network, each mesh of which in a cross section appears to surround a single follicle. This network has depth as well as length and breadth and surrounds the follicle in three dimensions. The arteries which supply the lobules, approach the lobules and immediately split up into capillaries without giving off any follicular arteries. The veins collect from the capillaries at a point somewhat removed from the arteries often directly opposite them, but soon approach them and follow the same general course. Vascular System of the Thyroid Gland. 489 This description of the thyroid blood-supply must be taken only as indicating a general scheme. Certain variations will, of course, be noted. The size of the lobules, depending upon the number of follicles composing it, will, of course, vary and with it the number of arterial branches supplying it. Also the blood-supply of the individual follicles is subject to certain variations. In many cases a single follicle, as shown in Fig. 9, besides receiving its blood-supply from what might be termed its own follicular artery, receives small branches from an artery which supplies an adjacent follicle. The veins while in general following the course of the arteries, also show many variations. Often the vein which springs from the follicular network, instead of passing back side by side with the artery, empties into a vein which follows the course of an artery supplying follicles on the far side of the lobule. Such a picture is seen in Fig. 6. The capillary network surrounding the follicle anastomoses very commonly with that of an adjacent follicle. Thus it is seen, that in the thyroid, too, we have a definite system of blood-supply, a definite system of vascular units, which repeat themselves with a greater or less constancy throughout the entire organ. These vascular units correspond in most instances very closely with the structural units. The smallest vascular unit present is the follicular unit, which consists of the follicular network, each in the case of the dog and man, with its own artery and vein. In the cat, as already stated, this network is not so rich and distinctive, and follicular arteries and veins are not present. Yet, the large mesh containing the follicle is the homologue of the network and may be regarded as the small- est unit present. This vascular unit corresponds to the histological unit of the individual follicle. The next vascular unit in size is the lobular unit. This is com- posed of (1) the arteries of the fourth order which run over the clumps of follicles having as their direct branches the follicular ar- teries, and (2) the arteries of the third order which pass between the lobules. This vascular unit corresponds to the structural unit of the lobule. 490 Ralph H. Major. The next vascular unit which comes into consideration is what might be termed the lobar unit, and is formed by the arteries of the second order, which surround collections of lobules or lobes and give off arteries of the third order. The corresponding structural unit is not so easily determined as are the lobules, but they may be considered as a collection of lobules, which is marked off from a similar collection of lobules by denser septa. The term lobe as used here is the microscopic lobe and does not refer to the lobe of gross anatomy, the term which is appled to a much larger anatom- ical division of the gland, for in the human the gland is considered as composed of a median and two lateral lobes, and in the dog the term lobe is applied to what is really gross-anatomically considered a right and a left thyroid gland. As arteries of the first order, for the sake of simplicity, have been grouped together, the branches of the thyroid arteries which ramify over the surface of the gland, supply definite regions and penetrate it giving off branches of the second order. Finally as the largest unit present, the prime unit, we have the thyroid gland itself, supplied by the thyroid arteries, superior and inferior, which differentiate it from the standpoint of vascularization from surrounding structures such as the thymus, submaxillary gland, ete. An exhaustive study of the lymphatics of the thyroid does not he in the scope of these notes. Many observers, among them Baber (5) and more recently Renaut and Petijean (16), have described them and most of the text-books refer to their presence, their extreme richness and their general distribution. Yet a short consideration of their relation to the blood vessels may be of interest. The lymph spaces surround each follicle just outside the capillary network, filling in as it does the interstices left between the follicles. The relation between the capillaries and the lymphatics also indicates how the individual follicles are surrounded by lymphatic spaces. These spaces connect with larger trunks which definitely run in between the different lobules. These trunks in turn run into larger ones be- tween the lobes which follow fairly closely the course of the blood vessels and becoming larger finally unite to form a network of Vaseular System of the Thyroid Gland. 491 lymphatics beneath the capsule. From there they empty into lymph- atics draining the gland, which usually follow the blood vessels out, one trunk passing upwards towards the submaxillary gland and the other passing to the lower cervical region, as described by Baber and figured by Bartels. In one injection the lower trunk of the left gland was seen to pass directly into the left subclavian vein. These observations were made upon the dog alone. In connection with the enormous blood supply of the thyroid gland, an anatomical study shows conditions favorable to a rapid and consequently a rich blood supply. The numerous arteries, the fact that each artery does not terminate until it reaches the follicle, the ultimate unit of the gland, would, theoretically considered, aid rather than retard a rapid circulation through the gland. It is rather interesting to note that the thyroid with its follicular artery and vein, resembles to a certain extent the kidney with its glomerulus and vasa afferentes and efferentes, and that the kidney, according to Tschuewsky, shows a blood supply exceeded among the organs he examined, only by the thyroid gland. BIBLIOGRAPHY. 1. TSCHUEWSKY, ‘Der Blutstrom in der Schilddrtise.”’ Pfitiger’s Archiv, Bd. 97, July 6, 1903. 2. Cyon, quoted by Howell ‘‘Text-book of Physiology,” p. 775, 1906. 3. HALSTED AND Evans. “The Parathyroid Glandules, ete.’ Annals of Sur- gery, Vol. XLVI, No. 4, Oct., 1907. 4, BaBer. “Contributions to the Minute Anatomy of the Thyroid Gland of the Dog.” Philos. Trans. Roy. Soc., Lond., 1876. 5. Baper. ‘Researches on the Minute Structure of the Thyroid Gland.” Philos. Trans. Roy. Soc., Lond., 1881. 6. LANGENDORFF, “Beitrige zur Kenntnis der Schilddrtise.” Archiv fiir Anat. und Physiologie, Phys. Abt., 1889, Suppl. . WOLFLER. “Bau und Entwicklung der Schilddrtise.” Berlin, 1880. 8. Lustic. “Contribution 4 la connaissance de Vhistogénése de la glande thy- roide.” Archiv. italiennes de biologie, 1891. 9. Bronpr. Berliner klinische Wochenschrift, 19 Noy., 1888. 10. Htrrtr. “Beitriige zur Kenntnis der Sekretionsvorgiinge in der Sehild- driise.” Pfliiger’s Archiv, Bd. 56, 1894. 11. ANDERSSON. “Zur Kenntnis d. Morphologie der Schilddriise.”’ Archiv fiir Anat. u. Phys., Anat. Abt., 1894. 492 Ralph H. Major. 12. 138. 14. 15. 19. Koun. ‘Studien tiber die Schilddriise.” Archiv ftir mikroscopische Anat- omie 1895 und 1896. Srretr. ‘Ueber die Form der Schilddriisenfollikel des Menschen.” Archiv fiir mikroscopische Anatomie, Bd. 48, 1896. Fuint. ‘Note on the Structure of the Thyroid Gland.” Johns Hopkins Hospital Bulletin, 1908. Coco. ‘Contributo all’ istologia della glandula tiroide.”’ Anatomischer Anzeiger, Feb. 28, 1901. . REGAUD ET PETITJEAN. “Recherches comparatives sur l’origine des vais- seaux lymphatiques dans la glande thyroide,” ete. Bibliographie Anato- mique, Tome XIV, 1905. . KouHrrAuscH. ‘“Beitriige zur Kenntnis der Schilddriise.” Miiller’s Archiv, 1853. . STRECKEISEN. “Beitrige zur Morphologie der Schilddrtise.’ Virchow’s Archiv, Bd. 103. LANDSTROM. Dissertation, Stockholm, 1907. THE STRUCTURE OF SMOOTH MUSCLE IN THE REST- ING AND IN THE CONTRACTED CONDITION. BY CAROLINE McGILL, Instructor in Anatomy, University of Missouri. WiTH 7 TEXT-FIGURES AND 7 PLATES. PAGE PS ALE OOMELTOME Sais ese, a acsvehe oo mage, Seah a OMe ROPE COG de naar Ate ea ea 494 a. Matter atures Review so. :r. stn, s cnt on nen ek ee re ee eee 495 i On'the structure of resting smooth musele.: 3... uae - nes 495 2. On the structure of contracted smooth muscle.............. 496 Ti.” )- Material: and: Methods? 0 010 spa ot evsntecrsieteue 4 © a olaneev ete olan 500 ae Miaberial asec te of yro'. ye aim te aN eke race ne Re OI eee 500 ye Xa bOnwOL resting MUSCles aan tte retaken ack ae 501 a.) dixatlon OrecOni Laced: MmUSCle: iri aia are eee 3 502 4. Methods of fixation, embedding and staining .............. 505 LV.) Structure of Resting Smooth Muscle” .2-7 Sas... citar eee Re) 7 i Generali structure of ‘smooth muscle) tissue = 5-2 ee oe a 507 a. Smooth muscle with complete syncytial structure..... 509 b. Smooth muscle with end to end anastomoses of fibers. 511 c. Smooth muscle with apparently isolated fibers........ 513 Pepe yoy il oN Gr Ves ane a ee EIEN OS Good UO D OUD UGS bo BGitcow or 514 livia: och oualorabs Googe pocoscvsce sco OE EOD OO DUOTD Ese 515 Ome Conrsermyonlbrillasi 7. si) sacar ves oh ehsehoe ae eee 515 OP MING @ite Beneete te he cicten e tance, Sites BYR TORS ORO RE Sa eh ee era eee 516 Ge lboyrereSiitiene ul Goa enhye! WISE 9G 60 Gano cceubdnsoocosatootEG.: 517 VY. Gross Changes in the Muscle Coats During Contraction........ SS i eimetheroieestives tract, (Ay aise cen. somisei hae Leet ence & 518 Die SEM CREDO S ue saps 2%... fn acute 4 cae ae eae ee ne Ao Oceus Ded 520 Vi Horms or Contraction in Smooth Mnscle eee yen cette lers eon 520 i Berigtalnie Com iracioniv sei term ets retire eke ekepere: eee ate ce 520 ay) Thestormyorsihes contracwowm waves cri. etl 521 O30 Lherform of thevcontraction nodes en. ees eae eae 524 ee Rotel: Contractions cers ws olois ois erat or craye aielor oles ePe Seeley 527 THH AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. 494. Caroline McGill. VII. The Behavior of Myofibrille During Contraction ............. Spe FEO) 1. The continuity of the myofibrille through the contraction 101010 (eS ROT CIR OCS ata On. G Garo Or oe eeu 6 529 2. The increase in the thickness of the myofibrillz during con- RA CUION ele alee 545 I. Inrropuction. In a preliminary paper, McGill, (3) 1907, were described briefly the finer structural changes which take place during the contraction ot the smooth muscle in the intestine of Necturus and some of the raammals. In the present paper a more detailed account of contrac- tion, not only for the intestinal muscle, but for smooth muscle in gen- eral, ig given. The structure of resting smooth muscle is described, in the main, from the standpoint of its syncytial arrangement. The general histology of resting smooth muscle is discussed at length by Heidenhain, 1900, and will be considered only briefly in this paper. The structure of contracted smooth muscle was studied in both living and fixed material. The general form of the contraction area is here described, together with a more detailed account of the behavior of the myofibrille and of the nuclei. The myofibrillee have long been considered the contractile elements of smooth muscle. There has, however, been little proof that they are such. It has therefore seemed worth while to study them carefully in an effort:to obtain some evi- dence along this line. Furthermore, the form of the contracted smooth muscle nucleus has recently been much disputed. In this paper an attempt is made to throw some light on this subject also. This paper is confined to a consideration of vertebrate muscle. Enough work has been done on invertebrate muscle to show that it is Structure of Smooth Muscle. 495 very favorable material, especially for experimental work. The description of contraction in invertebrate smooth muscle along with a review of the literature on the subject is reserved for a later paper. This work was done in the Anatomical Laboratory of the Uni- versity of Missouri under the direction of Prof. C. M. Jackson, to whom I am indebted for many valuable suggestions. Il. Lirerature Review. 1. On the structure of resting smooth muscle. For the literature on the general structure of smooth muscle, the reader is referred to the excellent review given by M. Heidenhain, 1900. Since 1900 several papers have appeared. Henneberg, 1901, Heiderich, 1902, Forster, 1904, Schlater, 1905, and Soli, 1906, describe smooth muscle as though made up of entirely separate and distinct cells. The following are the main references to the syncytial structure of smooth muscle: Drasch, 1895, in the skin of the salamander, found around the periphery of the large poison glands a complete layer of smooth muscle fibers, united by wide protoplasmic anastomoses into a syncytium. Schaper, 1902, in Urodela, found the smooth muscle fibers in the mesentery anastomosing more or less and mentions the probability that the tissue forms a syncytium. Rohde, 1905, in a comparative study of the smooth muscle in both vertebrates and invertebrates, described protoplasmic connections not only between smooth muscle celJs, but also between muscle cells on the one hand, and epithelium, ganglion cells and connective tissue cells on the other. Rohde makes the assertion that these intercellular bridges represent the remains of an embryonic syncytium. He gives, however, neither figures nor descriptions of embryonic material to support this view. McGill, (1), 1907, in the digestive tract of the pig found that smooth muscle arises, in common with the interstitial connective tis- sue, from the mesenchymal syncytium surrounding the endodermal tube. Some of the mesenchyme cells in the area of muscle formation dc not elongate, but persist as the connective tissue cells, connected by 496 Caroline MeGuill. protoplasmic strands. with the muscle protoplasm. Often in a single protoplasmic mass connective tissue fibers and myofibrille differen- tiate side by side. In later development most of the connective tissue fibrils are crowded out of the muscle bundles by the rapidly developing myofibrillee, though some may, even in the adult, retain their primitive relation. As the myofibrille form, they tend to run in longitudinal bundles, but always show marked side anastomoses with neighbor- ing bundles. Throughout development, and in many instances in the adult, this syncytium persists. In the adult the syncytial arrangement was demonstrated in the muscle of the digestive tract of Necturus, dog, cat, and pig. 2. On the structure of contracted smooth muscle. The literature on the structure of contracted smooth muscle is reviewed in detail by Heiderich, 1902, consequently only the more important references dealing directly with the subject will be given in this paper. a The earlier writers on the structure of contracted smooth muscle held that the tissue shortens by a zigzag folding of the fibers. Among the investigators who supported this view may be mentioned Prevost and Dumas, 1823, Remak, 1843, Leydig, 1849, Mazonne, 1851, Meissner, 1858, Schwalbe, 1868, Arnold, 1871, Rouget, 1881, and Marshall, 1887. Shultz, 1895, considered that all zigzag folding and wrinkling of smooth muscle fibers indicate contraction in the absence of tension. In opposition to the zigzag theory of contraction, Kéllker, 1849, working on the smooth muscle of the ureter, prostate and small intes- tine of several forms, described knotlike thickenings in the contracted fibers which he considered contraction areas. Heidenhain, 1861, found two types of contraction in smooth muscle: (1) A peristaltic, where the fibers show knotlike contraction areas with uncontracted areas between; (2) a general or total where there is a shortening and thickening of the entire fiber. The latter type only he held as normal. Margo, 1862, found, as did Meissner: a cross-striation of the con- tracted smooth muscle fiber, not due, however, to wrinkles, but to rows of small granules analogous to the sarcous elements of striated muscle. Strueture of Smooth Muscle. 497 Van Gehuchten, 1889, deseribed nuclei in the smooth muscle of the frog, which show a distinct spiral form. Regarding their significance he says nothing. Ranvier, 1889, by injecting lemon juice into the gall-bladder of the guinea-pig and afterwards fixing in osmic acid obtained distinct cross-striations in the smooth muscle fibers which he interpreted as contraction phenomena. Apathy, 1890, in support of Englemann’s inotagmen theory for the contraction of striated muscle, proposed a similar theory for smooth muscle. According to his theory during contraction the chemical composition of the sarcoplasm immediately surrounding the nucleus is so altered that the interfibrillar sarcoplasm draws water from it. In consequence the myofibrille embedded in this sarcoplasm swell, becoming at the same time shorter. This in turn causes the shorten- ing and thickening of the entire fiber. Relaxation is the reverse phenomenon. Klecki, 1891, found that the intercellular bridges are more numer- ous and longer in contracted than in uncontracted muscle. At times in uncontracted muscle they are entirely absent. He gives no further description of contraction. However, in his figures he shows some fibers staining lightly, others darkly, in cross section. The dark fibers are the smaller. Which be considers the contracted fibers he does not state. Roulé, 1890-1891, studying contraction in smooth muscle, came to the conclusion that it is brought about by the myofibrille, which decrease in length and increase in thickness. In his article on intercellular bridges in smooth muscle, Barfurth, 1891, did not discuss contractility. However, his figures taken from cross-sections of the fibers show dark and light fibers, both of equal thickness. Werner, 1894, found that smooth muscle fibers, left some time in a warm oven, show distinct cross-striations and strongly wrinkled con- tour. Drasch, 1895, in the smooth muscle of the poison-glands of Sal- amander maculosa, in contracted fibers, described cross-striations. He thought they were due to a wrinkling of a membrana propria and 498 Caroline MeGill. not of the fiber itself. However, in portions of the gland having only scattered muscle cells, he found distinct knot-lke enlargements of the fibers, which he attributed to contraction. He also observed that in the contracted fibers, the fibrillar structure is lost, and that such fibers show marked affinity for eosin. Schaffer, 1899, studying fresh preparations of the intestinal muscle of the horse, found knot-like swellings on some of the fibers which he considered pathological. In these enlargements no myofi- brillze were oberved. Schaffer, in normal contraction, described the entire fiber becoming shorter and thicker, and at the same time losing its fibrillated structure. Heidenhain, 1900, discusses the behavior of the smooth muscle nucleus. During contraction the nuclei become distinctly shorter and thicker. In certain degrees of contraction this is the only change. In very strong contraction, however, the nuclei are variously folded and twisted. If the resting nucleus is very slender, as in the muscle in the blood vessels, it may, when contracted, wrap up into a spiral. Less elongated nuclei, in firm contraction, show various sorts of fold- ing and wrinkling. Heidenhain considers both the “Grenzfibrillen” and the “Binnenfibrillen” contractile elements, though he gives no direct evidence that they are such. Henneberg, 1901, studied the smooth muscle of the carotid artery of the ox, both in the resting and in the qontracted condition. When the carotid is cut, the proximal end contracts very firmly, the distal end relaxes completely. Small pieces from any part of the artery, if cut out and warmed, slowly contract, if cooled, relax. By quickly removing very small pieces and throwing them into hot water, Henne- berg was able to fix the tissue rapidly enough to prevent contraction of the expanded muscle. In material so preserved he found two types of fibers; (a) Long, slender, band-like fibers with no myofibrille, with protoplasm staining deep red in eosin and black in iron-hema- toxylin. In such fibers the nuclei are long and rod-like; (b) spindle- shaped fibers, thicker in cross-section than type (a), with slightly staining, fibrillated protoplasm and short, thick nuclei. Between these two types he found all transitions. The deeply staining, homo- geneous fibers, he considered the resting; the lightly staining, fibril- Structure of Smooth Muscle. 499 lated fibers, the contracted fibers. In firmly contracted muscle, fibers of type (b) predominate ; in resting muscle, fibers of type (a). Heiderich, 1902, studied contraction of smooth muscle in the in- testine, urino-genital tract and blood-vessels of a number of mammals. He obtained contracted muscle by heating the tissue and by injecting apomorphine. Heiderich’s conclusions are almost diametrically opposed to those of Henneberg. He found in contracted muscle, as did Henneberg, two types of fibers: (a) fibers with homogeneous protoplasm, showing marked affinity for eosin, (b) fibers with fibril- lated protoplasm, showing little affinity for eosin. The former he considered the contracted, the latter the expanded fibers. In fixed and stained material after certain fixatives the homogeneous fibers have smaller diameter than do the fibrillated fibers. In fresh material this is not the case. Consequently Heiderich concludes that certain reagents must cause more shrinkage in the contracted than in the relaxed fibers. In the homogeneous fibers the nuclei are shorter and thicker than in the fibrillated fibers. The elastic fibers in the neigh- borhood of the homogeneous fibers run a wavy course, elsewhere they are straight. . These facts lend further evidence that the homogeneous are the contracted and the fibrillated the resting fibers. Heiderich describes quite fully the two types of contraction mentioned earlier by Heidenhain, 1861. These types are (a) the peristaltic, occurring in the smooth muscle of digestive and urino-genital tracts and (b) the total, occurring in the blood-vessels. In type (a) the contraction passes over the fiber in a series of waves so that several thickened, hemogeneous nodes may appear in each fiber. Between the nodes the myofibrille are distinctly seen. In type (b) there is general shorten- ing and thickening of the entire fiber. In explaining the contraction of smooth muscle Heiderich supports the inotagmen theory of Apathy. He found nothing in the structure of the myofibrille, how- ever, to indicate their relation to contraction. Schaper, 1902, in the muscle fibers in the mesentery of Urodela found, at times, spindle-shaped enlargements of the fibers. The myo- fibrillee in the mesenteric muscle are frequently segmented, made up of alternate dark and light bands. Schaper mentions the possibility that the segmentation may be due to contraction set up by the fixative. 500 Caroline McGill. Benda, 1902, described in smooth muscle two types of myofibrille, coarse and fine, corresponding somewhat closely to the “Grenzfibril- len” and “Binnenfibrillen” of Heidenhain. He considered only the fine myofibrille contractile elements. The coarse myofibrille he believed to be elastic or supportive structures. Forster, 1904, studied contraction in amphibian and mammalian muscle. According to his description, the muscle cell as a whole con- tracts in such a way that it is rolled into a spiral. The nucleus follows this spiral winding. From the amount of spiral winding of the nucleus the amount of contraction of the muscle fiber may be deter- mined, Schlater, 1905, like Forster, described the smooth muscle nucleus as undergoing a spiral winding during contraction. THe is of the opin- icn, however, that the nucleus is entirely passive, but believes that, at the same time, there may be an active decrease in length and increase in diameter of the nucleus. Soli, 1906, 1907, in the smooth muscle of the stomach of birds, found practically the same conditions described by Heiderich for the peristaltic form of contraction. There is this difference, that in all of his material he found the contraction nodes of greater diameter than the uncontracted internodal segments. Verzar, 1907, in the smooth muscle cells of the amnion of the chick found with ordinary hematoxylin-eosin stains, very distinct bound- aries to the cells. When stained with iron-hematoxylin, no distinct boundaries were present, and the myofibrille, both coarse and fine, apparently run from one cell to another. McGill, (3) 1907, in a preliminary paper described briefly the fibrillar and nuclear changes which are given in more detail in this present paper. III. Marerrat aAnp Mernops. 1. Material used. Smooth muscle from the following vertebrate sources was the mate- rial used in this investigation: among Amphibia, from the alimentary canal and urinary bladder of the frog and of Necturus; among birds, from the alimentary canal of the chicken; among mammals, from Structure of Smooth Musele. 501 several regions of the alimentary canal and urino-genital tract and from the blood-vessels of dog, cat, ox, pig and man. ~ 2. Fixation of resting muscle. To obtain smooth muscle in the resting or relaxed condition several methods were employed. When the tissue was found completely extended, as it frequently is in the various organs of the alimentary canal, pieces could be fixed in that condition. To do this, very small pieces of the resting muscle were clipped out with sharp scissors and thrown quickly into the fixative. The fixative should be one that acts rapidly, such as hot water, hot sublimate solution or hot Zenker’s fluid. In using this method the work should be done rapidly or the mere mechanical stimulus of handling the muscle will often cause it to contract. When the fixative used penetrates rapidly, as does hot Zenker’s fluid, larger pieces may be fixed without undergoing appreciable contraction. In the large intestine of mammals, where, after stimulation, the contraction proceeds very slowly, whole seg- ments of expanded muscle were removed and fixed in Zenker’s fluid without producing contraction. In the small intestine of mammals the smooth muscle is so irritable that even the slightest stimuli set up very rapid contractions. Hence it was found impossible to fix the tissue rapidly enough to prevent some contraction. Even here, how- ever, while small areas were contracted, there were many areas of entirely resting muscle, unless the stimulus was strong and contin- uous. In the alimentary canal of birds the tissue is so easily stimu- lated that the pieces could seldom be fixed without showing some con- traction. Here the only means of obtaining entirely relaxed muscle was by employing narcotics. In Amphibia, especially during the win- ter, the irritability of the smooth muscle of the alimentary canal is so low that whole segments were removed and placed in any of the ordin- ary fixatives without producing contraction. Tn the large arteries, upon section, the portion next to the heart con- tracts firmly, that away from the heart, relaxes completely. Small pieces, taken from the distal end of the cut carotid a few minutes after the artery had been severed, were fixed in hot water or hot Zenker’s fluid. They show completely extended muscle. This method was successfully used by Henneberg, 1901, and by Heiderich, 1902. 502 Caroline McGill. By the use of narcotics, smooth muscle can be made to relax entirely, and will remain in this condition long enough to be studied fresh or to be fixed and sectioned. Small pieces placed for a short time in 0.5 per cent to 4 per cent cocain solution in normal saline became completely extended. Very weak solutions of atropine sul- phate, 0.00005 per cent to 0.001 per cent, had a similar effect. The nareotized tissue was either studied fresh by teasing or by frozen sections, or was fixed and sectioned. Lastly, living smooth muscle was studied directly under the micro- scope. Small pieces of the muscularis from the intestine of Necturus, chicken and dog were removed from the recently killed animal and were mounted in either lymph or Ringer’s solution on a warm slide, and were examined immediately. At first, due to the stimulation of cutting and mounting, the tissue is firmly contracted. Upon stand- ing a few minutes, however, the fibers competely relax. Though the finer details of structure cannot be made out from living material, yet the general form of the fiber and even of the nucleus and myo- fibrillee can be easily studied. 3. Fixation of contracted muscle. Contraction of smooth muscle tissue was studied from material prepared in a number of ways. In the muscle of the alimentary canal of mammals, especially in the small intestine, the tissue is usu- ally in peristalsis so that pieces taken at random at some point contain contracted fibers. Then the mechanical stimulation of removing the tissue sets up more marked contraction waves. This, together with the stimulating action of the fixative makes it often impossible, without the use of narcotics, to prepare sections of the smooth muscle of the small intestine, which do not show at least a part of the fibers contracted. However, to get very firmly contracted smooth muscle from this region the tissue was specially stimulated, either mechanic- ally or electrically. In preparing the material the animal was anesthetized and the abdominal cavity quickly opened; then the stimulus was supplied to one portion of the intestine and continued until an area was com- pletely contracted throughout the whole circumference. The entire segment was then removed and fixed in hot Zenker’s fluid. Ocecasion- Structure of Smooth Muscle. 503 ally small pieces were fixed, but they did not give as good results as did the larger pieces. This was due to the fact that in small isolated pieces of muscle the tension is removed and consequently the fibers in contracting take an abnormal wavy or zigzag course or become otherwise distorted. In every instance when placed in Zenker’s solution the larger pieces were perfectly fixed throughout. When relaxed muscle is placed into fixatives which act slowly the chemical stimulus itself is often sufficient to produce very firm con- traction waves. On opening the abdominal cavity so that the alimentary canal is exposed to the air for even a short time, strong peristalses appear. Tn the small intestine the waves usually involve only a small part of the entire circumference of the tube. In the large intestine, however, the tube may contract firmly throughout the whole diameter and is easily fixed in this condition. On removing the intestine from a recently killed animal and placing it in warm Ringer’s solution (387° C.) contractions, similar to those caused by exposure to air, though stronger, are set up. The large intestine of dog and cat was found to be one of the most favorable places to obtain both contracted and resting smooth muscle. When this portion of the alimentary canal is stimulated, at the point where the stimulus is applied, it contracts very firmly throughout the entire circumference. Areas between the point of stimulation are just as markedly relaxed. Furthermore, it requires considerable stimulus to start peristalsis and the tissue contracts very slowly. Hence whole segments were removed and fixed with portions remain- ing entirely relaxed, others firmly contracted. This was consequently excellent material for a comparative study of resting and contracted muscle. When the large intestine of the dog is filled with feces, the muscle normally contracts in rings around the feces. At other points it is relaxed. From such areas with no special stimulus entirely con- tracted and entirely relaxed muscle was obtained. In the chicken the muscle of the alimentary canal is highly irritable so that the shghtest stimulus induces contraction. The fixative alone is sufficient to produce strong peristaltic waves. Contracted muscle from the stomach, small intestine and eeecum was studied. 504 Caroline McGill. In Amphibia, as previously mentioned, the muscle of the digestive tract is very inactive. In order to obtain contracted muscle from these forms the tissue was stimulated for some time with a rather strong electric current. It was often possible on the side cf the intestine where the stimulus was applied, to obtain a very firmly con- tracted area while the remainder of the muscle in the circumference of the tube was entirely relaxed. For contracted arterial muscle, the proximal end of the previously severed carotid was taken and rapidly fixed in hot Zenker’s fluid. Only mammalian blood-vessels were studied. In the urino-genital tract no special effort was made to get con- tracted muscle, but usually in fixed material contraction waves were seen. The morphological changes in smooth muscle produced by a number of drugs which cause contraction were studied. The tissue was placed in a physiological solution of the drug until contracted, and then examined, either living or after fixation in sections. The drugs used were pilocarpine, apomorphine and strong solutions of atropine (0.01 per cent,- or stronger). The drug effects were tested on intestinal muscle of Necturus, cat and dog. Contraction in smooth muscle was also examined directly in fresh muscle. Small pieces of muscle from the intestine of Necturus, chicken, dog and cat, and from the carotid artery of ox were clipped out with sharp scissors and mounted in normal saline, Ringer’s solution, or blood serum, upon a warm slide. The slide was fitted with foil electrodes for electrical stimulation. Thin longitudinal strands of the muscle were placed between the electrodes, then a cover-glass supported by thin strips of filter paper was adjusted. The tissue was examined directly under the microscope. Low power lenses were usually adequate, but for finer structures a water-immer- sion (Zeiss H lens) was used. After mounting, the tissue was allowed to stand for a few minutes, or until it relaxed completely, then a weak electric current was passed through. As the stimulus was applied the muscle. fibers contracted. The course of the con- traction waves and some of the finer structural changes accompanying them could then be observed. Structure of Smooth Muscle. 505 In the peristaltic type of contraction there passes simultaneously over neighboring fibers a very characteristic sort of contraction wave. It has seemed important to determine whether the simultaneous oceurrence of contraction in contiguous fibers is due to nerve control or to some morphological connection between the fibers themselves. To determine this it was necessary to exclude stimulation of the muscle fibers through the nerves. This: was done by im- mersing the tissue for some time in very weak solutions of atropine sulphate. After such treatment, the tissue could still be mechanically stimulated to contraction. Atropine is supposed to cause relaxation of smooth muscle cells by paralyzing the motor nerve endings in the fibers. This point has not been entirely proved, but most recent work seems to confirm the statement. Unger, 1907, reviews the literature on this subject and gives additional data. In testing the atropine effect the methods of Unger were used. The development of the power of contraction in smooth muscle was studied in a series of pig and chick embryos, together with a few observations on an eight months human feetus. In most instances the fixative was relied upon to furnish the necessary stimulus. How- ever, some experimental work was done. The musele from the small intestine of pig embryos ranging in length from two centimeters to thirty centimeters was stimulated in solutions of pilocarpine and apomorphine, then fixed and sectioned. Tissue from the muscular stomach and small intestine of the chick embryos from five days to twenty days old, was studied both living and after fixation. The fresh muscle was studied directly under the microscope. The fixed muscle was stimulated mechanically before fixation. 4. Methods of fixation, embedding and staining. The muscle was fixed in several different reagents, including alcohol, aleohol-formalin, saturated aqueous solution of corrosive sublimate, hot water, Gilson’s, Flemming’s and Zenker’s fluids. Zenker’s fluid proved most satisfactory both for general structure as well as for bringing out cytological details. The rapidity with which it penetrates even large pieces of tissue makes it especially valuable in this work. With it, the muscle fibers could be fixed in any stage of contraction desired, the: reagent itself not acting as a stimulus. 506 Caroline McGill. For demonstrating the gross form of the muscle fiber, macerating fiuids were occasionally used. Those giving the best results were 30 per cent ethyl alcohol, 30 per cent methyl alcohol, 20 per cent nitric acid and various strengths of potassium hydroxid solutions. They of course served as poor fixatives of the cell protoplasm as well as macerating solutions. Most of the material was embedded in paraffin and cut in sections from three micra to ten micra thick. To bring out the general form of the smooth muscle fiber the thicker sections were often most useful. Some tissue was embeddéd in celluloid, but only for comparison. Fresh material cut on a freezing microtome immediately after removal from the body gave for some purposes quite favorable results. Fresh material unstained was found to show much of the finer structure of the tissue; however, for the more detailed work stains were indispensable. Fresh muscle stained intra vitam by methylene blue or neutral red gave good results in some respects. Fixed material sectioned and stained was used for most of the work. For all general staining, as well as for the demonstration of contraction waves and myofibrille, Heidenhain’s iron-hematoxylin with a counter-stain of eosin or Congo red was found to give excellent results. With this method, by staining and decolorizing for various lengths of time, very different effects may be produced. Moreover, structures which are not shown by the ordinary method may be demon- strated by repeated immersion in the hematoxylin, followed in each ease by the extraction of the hematoxylin in iron-alum. Iron- hematoxylin was especially valuable for staining areas of contraction and for tracing the myofibrille through such areas. When the hematoxylin is only partially extracted by the iron-alum the contrac- tion waves are stained uniformly black throughout. Further extrac- tion brings out the continuity of the myofibrille through such areas. When the sections are further decolorized the hematoxylin is entirely removed from the contraction nodes before it leaves the nuclei. Such sections counterstained in either eosin or Congo red show the contrac- tion waves intensely red. Delafield’s hematoxylin, Hansen’s hematoxylin, and Flemming’s triple stain were also used as general stains. Structure of Smooth Muscle. 507 For differentiating the connective tissue in which the smooth muscle cells are embedded, Mallory’s anilin-blue connective tissue stain, Van Gieson’s picrofuchsin stain, Weigert’s elastic tissue stain and various iron-heematoxylin mixtures gave good results. Mallory’s stain, at the same time, was found to be a good differential for muscle. With this stain the myofibrille of relaxed muscle stain intensely red. In con- tracted muscle the entire contraction node stains yellow. The nuclei are yellow, all collagenous fibrils blue. Van Gieson’s mixture stains contraction nodes intensely yellow, collagenous fibrils bright red. Van Gieson’s stain at the same time is an excellent nuclear stain. Elastic fibers were brought out best by Weigert’s resorcin stain, though iron-hematoxylin often showed them distinetly. IV. Structure or Resting SMootH MUSCLE. 1. General structure of smooth muscle. No attempt will be made here to go into the details of the structure of resting smooth muscle. In this paper the subject will be considered chiefly from the standpoint of the histogenesis of the tissue, which was not considered by Heidenhain. Hence before discussing the histol- ogy of the adult tissue it has seemed advisable to give a short resumé of the process of development. For a review of the literature on this subject, together with a detailed description of the histogenesis of smooth muscle, the reader is referred to a paper by the author, McGill (1), 1907. In the digestive and respiratory tracts of the pig, smooth muscle arises in common with the interstitial connective tissue, from the mesenchymal syncytium surrounding the endodermal tube. The differentiation of smooth muscle begins in the mid-cesophagus of the 5 mm. pig embryo. 650. The idea that smooth muscle is a syneytium is not new. However, most authors who have described it have found the cells united only by very delicate protoplasmic strands, the so-called intercellular bridges. Among these investigators may be mentioned Leydig, 1849 ; Wagner, 1869; Flemming, 1878; Kultschitzky, 1888; Barfurth, 1891; Heidenhain, 1893; Werner, 1894, and Bohemann, 1895. The intercellular bridges were considered by most of these writers as of secondary origin and not as the result of the persistence of an embryonic syncytium, The improved connective tissue stains of recent years, such as Mallory’s anilin-blue mixture, Van Gieson’s mixture, and Mallory’s phosphotungstic mixture, have shown that these fine anastomoses between the muscle cells are chiefly connective tissue strands, Figs. 20, 23, 27 and 34, and not true protoplasmic bridges. 510 Caroline McGill. The possibility that muscle protoplasm may surround the con- nective tissue strands and thus be continuous from muscle cell to muscle cell must not be forgotten. The sarcoplasm between the myo- fibrille is extremely hard to differentiate. A small amount of it may continue along the collagenous fibers and still not be seen in preparations. The collagenous fibers between the muscle cells are surrounded by protoplasm during development. In a few places this was seen in the adult. Where there is such an arrangement, the collagenous strands with surrounding protoplasm do represent true cell bridges. A number of investigators have described a smooth muscle syn- cvtium for adult muscle, where the fibers are united by wide protoplasmic strands. These references have been abstracted in the literature review. Drasch, 1895, in the skin glands, and Schaper, 1902, in the mesentery of Urodela, Rohde, 1905, in a number of vertebrates, and McGill (1), 1907, in the pig, have all described such syncytia. Among Amphibia rather complete syncytia were found in the urinary bladder and the digestive tract of Necturus and frog. Such ar anastomosis is shown in Fig. 7, drawn from the intestine of Necturus. In the alimentary canal of chicken only occasionally are there marked side anastomoses of the fibers, though end to end union is common. In mammals marked side anastomosis of muscle fibers was observed in some regions in the pig, dog, cat and man. In some of these forms it is very pronounced, in others it is only occasionally met with. In the muscularis muscose of the small intestine of the pig is the most complete syncytium among the mammals studied, Fig. 14. The muscle syneytium of the digestive tract of adult pig is made up of the much elongated muscle nuclei, each surrounded by a granu- lar protoplasmic reticulum, and outside of this a layer of myo- fibrillee embedded in clear protoplasm, Figs. 14, 15. The myo- fibrillee are mainly arranged in spindle-shaped bundles, but from these bundles many of them pass in broad end and side anastomoses, into neighboring groups. Structure of Smooth Muscle. 511 The granular protoplasmic reticulum surrounding the nucleus is quite variable in amount. In the two layers of the muscularis of the small intestine it is restricted to a small mass at the pole of each nucleus, Fig. 16. In the muscularis mucose of the small intestine there is, surrounding the nucleus, a large spindle-shaped mass of reticular protoplasm, connected by wide anastomoses with that sur- rounding neighboring muscle nuclei, Fig. 14 a; occasionally it is con- nected by similar anastomoses with the stellate-shaped connective ee ee ee Ee Se a ___ Sars Text Kie. 2. End to end anastomosis of smooth muscle cells. Intestine of pig. > 650. tissue cells, Fig. 14 b. Fig. 17 shows a cross section through such muscle tissue, in which the wide area of reticular protoplasm around each nucleus is clearly indicated. In the muscularis of the intestine of the pig the main mass of muscle consists of the myofibrille. They are arranged in a heavy luyer around the nucleus, Fig. 16, and in heavy bundles connected with adjacent nuclei, Fig. 15 a. In the muscularis mucose there Text Hig. 3: An isolated smooth muscle cell from the intestine of pig. >< 650. is often only a thin layer of myofibrille around the margin of the reticular protoplasm, Fig. 13, 14, 17. Between these two types there exist all transitions. Fig. 7 shows a portion of a smooth muscle syncytium from the intestine of Necturus. There is direct continuity between the reticular protoplasm surrounding the nuclei as well as between the myofibrille. b. Smooth muscle with end to end anastomoses of fibers. 512 Caroline MeGill. As smooth muscle develops, in many places the wide side anas- tomoses of the cells become less apparent, but the end to end union still persists, text Fig. 2, Figs. 10, 11, 12. The loss of side anas- tomoses is probably due to a rapid elongation of the central part of the fiber between the anastomoses, thus pushing the anastomoses to the ends of the fibers. It may possibly in places be due to an actual dis- appearance of the anastomoses themselves. Much of the muscle of = Fic. 4. A completely relaxed cell. Fic. 5. A cell contracted at one end. Fic. 6. A cell contracted at each end. ee Fic. 7. A completely contracted cell. Text Wes. 4-7. Muscle cells in various stages of contraction. Carotid of ox. X 650. the pig’s intestine is in this condition. It occurs commonly in the muscle of the digestive and urino-genital tracts of dog, cat and man, and was occasionally observed in the carotid of the ox. In this type there may be continuity from cell to cell of the reticular protoplasm, Fig. 13. Most often, however, only the myofibrillee form the anas- tomoses, Fig. 11. The continuity of myofibrille from fiber to fiber was long ago described by Rouget, 1863, and has since been mentioned by several writers. Structure of Smooth Muscle. 513 e. Smooth muscle with apparently isolated fibers. by far the majority of writers on the structure of smooth muscle have described it as everywhere made up of entirely free and inde- pendent spindle-shaped elements, the so-called muscle fibers, muscle cells, or muscle spindles of Kolliker, 1849. Such an element is diagrammatically represented in text Figs. 3, 4. Recent advocates of this idea are Heidenhain, 1900; Heiderich, 1902; Forster, 1904; Schlater, 1905, and Soh, 1906. In the material studied in this investigation it was found exceed- ingly difficult to demonstrate isolated smooth muscle fibers. In the walls of the carotid of the ox some of the fibers appear to be free, Fig. 33. Here the fibers are long, slender structures, thicker in the vicinity of the nucleus than elsewhere. Because of their length and the curved course they take in surrounding the lumen (most of them are circular fibers) it is possible in only a few places to get them cut in exactly longitudinal section. It should be remembered that spindles appearing independent in such sections may appear so because the section is cut shghtly obliquely. In these sections anas- tomosis of fibers end to end would not show even if present. Further- more, a tangential section of a spindle-shaped fiber may look like an isolated fiber. Consequently it is not surprising that end to end union is hard to demonstrate in arterial muscle fibers. In many instances end anastomoses do occur, so even here there is at least a partial syncytium. It is probable, too, that the syneytium is much more nearly complete than sections would lead one to conelude. The development of arterial muscle was not studied in detail, but in both chick and pig the tissue arises as a complete syncytium. In all other muscle studied the syncytial structure in the adult is more apparent than in arterial muscle. Most of the work of earlier investigators on the general form of the smooth muscle cell was done on macerated material. In maceration the reagent usually destroys at least the peripheral myofibrille, so that fine anastomoses even if present are destroyed, leaving only the central spindle-shaped portion of the cell intact. In this way are undoubtedly obtained many of the spindle-shaped cells figured in the text-books. 514 Caroline MeGuill. The relaxed muscle fiber of the carotid of the ox is a long, spindle- shaped structures, much thicker in the center than toward the poles. Ai the ends it may branch and as before mentioned does anastomose with neighboring fibers. It is made up of a much elongated central nucleus, Fig. 82; outside of the nucleus is a small area of reticular protoplasm, and outside of this, forming, in most cases, the bulk of the cell, is a thick layer of longitudinally running myofibrille, Figs. 10, 34 a. If any sarcoplasm exists between the myofibrille it is not demonstrable by the ordinary stains, such as hematoxylin, eosin, ete. Just beneath the elastica interna many of the smooth muscle cells show a large amount of reticular protoplasm around the nucleus. In such fibers the myofibrillze are restricted to a thin peripheral layer, Fig. 9. These fibers closely resemble those already described in the muscularis mucose of the small intestine of the pig, Fig. 13. 2. Myofibrillee. In adult smooth muscle, just as in development, two types of myo- fibrille occur: (1) very fine fibrillee, evidently corresponding to the -elementary fibrils of Apathy, 1890 and 1891, and to the “Binnen- fibrillen” of Heidenhain, 1898 and 1900; and (2) coarse fibrille which seem to correspond to the primitive fibrils of Apathy. The latter, in some particulars, also resemble the ‘“Grenzfibrillen” of Hei- denhain, in others more closely the myofibrille of Benda, 1902. There are often in a single muscle cell fibrillee showing all gradations in size from the coarse to the fine myofibrille. In completely extended muscle the individual myofibrille are, throughout their entire length, of comparatively even caliber. All of the myofibrille stain intensely with protoplasmic stains and with iron-hematoxylin. During early development in the pig and the chick all of the myo- fibrillee begin as exceedingly coarse structures. These later break up, probably by longitudinal splitting, into finer fibrille. In the older foetus there is undoubtedly some formation de novo of fine fibrillee. In the adult muscle the coarse myofibrille represent either persisting embryonal structures, in which case they are usually entirely homo- geneous, Fig. 13, 15, or they may be formed by the subsequent adhesion or union of the fine myofibrille into bundles. In this last condition the finer fibrille entering into their formation may, at times, be demonstrated. Structure of Smooth Muscle. 515 The fine myofibrillz in some muscle become very numerous in the later stages, and in the adult the muscle fiber may be well filled with them. They are often the only fibrille present. This is usually the case in the intestinal muscle of Necturus, Figs. 6, 23, 24 and of the chicken, Figs. 2, 11. In the muscle of the digestive and urino- genital tracts of mammals at times there are present none but fine -myofibrille, Fig. 15. In cross section the fine myofibrillee appear as fine dots, Figs. 16, 26. In the adult smooth muscle coarse myofibrillee may or may not be present. When present they are occasionally the only myofibrille found, as in portions of the muscularis mucose of the small intestine of the pig, Fig. 17, and in the carotid of the ox, Fig. 4 a. Usually, however, they are associated with fine myofibrille. In this relation they were found in large numbers in the carotid of the ox, Fig. 5, and in the muscularis mucose of the esophagus of the pig. As occasional structures they occur throughout the muscularis of the digestive tract of dog, cat, pig, and man. No coarse myofibrille were found in the smooth muscle of Necturus notwithstanding the fact that in Salamander, a closely allied form, Heidenhain, 1900, found them in large numbers. When coarse myofibrille alone are present, they may be arranged as a peripheral layer, Fig. 17, or they may be scattered throughout the entire diameter of the fiber, Fig. 4 a. When associated with fine myofibrille, they may likewise occupy a peripheral position only, Fig. 4b. They then correspond to the “Grenzfibrillen” described by Heidenhain, 1900. More often the coarse myofibrille are scattered throughout the muscle cell. They then approach in arrange- ment more nearly the coarse myofibrillee described by Benda, 1902. In uncontracted muscle the ecarse myofibrille of the adult are of approximately even caliber throughout the entire length. They stain very intensely in eosin or in iron-hematoxylin. Tron-hematoxylin stains the coarse myofibrille long after it has been extracted from the fine myofibrillz so that usually in an iron-hematoxylin-eosin prepara- tion the coarse myofibrille are stained black, the fine myofibrille red. In the muscle syneytium both types of fibrille run past the limits of one cell, through the anastomoses into neighboring cells, Figs. 13-15. 516 Caroline MeGill. The coarse myofibrillz, especially those lying close to the periphery of the muscle cell, have to be differentiated from the elastic fibers which, in some muscle, lie in the connective tissue immediately sur- rounding the muscle protoplasm, or even embedded in the peripheral protoplasm itself (Fig. 25, McGill (1), 1907). This is especially true in material stained with iron hematoxylin. With this stain both coarse myofibrilla and elastic fibers are intensely black. In longitudinal section is it usually comparatively easy to tell them apart, for the elastic fibers are more wavy than the coarse myofibrille. Moreover, the elastic fibers are always around the periphery, while the coarse myofibrille may be scattered throughout the cell proto- plasm. With Weigert’s elastic tissue stains the elastic fibers can be differentiated from the myofibrille. 3. Nuclei. The nucleus of resting smooth muscle is a much elongated, rod- shaped structure many times as long as wide. In the material studied the nuclei vary in length from twenty micra in chicken intestine to eighty micra in Necturus intestine. Schultz, in a large number of forms studied, found the shortest nuclei in smooth muscle of the dove, thirteen micra, and the longest in the intestine of Pro- teus, seventy-two micra. The proportion of length to width varies extremely. In muscle of a primitive type, as in the syncytia shown in Figs. 7 and 14, the nuclei are very wide in proportion to their length. In arterial muscle the nuclei are extremely long and narrow, Figs. 5, 82. In muscle of the marked syncytial type the nuclei are usually located at the nodal points, the long axis parallel with the long axis of the bundles of myofibrille. In apparently isolated fibers, as in the muscle of the carotid of the ox, the nuclei usually lie close to the center of the fiber. Occasionally an eccentric position is observed. The nucleus may lie so close to one side of the fiber that one surface appears free from the cytoplasm. Figs. 19, 25. An eccentric posi- tion of the nucleus was frequently observed in intestinal muscle of Necturus, chicken, and dog. This position of nucleus was described by Kolliker in amphibian muscle and by Lenhossek in cat muscle. In the chicken the nuclei are often nearer one pole of the fiber than the Structure of Smooth Muscle. 51% other. Typically, however, the nucleus is always surrounded by myofibrille: The nuclear membrane consists of a heavy network of linin studded with fine chromatic granules. It is of smooth and even contour. Inside the nucleus is a delicate linin reticulum, with a slightly ecarser but still finely meshed chromatic reticulum. Both linin and chromatin are composed of fine granules, Figs. 5, 24, 27, 82, 90. From one to several plasmasomes are present. No spiral strand of chromatin, such as is deseribed by Miinch, 1903, was found in rest- ing muscle. Numerous granules are present in the reticular protoplasm imme- diately surrounding the nucleus, but none of them could be identified as centrosomes, such as were described by Lenhossek, 1899. 4. Interstitial connective tissue. The interstitial connective tissue of adult smooth muscle resembles as a rule ordinary areolar tissue. The connective tissue cells in some eases seem to retain their primitive relation to the protoplasmic syneytium, Fig. 14 b. The collagenous fibers may be arranged in a loose reticulum, Figs. 2, 24, 30, or as a denser reticulum, Figs. 15, 16. Where the muscle cells lie very close together the collagenous fibers may be crowded into thin membranes. Such membranes have been described by Watney, 1879, by Heidenhain, 1900, and were figured by the author in a previous paper (1), 1907. The collagenous fibers were stained by Mallory’s anilin-blue con- nective tissue stain and Van Gieson’s stain. With either of these stains they appear extremely fine, united in may places into bundles, Figs. 28, 29, 34. Here and there in the adult they still appear to run through the protoplasm of the connective tissue cells or even in among the myofibrillee. The elastic fibers are rather coarse, homogeneous structures, which vary greatly in thickness, Figs. 8, 12, 15. They frequently branch and anastomose. They lie for the most part close beside the muscle fibers. Rarely some of them are embedded in the muscle protoplasm among the peripheral myofibrille. The intimate relations of colla- genous and elastic fibers and the myofibrille are clearly understood 518 Caroline McGill. when their origin from a common protoplasmic syncytium is remem- bered. V. Tue Gross Cuances In Muscir Coats Durine ContTrRaAcrION. 1. In the digestive tract. The gross changes of the muscle coats of the digestive tract were studied in the small and large intestine of dog and Necturus. In the large intestine of the dog during contraction there passes over the organ from before backward a series of ringlike areas of contraction. These areas include the entire circumference of the intestine. All of the muscle in a cross section of a given segment is contracted about the same amount. The gut may, however, be very firmly contracted at one point and a neighboring segment may be completely relaxed. During contraction both layers of the muscularis thicken simul- taneously. Along with this there is such a decrease in the lumen that the whole diameter of the tract decreases. The two coats contract in equal ratio so that if before contraction the ratio of the thickness of the longitudinal coat to that of the circular coat is as one to two, after contraction this same ratio is maintained. The following data are from measurements taken on sections from closely lying seg- ments of the large intestine of dog: Intestine 1. a, contracted area b, uncontracted area cireular coat, 1.1 mm. circular coat, 0.38 mm. longitudinal coat, 0.6 mm. longitudinal coat, 0.17 mm. OIG Gs AES: Dale Sessa 8 reo Intestine 2. a, contracted area b, uncontracted area circular coat, 0.4 mm. circular coat, 0.2 mm. longitudinal coat, 0.12 mm. longitudinal coat, 0.065 mm. ON 2 3204 =A or. 0:065) 220!2) == 3. Intestine 3. a, contracted area b, uncontracted area circular coat, 0.61 mm. circular coat, 0.34 mm. longitudinal coat, 0.2 mm. longitudinal coat, 0.11 mm. OL OlGile—alererene 0.11 :0.384=1 :8. b. Smooth musele with end to end anastomoses of fibers. Structure of Smooth Muscle. 519 As the large intestine of dog contracts, aside from an increase in thickness of both muscular coats and a decrease in the lumen of the tube, there is an accompanying decrease in the length of the contract- ing’ segment. The decrease in lumen is due to contraction of cir- cular muscle, the decrease in length to the contraction of the longi- tudinal muscle. In the small intestine of Necturus, dog and eat the peristalses are very irregular. The waves are short and follow each other rapidly. Seldom does a wave extend entirely around the circumference of the intestine. Thus in a single cross-section of the small intestine all gradations from completely contracted to relaxed muscle may be found. Here, as in the large intestine of dog, a very constant ratio exists between the thickness of the longitudinal and that of the cir- cular muscle coat. In a preliminary paper (McGill (3), 1907), Fig. 2 is a cross section from a contracted area, Fig. 1 from an uncon- tracted area of the same section of small intestine of Necturus. Because of the incomplete contraction several measurements could be made on a single cross section. The following measurements are taken from cross sections of the small intestine of Necturus: Section 1. a, uncontracted area circular coat, 0.262 mm. longitudinal coat, 0.125 mm. b, partially contracted area circular coat, 0.2 mm. longitudinal coat, 0.1 mm. ec, uncontracted area circular coat, 0.162 mm. longitudinal coat, 0.081 mm. The ratio of thickness of the longitudinal to the circular coat in all three measurements is approximately as 1 to 2. A number of measure- ments were taken from different sections, all with like results. 520 Caroline McGill. As the small intestine of Necturus contracts the lumen decreases in diameter, due to the contraction of the circular muscle. The simultaneous contraction of the longitudinal muscle has, however, little effect upon the length of the intestine. A number of measure- ments were taken upon segments of intestine, both before and after ecntraction. In every instance the length remained nearly constant, though sections showed in the contracted segments distinct contraction waves in the longitudinal fibers. Though the longitudinal fibers were distinctly shortened at the contraction waves, they were unduly stretched between, which possibly accounts for no decrease in length. The work was done on animals which had been kept in captivity for some time and had been fed little, so contraction may not have been entirely normal. 2. In arteries. In arteries during contraction there is a marked thickening of the media with accompanying decrease in the size of the lumen. When the fresh carotid of the ox is severed the proximal end contracts and the distal end relaxes. As the muscle contracts the wall of the vessel thickens and the lumen decreases in diameter, as it relaxes the reverse changes take place. Since there is little longitudinal muscle there is little change in the length of the vessel. VI. Forms or Contraction 1x Smootu MuscuLe. In smooth muscle two main types of contraction have been de- scribed: 1. Peristaltic contraction, where one or more contraction nodes appear in the fiber, with uncontracted areas between; 2. Total contraction where the entire fiber shortens and thickens. In this study, muscle with contraction of type 1 was easily demonstrated. Muscle of type 2 was seldom found, though in blood-vessels this type is in places nearly approached. 1. Peristaltie contraction. Before discussing the changes which take place in smooth muscle during the peristaltic type of contraction, a few of the terms used will be defined. By contraction area is meant the entire mass of muscle, all or a part of the fibers of which have undergone active shortening Structure of Smooth Muscle. 521 and thickening. The term contraction wave will be restricted to the deeply staining so-called homogeneous bands of firmly contracted muscle which pass irregularly across the contraction areas, Figs. 1, 2, 11, 21, 30, 37. Contraction node refers to the deeply staining thickened area in the individual muscle fiber, and internodal segment to the fibrillated, uncontracted or weakly contracted portion of the fiber between the contraction nodes. a. The form of the contraction wave. As the muscle of the digestive tract contracts, contraction waves appear. They are irregular in outline and branch and anastomose, Fig. 1, 30, 37. In Fig. 1 they are shown in a cross section through the contracted large intestine of dog. They are seen in the circular coats of the muscularis as irregular bands extending in places across the entire thickness of the muscle coat. The section of course shows them only in two dimensions. In serial sections their extent im the third dimension was determined. In this direction up and down the intestine they extend as irregular bands of about the same width as shown in the cross-section of the intestine. In the longitudinal coat the waves-are similar in form to those found in the circular coat. In fact, usually when a contraction wave in the circular coat reaches the outer margin, there is opposite it in the longitudinal coat a similar wave, Fig. 1. When the contraction waves are cut in cross section of the muscle they appear as irregular polygonal areas studded thickly with the cut ends of contracted muscle fibers, Fig. 1, 1, m. The contraction waves may extend quite obliquely across the area of contraction, Fig. 1 ¢. In places the contraction waves are of even contour, in other places of very diffuse and irregular outline. Not all contraction waves pass across the entire thickness of the mus- cularis. They may involve only a few fibers or when just beginning to form only a portion of a single fiber. In the large intestine of dog and in the small intestine of Necturus, in tissue firmly contracted the contraction waves are quite broad, involving from one-fourth to one-half of the length of the muscle fiber, Figs. 21, 23, 30. In muscle not so firmly contracted they may be extremely narrow, Figs. 35, 39, 40. There are from one to three of these waves in each contracted fiber. 522 Caroline MeGuill. In the small intestine of chicken, Figs. 2, 11, and of mammals the contraction waves are narrower and closer together. There may be several traversing each fiber. In fact they are often so numerous as to give the fiber a distinctly cross-striated appearance. It was prob- ably this that led many earlier writers to describe a cross-striated involuntary muscle in portions of the digestive tract. Here and there the peristaltic type of contraction does not produce contraction waves. In such muscle there is apparently no relation whatsoever between the contraction nodes of neighboring fibers. This seems to be largely true for the smooth muscle of the urino- genital tract. The conclusive way to study contraction phenomena is in fresh muscle. Small pieces of living muscle from the small intestine of Necturus, from the small and large intestine of dog, and from the muscular stomach of chicken were mounted as already described, in Ringer’s solution or blood serum, over small electrodes on a slide and examined under the microscope. When the tissue was stimulated it contracted and the contraction waves could be observed passing over the muscle fibers, causing distinct enlargements of the fiber as they passed. The contraction is initiated almost simultaneously in neighboring fibers. Thus arise the contraction waves. To determine whether the form of the contraction wave is due to nerve regulation or whether it is the result of some morphological connection between the muscle fibers themselves, portions of the intestine of cat were treated with atropine until the nerve endings in the smooth muscle were completely paralyzed. They were then stimulated mechanically. Marked contraction took place. Sections of such contracted material, as well as the living muscle examined directly showed identically the same form of contraction wave as did sections from the normal material used for control. The methods used are described under material and methods, so need not be de- scribed here. The following data from a single experiment show the effect of atropine: March 14, 1908. A large cat weighing 2700 em. was decapitated, the abdominal eavity quickly opened and the intestines removed by clipping the Structure of Smooth Muscle. 523 mesentery close to the tube. They were placed in a jar containing 2000 e. c. of Ringer’s solution, kept at a temperature of 37° C. 2.15 p. w.—Placed the intestine in Ringer’s solution. 2.45 p. m.—The intestine was contracting rhythmically, so added 4 mg. of atropine. The peristalses stopped almost immediately. 3.45 p. m.—The intestine was still quiet. Added 10 mg. more of atropine. 5.45 p. m.—The intestine was still quiet. Added 10 eg. of atro- pine. Contraction began again in a few minutes, probably due to the overdose. During the progress of the experiment pieces of the intestine were removed, stimulated mechanically until completely contracted, then fixed in Zenker’s fluid. Normal intestine from the same animal was similarly fixed for control. Sections from both the normal and atropinized muscle as well as the living muscle examined directly all showed contraction waves of exactly the same form. The above experiment would lead one to conclude that the contrac- tion waves do not depend upon nerve control. They must be trans- mitted to neighboring fibers through some other connection between the cells. Since the contraction waves are most distinct in muscle with most complete syncytial structure, it is possible that’ they are transmitted through the anastomoses between the fibers. However, in muscle with only end to end anatomoses, the contraction waves are frequently well marked. There is a possibility that the interstitial connective tissue may transmit the waves. The continuity of the contraction waves from circular to longitudinal coats, between which there is often a rather thick layer of connective tissue, would rather seem to support this view. The results of experimentation with atro- pine are not absolutely conclusive, for the paralyzing effect of atro- pine has been questioned. This was discussed under material and methods. The staining reactions of contraction waves have already been described. In Figs. 1, 2, 11, 23, they are shown in material stained in Mallory’s anilin-blue connective tissue stain, in Figs. 21, 37, in material stained in Delafield’s heematoxylin eosin. 524. Caroline MeGill. The behavior of the interstitial connective tissue during contrac- tion is worthy of mention. All the changes noted are purely passive ones, caused by the decrease in length and increase in thickness of the nearby muscle fibers. During contraction the changes are similar to those described by Heiderich, 1902. In uncontracted areas the col- lagenous fibers form a loose reticulum, Figs. 2, 13, 15, 24, ete. ; elastic fibers run straight, Fig. 12. In the contraction waves the collagenous fibers become much condensed, Figs. 20-23, 28-30. This explains why, by earlier workers on intercellular bridges, all of whom considered the collagenous fibers protoplasmic connectives, the bridges were described as being more numerous and larger in contracted than in uncontracted muscle. If the pressure of the contracting fibers in the contraction waves be great enough the collagenous fibers become packed into distinct membranes, Fig. 31. The elastic fibers where contraction waves pass over the muscle become distinctly wavy, due to passive shortening, Fig. 12. Well defined contraction waves do not occur in arterial muscle. Frequently a contracted fiber will be entirely surrounded by uncon- tracted fibers. More often an irregular area, including anywhere from a few fibers to the entire thickness of the muscle coat, is con- tracted, while neighboring muscle is relaxed. In firm contraction practically every fiber in the arterial wall may be contracted. b. The form of the contraction node. In peristaltic contraction the muscle fiber is traversed by one or more thickened areas, the contraction nodes. They represent the portion of the fiber included within the contraction wave, conse- quently they are just as numerous in the fiber as are the contraction waves passing over it. In the large intestine of dog and the small intestine of Necturus, when firmly contracted the single contraction nodes may include as much as one-half of the entire length of the fiber, Figs. 21, 23, 30. When less firmly contracted the contraction nodes are not so wide, Figs. 35, 40. In the smooth muscle of the small intestine of mammals and in the muscular stomach of chicken the contraction nodes are shorter and more numerous. Fig. 11. In the muscle of the cesophagus of the pig they are often so close together as to make the fiber appear distinctly cross-striated. Or Structure of Smooth Muscle. 52 While the internodal seginents of the fibers are lightly staining and distinctly fibrillated, as in Figs. 2, 5, 11, 20-22, 35, 36, the contrac- tion nodes in ordinary material appear homogeneous and are deeply staining. This condition has been described by numerous investi- gators and by them has been variously interpreted. Iolliker, 1849 ; Roulé, 1890, 1891; Schaffer, 1899; Heiderich, 1902, and Soli, 1906, 1907, considered the homogeneous nodes the contracted por- tions of the fiber. NHenneberg, 1901, because he found that these nodes in his fixed and stained preparations were usually of smaller caliber than the internodal segments, described them as uncontracted, the internodal segments as the contracted portions of the fiber. His work was done upon the carotid of the ox. Heiderich demon- strated quite conclusively on the same material that the homogeneous nodes are nodes of contraction. That Henneberg found them smaller in cross section of fixed material than the internodal segments Hei- derich explained as due to their being more subject to shrinkage in some reagents than are the fibrillated internodal segments. He tested a large number of fixatives. With some the internodal seg- ments were larger than the homogeneous areas, with others smaller. In fresh material the homogeneous areas were always of greater ealiber. The fact that the nuclei shorten and thicken at the homo- geneous areas while the elastic fibers run more wavy and the colla- genous fibers are more condensed at these points he took as additional evidence that the deeply staining homogeneous nodes are the contrac- tion nodes. Soli, 1906, 1907, in the stomach muscle of birds found in fixed material that the deeply staining homogeneous areas are invari- ably of greater caliber than are the fibrillated portions of the fiber, so he considers the former the contraction nodes. In most of the material investigated by the author the contraction nodes resemble those described by Soli, 1906. That is, they occur as deeply staining thickened nodes of the fiber, Figs. 2, 11, 12, 20-22, 30, 87. With ordinary stains, such as hematoxylin eosin or in iron- hematoxylin, after the usual differentiations they appear homogene- ous. Muscle of this type was found throughout the digestive tract of chicken and mammals and in the urino-genital tracts of mammals. The most pronounced thickening of the fiber at the contraction node 526 Caroline McGill. was found in the muscle of the digestive tract of chicken, Fig. 2, of dog, Fig. 21, and in that of the urino-genital tract of man. Figs. 3 and 31 show the marked thickening which may take place in the fiber during contraction. In Fig. 3 the contraction nodes are stained black, in Fig. 31 orange. The mortising of the contraction nodes of neighboring fibers together, as is shown in the contraction wave in Fig. 2, is further proof of the large amount of thickening of the fiber which takes place at this point. The occurrence of thickening of the contraction nodes in fixed material is probably due to the fact that the fixative used (Zenker’s fluid) did not produce the shrinkage of the contraction nodes which was obtained by Henneberg and Heiderich in their work. When the contraction nodes are seen in cross section many of them are oval or elongated, indicating that the fiber at this point is dis- tinctly flattened. The flattening is probably due to unequal pressure of neighboring nodes. It is seen most frequently where there is little connective tissue separating the nodes. In the circular muscle of both intestine and blood-vessels especially nearest the lumen the contracted fibers show this flattening. In cross section of these fibers the long diameter extends from nearest the lumen outward. Since, when the muscle of these tubes is contracted the lumen is nar- rowed, this direction of flattening is just what one would expect if it be due to pressure. The uncontracted fibers, internodal segments and even the contraction nodes, when they are separated by much loose connective tissue, are typically round in cross section. In the musele from the contracted areas of the small intestine of Necturus the deeply staining contraction nodes are of just about the same thickness as the internodal segments. In this the muscle approaches that described by Henneberg. But even here when the nodes and internodal segments of the contracted areas, Fig. 23, are compared with the fibers in completely relaxed muscle, Fig. 24, it will be seen clearly that both are of much greater caliber than are the relaxed fibers. Furthermore, from the shortening of the nuclei and the condensation of the connective tissue, it will be seen that the fibrillated internodal segments have also undergone some contraction. So even in Necturus muscle it seems that at all times the contracted fibers are of greater caliber than are the relaxed fibers. Structure of Smooth Muscle. 527 The contraction nodes can be demonstrated in fresh material. Small pieces of muscle from the stomach of chicken, mounted in physiological solutions and examined under the microscope show them even when the muscle is not stimulated electrically. With some electrical stimulation they may be demonstrated in almost any living smooth muscle. ‘They appear under the microscope as marked homo- geneous thickenings of the fiber. Though all previous investigators of contractility in smooth muscle have described the contraction nodes as homogeneous, it is possible in material stained in iron-hematoxylin and properly differentiated, to trace the myofibrillee through them and to show the continuity with the myofibrille in the internodal segments, Figs. 35, 40. This will be discussed more fully later when the behavior of the myofibrille during contraction is described. As proof that the homogeneous, deeply staining nodes are the con- tracted portions of the smooth muscle fiber may be mentioned the following: In hving material they show as distinctly thickened areas. In fixed material when the fixative used is one which does not produce unequal shrinkage of the nodes and the internodal segments, the homogeneous nodes show (as in fresh material) as thickenings of the fiber. In both fresh and fixed material the nuclei are drawn closer together and are shorter and thicker in the contraction nodes than in the internodal segments. Figs. 20-22, 87. Around the con- traction nodes the collagenous connective tissue is condensed. Figs. 20-23, 30. In the neighborhood of the nodes the elastic fibers run a very wavy course, while through the internodal segments they are comparatively straight, Fig. 12. And, as will be described more fully later, the myofibrillee, when they can be traced through the con- traction nodes, run straight and are thicker there than they are in uncontracted internodal segments. A spiral winding of smooth muscle during contraction, such as is described by Forster, was not observed. 2. Total contraction. Total contraction, as it has been described, is characterized by the decrease in length and increase in thickness of the entire muscle fiber, text Figs. 4, 7. This form of contraction was observed by Kolliker, 528 Caroline McGill. Heidenhain, Henneberg and Heiderich in the muscle of blood- vessels. It is not the only type of contraction described for blood- vessels. Heiderich, 1902, in the umbilical vessels found peristaltic contraction. In the material studied in this investigation typical total contrac- tion was not observed. In the smooth muscle of the blood-vessels of dog, cat, pig, ox and man and in the sphincter pylori of Necturus and dog, the contraction approaches the total type, Figs. 33, 34. The contraction often involves all but the tips of the fibers, Fig. 33. Here, as in determining the form of arterial muscle, the interpreta- iton of sections is difficult. A fiber contracted in the middle, as in Fig. 33 and Fig. 34 a, if cut through slightly obliquely or tangenti- ally, would appear in section as a short, completely contracted fiber, Fig. 34 b. In rare instances the contraction may involve the entire fiber. More frequently the contraction passes over the fiber in broad nodes involving from one-half to two-thirds of the fiber, Fig. 34, text Fig. 5. Such fibers are well differentiated in material stained in Mal- lory’s anilin-blue connective tissue stain. The contraction node stains orange, the uncontracted portion of the fiber red. It seems highly probable that arterial contraction is only a modified contraction where the contraction nodes are much longer and involve more of the fiber than is usual in the peristaltic contraction in the muscle of the ali- mentary canal or the urino-genital tract. In some places in the carotid of the ox two or even more contraction nodes are present in a single muscle fiber, thus producing a typical partial contraction, Fig. 5, text Fig. 6. The contraction nodes in the muscle fibers of blood-vessels are not as deeply staining as are those in the muscle of the digestive and urino-genital tracts. Consequently the fibrillee in material stained in either iron-hematoxylin or Mallory’s anilin- blue connective tissue stain can in almost every instance be traced through the contraction nodes, cn in Figs. 33, 34. Fig. 34 shows them in cross section of a contraction node. ‘The eut ends of the fibrille show as fine dots. It is only when overstained that the contraction nodes appear homogeneous as described by Henneberg and Heiderich. Structure of Smooth Muscle. 529 VII. Tur Benavior oF THE Myorrprittar Durtna Contraction. Although the myofibrille have long been considered the contrac- tile elements of smooth muscle, previous investigators have not demonstrated that they are such. The myofibrille can be easily seen, even in fresh material, in uncontracted muscle and in the inter- nodal segments of contracted muscle, Fig. 2, 5-36. The contraction nodes, even by recent workers on smooth muscle, have been described as entirely homogeneous, Henneberg, 1901; MHeiderich, 1902; Soli, 1906. The author in a preliminary paper, McGill (38), 1907, showed that in material properly differentiated the myofibrille may be traced through the so-called homogeneous nodes, and that they apparently thicken as they pass through. In material fixed and stained in the ordinary manner these nodes do appear perfectly homogeneous, Figs. 2, 3, 5, 11, 20, 22, 31. However, in muscle fixed in Zenker’s fluid, then over-stained in iron-hematoxylin and the hematoxylin carefully extracted, it is pos- sible in many instances in the contraction nodes to show a distinct fibrillation, cn in Figs. 6, 18, 33, 34, 35, 36, 39, 40. When exam- ined under high magnification, the individual fibrille may some- times be traced continuously through one contraction node and inter- nodal segment into the next contraction node, Fig. 40. The myofibrille when they can be traced through the contraction nodes run just as straight a course as in the uncontracted muscle or in the internodal segments of contracted muscle, Figs. 6, 33, 34, 39, 40. If they were not contractile elements one would expect them to be folded and wavy as are the elastic fibers when the muscle fiber shortens. When uncontracted muscle fibers are caught between contracted fibers the myofibrille, as well as the whole uncontracted fiber, may be thrown into zigzag waves due to passive shortening. Likewise when small pieces of muscle are removed and allowed to contract in the absence of tension, the fiber and consequently the myofibrille even in contracted muscle may take a wavy course. But in muscle contracted with normal amount of tension, then fixed and sectioned, the myofibrille in both the contracted and uncontracted fibers run comparatively straight. The zigzag form of contraction so frequently 530 Caroline McGill. observed and described by earlier workers was as a rule undoubt- edly brought about by removal of the tension under which the fibers normally contract. They studied only small isolated bits of tissue, so their results are easily explained. As the myofibrille enter the contraction nodes they often appear to thicken distinctly, Figs. 5, 6, 35, 40. The amount of thicken- ing depends upon the amount of contraction. The nodes labeled a in Figs. 39, 40, are just beginning to contract, so there is only a slight increase in diameter of the myofibrille. Nodes labeled 6 in Figs. 35, 40, are more firmly contracted and show more marked enlargement. In muscle where the contraction nodes come very close together, as in pig’s cesophagus when the tissue is just beginning to contract, the myofibrille may appear segmented, as shown in Fig. 26. Even in wide contraction nodes, as in those of arteries, the contracting myofibrille may thicken unequally, giving a segmented appear- ance, Fig. 18, from the carotid of ox. Where this condition is marked it may give an appearance closely simulating the myofi- brillee of striated muscle. The segmentation of the myofibrille of the smooth muscle in the mesentery of Urodela, described by Schaper, 1902, was probably due to mild contraction. However, it should be remembered that iron-hematoxylin and orange G, the only stains by which the myofibrille of contracted muscle could be demonstrated, are largely physical stains. Instead of there being a true enlargement of the myofibrille themselves at the point of contraction, the physical condition of the inter-fibrillar substance immediately surrounding the myofibrille may be so altered as to make it stain as intensely as the myofibrille, and thus pro- duce an apparent though not a real thickening of the fibrille. Were the physical condition of all the inter-fibrillar substance so altered, the stained material would show the whole contraction node homo- geneous. On the other hand, the homogeneous appearance of con- traction nodes may be due to the crowding together of enlarged myofibrille. If closely packed, in material deeply stained, the myofibrille would not be demonstrated. Structure of Smooth Muscle. 531 VIII. Tuer Benavior or tHE Nucuer Durine ContTRAcTION. 1. The form of the contracted nucleus. Two ideas have been advanced as to the form of the contracted nucleus. The first is that during contraction there is an active shortening and thickening of the nucleus, so that it changes from a rod-shaped to an oval-shaped structure. This is maintained by Henneberg, Heiderich and Soli, among recent workers. The other is that during contraction the nucleus is passively or, perhaps actively folded or twisted into a spiral. This type was described by Forster and Schlater. In all the smooth muscle studied, during contraction the nuclei are drawn closer together in the contraction waves than in the uncontracted areas. They undergo distinct decrease in length and increase in thickness, Figs. 22, 37. This can be observed as well in fresh as in fixed material. In fact when living smooth muscle is stimulated to contraction under the microscope the contraction of the nucleus can be observed. The nuclei (with the possible exception of the extremely long nuclei in arteries) do not undergo folding or spiral twisting during contraction. However, in normal contraction, when an uncontracted fiber is caught between fibers that have contracted, both it and its nucleus may be passively twisted or folded. Frequently in the walls of contracted arteries spiral nuclei were observed, Figs. 81-84, from the carotid of ox. But Figs. 60, 61, 78-80, show conclusively that shortening and thickening of the nuclei do occur. It is probable that when both nucleus and fiber contract at equal rates there occurs only the type of contraction shown in Figs. 78-80. If, however, as undoubtedly may happen in muscle with extremely long nuclei, the fiber contracts more rapidly than does the nucleus, various forms of folding or twisting of the nucleus will result. Figs. 81, 82, show nuclei, which are shorter and thicker as well as spirally wound. Of course, this might be due to passive shortening of a partly contracted nucleus. Many of the spiral nuclei belong to fibers passively contracted. However, they do occur in the contraction nodes of actively contracted fibers. 532 Caroline McGill. Figs. 27-29 and 85-89 show two. series of nuclei from the smooth muscle of the intestine of Necturus, which illustrate well the de- erease in length and increase in thickness which occurs during con- traction. The following measurements taken from nuclei of both contracted and uncontracted smooth muscle of Necturus, serve as further illustration of the point in question: Length of Nucleus. Greatest Width of Nucleus. Wore wees 82 micra 5 micra ING@is 2 en eee 72 micra 8 micra TGR ee PENN cot 62 micra 9 micra IN Gideon es oee 55 micra 9 micra INO ue ea cizear ee 43 micra 10 micra ING OS AAs tO 35 micra 15 micra IN Ge tiene sate 30 micra 17 micra ING Seton te Leas 29 micra 16 micra SUCH eae re 28 micra 18 micra Figs. 42-53 is a similar series from the smooth muscle of the large intestine of dog, Figs. 55-58 from a small artery from the mesentery of pig, Figs. 65-67 from the bladder of cat, Figs. 68-72 from the muscular stomach of chicken, and Figs. 77-80 from the carotid artery of ox. During contraction the nucleus changes from rod-shaped to oval- shaped or elliptical. The nuclear membrane which in the resting nucleus is of very even contour, in the contracted nucleus is often distinctly serrated at the ends, Figs. 28, 29, 88, 89. It often has the appearance of being indented by the contracting fibrille. Whether this is actually the case is uncertain. The contraction node passes along the smooth muscle fiber, caus- ing a distinct enlargement as it goes. As it approaches a nucleus, the nucleus begins to thicken at the end nearest the node, Figs. 20, 21, 28, 50, 61, 86. When the whole nucleus is included in the con- traction node, it assumes its completely contracted, oval form, Figs. - 19, 21, 23, 29, 89. Occasionally each end of a nucleus may be caught in a contraction node while the middle lies in an internodal Structure of Smooth Muscle. 533 segment. Then the ends of the nucleus contract, while the center remains unchanged. This frequently occurs in the intestinal muscle of Necturus, where the nuclei are extremely long, Fig. 28. 2. The behavior of the chromatin during nuclear contraction. In the resting smooth muscle nucleus the chromatin is arranged as a very delicate central reticulum and as a thin layer just beneath the nuclear membrane, Figs. 22, 24, 27, 42, 77, 85. During con- traction the chromatin is massed at the two ends of the nucleus. There is left a space relatively free from chromatin at the middle of the nucleus, Figs. 28, 29, 51, 89. As the chromatin masses at the two ends of the nucleus there is a streaming and rearrangement of the meshes. At the same time it becomes much more deeply staining. The massing of the chromatin at the ends of the contracted nuclei is most marked in the intestinal muscle of Necturus and dog, Figs. 28, 29, 51, 52. Necturus material is very favorable for study be- cause the nuclei are so extremely large. As the nucleus begins to enlarge at the point of contraction the chromatin reticulum breaks up into very fine threads, Fig. 86. These pass, as if by distinct streaming, toward the poles of the nucleus where they arrange themselves in loops or festoons, Figs. 28, 87, 88. As the process continues the fine strands of chromatin fuse to form exceedingly coarse threads. These may remain in a loose festoon at the end of the nucleus or else break up there into a coarse reticulum, Figs. 29, 59, 89. At the same time some of the chro- matin collects in a heavy layer just beneath the nuclear membrane. The strands of chromatin in the fully contracted nucleus are much coarser than are those in the resting nucleus, Figs. 27, 28, 29. Figs. 93-95, 96-99, show similar chromatic changes in the nuclei of Necturus where the smooth muscle had been stimulated to contrac- tion by pilocarpine. The chromatic changes in the nuclei of smooth muscle of dog intestine are quite similar to those observed in Necturus, Figs. 42-63. However, in resting nuclei the chromatin strands tend to run as fine longitudinal fibrils, Fig. 42. These are more pronounced in partially contracted nuclei, Figs. 43-46. As the nucleus contracts 534 Caroline McGill. these fibrils become coarser, Figs. 47-49. Instead of running straight, as they do in resting nuclei, they may run a wavy course or even twist up into distinct spirals, Figs. 48-49. Finally these chromatin strands collect at the ends of the nuclei and fuse there to form a coarse reticulum, Fig. 51. The chromatin spirals are never as distinctly marked as were those described by Miinch, 1903. In the stomach of the chicken the nuclei contract in about the same manner as described for Necturus, Figs. 68-72. In the nuclei of the smooth muscle fibers of arteries there are fewer chromatic changes than were observed in other muscle, Figs. 54-64, 77-80. The dark bands shown in Figs. 81-84 are due to folding of the nuclei and not to condensation of the chromatin. The whole behavior of the chromatin of the nuclei during con- traction indicates that the contraction of the smooth muscle nucleus is a very active process. It is highly improbable that the changes deseribed could be brought about passively by the contraction of the extra-nuclear portion of the muscle fiber, 3. The effect of contraction on the volume of the nucleus. An attempt was made to determine by actual measurement whether there is change in the volume of smooth muscle nuclei dur- ing contraction. Not enough data have been obtained to determine the point definitely. From the results at hand it seems that there is no change in the volume during contraction. 4. The effect of fatigue on the nucleus. Gilman, 1903, showed that when striated muscle is completely fatigued the nuclei are shrunken, crenated, more lghtly staining and less granular than in muscle not so fatigued. Similar experi- ments were made by the author on smooth muscle. Strips from the muscularis of the intestine of Necturus were suspended in a moist chamber, arranged for electrical stimulation, and were stimulated until no further contractions could be obtained. Forty contractions was the maximum number obtained from one piece. When the strip was completely fatigued, it was fixed and sectioned. The sections were studied, along with like sections from control muscle, for comparison. In none of the material was there any indication of shrinkage or crenation of the nuclei. In every instance the Structure of Smooth Muscle. 535 nuclei in the fatigued and in the control muscle showed precisely similar, structure. At the same time there was no apparent change in the myofibrille, the exhausted muscle appearing in no wise dif- ferent from resting muscle in structure. 5. The effect of drugs on nuclear contraction. The effect of a number of drugs on the contraction of smooth muscle nuclei was observed. Among those employed were cocaine, pilocarpine, apomorphine, adrenalin and atropine. Atropine and cocaine are in certain doses muscle narcotics. In muscle placed in these solutions until the tissue is completely relaxed, the nuclei show the structure of typical resting nuclei. Figs. 90-92 are nuclei from intestinal muscle of Necturus, relaxed by placing in a 1 per cent cocaine solution. Pilocarpime was the usual stimulant em- ployed. Figs. 93-95, 96-99, are from intestinal muscle of Necturus contracted by placing in pilocarpine solution. The changes brought about by contraction with this drug are precisely similar to those caused by the other stimuli used. TX. Cuyemicat CHances In SmootH Muscrit Durine CONTRACTION. The deep staining of the entire contraction node as compared with the hght staining of the internodal segments would seem to indicate that during contraction there is more change in the muscle fiber than can be attributed te increase in the thickness of myo- fibrille alone. The difference in staining reaction between the contracted and uncontracted muscle is so striking as to indicate a marked chemical change. With iron-hematoxylin the contraction nodes stain readily and intensely black. Figs. 1, 2, 3, 5, 11, 23. Of course since iron-hematoxylin is largely a physical stain, this may be due to the condensation of the fiber and swelling of the myo- fibrille at the contraction node. But the contrast seems entirely too sharp to be due to this alone. With Delafield’s hematoxylin and a counterstain of eosin the contraction nodes stain much more intensely with eosin than do the internodal segments. Figs. 20-22. The picric acid in Van Gieson’s mixture gives a like effect, Fig. 28. Mallory’s anilin blue connective-tissue stain is a valuable dif- 536 Caroline McGill. ferential. With it the myofibrille of the uncontracted portions of the fiber stain red in the fuchsin, the contraction nodes bright yellow with orange G. Figs. 30-34. This stain cannot be entirely relied upon as a chemical test, since orange G. is also known to be a physi- eal stain. However, the exceedingly sharp differentiation of the contraction nodes with all the above stains indicates marked chem- ical changes in the smooth muscle fiber during contraction. An attempt was made by using neutral red and phenolphthalein in physiological solution as indicators, to determine whether there is a change in the alkalinity of smooth muscle during contraction. If there be it should show at the contraction nodes. Small pieces of the fresh tissue were mounted on a slide in solutions of the indi- eator. When stimulated the tissue contracted, but there was no apparent change in the color of the indicator. It should be remem- bered that none of the indicators, neutral red, rosolic acid, ete., in use at present are delicate enough for microscopic tests unless the reactions be very marked. Schultz, 1907, in studying this same question in smooth muscle en masse determined that there was no decrease in the alkalinity during contraction, such as has been pointed out for striated muscle. X. DEVELOPMENT OF CONTRACTILITY. The development of contractility in smooth muscle was studied in the digestive tract in a series of chick embryos ranging in age from three days to twenty-one days of incubation. First a series of sections of embryos at different ages was made to determine when smooth muscle arises. Smooth muscle begins to form between the third and fourth day by an elongation of the cells in the mesenchy- inal syneytium surrounding the epithelial tube. By the fifth day the myofibrille are rapidly. forming as thick varicose structures. They run for long distances through the cytoplasm without regard for cell boundaries. By the seventh day the muscle nuclei are well elongated. The tissue is still a distinct syneytium and remains as such throughout development. The structure of the smooth mus- cle in the chick after hatching was not studied, so later development remains to be treated. Structure of Smooth Muscle. 537 Next was determined experimentally when contraction begins. The interesting question to decide here is whether or not contrac- tion is dependent upon the myofibrille. If it be, one would expect it not to appear until after the myofibrille are formed. The intes- tine and muscular stomach of the chick embryo were removed from the living embryo, kept at a temperature of 37° C., and stimulated mechanically, electrically or by heating. The tissue was stimulated while in focus under the low power of the microscope. The first contraction of the intestine observed occurred on the seventh day. It was merely a very slow contraction arising after marked stimu- lation. By the twelfth day, when the temperature is raised to 43° C., the intestine contracts rhythmically. By mounting small living pieces on a warm slide and examining under the high power of the microscope it was hoped that the finer details of contraction could be made out. Until the late embryo there are so many yolk granules present that this was not accomplished. Sections from the stimulated intestine were fixed and examined microscopically. No contraction changes were observed in the fiber until the twelfth day. Sections from contracted muscle at this time show many of the nuclei slightly shorter and broader than in uncon- tracted muscle. Figs. 73-76 show the degree of shortening observed in the contracted intestine of a fourteen-day chick embryo. On the twenty-first day, aside from shortening of the nuclei, the muscle of the contracted intestine shows in many fibers irregular, areas staining slightly deeper with eosin than does the rest of the fiber. In these areas the nuclei are shorter than elsewhere. They probably represent developing contraction nodes. At this time there are no nodes which stain differently with iron-hematexylin or Mallory’s anilin-blue connective-tissue stain. Everywhere the myofibrille can be traced uninterruptedly through the muscle syncytium. The author hopes soon to be able to trace the later development of con- traction in the chick after hatching. Soli, 1907, traced the development of smooth muscle in 1 the stom- ach of the chick. He obtained the first elongation of mesenchyme on the seventh day of incubation. He did not observe fibrille until the seventeenth day. Contraction nodes he found appearing on the ninth day after hatching. 538 Caroline McGill. In a section through the large intestine of an eight-month human foetus the author observed rather distinct contraction nodes. At that age the contraction waves are quite irregular in outline. SuMMARY. A. Structure of resting smooth muscle. 1. Smooth muscle in chick, pig and man arises as a syncytium and the syncytial structure persists in most instances in the adult. 2. Adult smooth muscle may show one of two and possibly of three types of structure. In type 1 there is a very distinct syncytial arrangement. The fibers are joined by both side and end anasto- moses. In type 2 the muscle fibers show few side anastomoses, but end to end union still persists. There is possibly a third type of muscle with no anastomoses between the fibers, each forming an individual cell. 3. Muscle of type 1 was found in portions of the digestive and urino-genital tracts of adult Necturus, chick, pig, cat, dog and man and of the arterial muscle of pig, ox and man. The tissue consists of the much elongated muscle nuclei, each surrounded by a granular protoplasmic reticulum and outside of this a layer of myofibrille embedded in clear protoplasm. Both granular protoplasm and myo- fibrillee may be continuous from cell to cell. Where there is much granular protoplasm there are few myofibrille and vice versa. 4. In muscle of type 2 the loss of side anastomoses is probably due to rapid elongation of the central part of the fiber during his- togenesis, crowding the anastomoses toward the ends. This type was found here and there in all muscle studied. The myofibrille pass continuously from cell to cell through the broad end anastomoses. Where there is much granular protoplasm it may also help to form the anastomoses. 5. Muscle of type 3, though it is the type usually described, was found hard to demonstrate. Frequently, especially in arterial mus- cle, what appear to be isolated muscle cells are seen. They are long, spindle-shaped elements, each with a central nucleus and surround- ing this the sarcoplasm filled with myofibrille. The cell body is Structure of Smooth Muscle. 539 thickest in the neighborhood of the nucleus and tapers toward the poles. When such isolated fibers appear in section, absence of end anastomoses may be due to the fact that the section is cut slightly obliquely. In macerated material the anastomoses are usually destroyed, and there results the spindle-shaped smooth muscle cell deseribed in the text-books. 6. In adult smooth muscle, just as in development, two types of myofibrille occur; very fine fibrillee corresponding to the elementary fibrille of Apathy and to the “Binnenfibrillen’” of Heidenhain ; coarse fibrillee similar to the primitive fibrille of Apathy which in some respects resemble the ‘“Grenzfibrillen” of Heidenhain, in others more nearly the coarse myofibrille of Benda. Some muscle fibers have only fine myofibrille, others only coarse myofibrille, while still others have both types. When coarse myofibrille are present they may be arranged as a peripheral layer similar to the “Grenzfibril- len” of Heidenhain or they may be scattered throughout the sarco- plasm, as are the coarse myofibrille of Benda. Each myofibrilla throughout its length is fairly uniform in caliber. The myofibrille run continuously from cell to cell through the anastomoses. 7. The nucleus of resting smooth muscle is a much elongated spindle-shaped structure ranging in length in the material studied from twenty micra in the digestive tract of the chicken to eighty micra in Necturus. An eccentric position of the nucleus was fre- quently observed. The nuclear membrane is of even contour. The chromatin is arranged in fine granular reticulum supported by a fine linin network. From one to five plasmasomes are present. 8. The connective tissue of resting smooth muscle appears like ordinary areolar tissue, in the meshes of which are embedded the muscle fibers. The branched connective-tissue cells anastomose with each other and occasionally with the muscle cells. The collagenous fibers are arranged as a loose reticulum, as a heavy reticulum or as distinct membranes. The elastic fibers form a loose network. The fibers which run parallel with the muscle fibers are compara- tively straight. B. The structure of contracted muscle. 1. During the contraction of the digestive tract both layers of 540 Caroline McGill. the muscularis contract simultaneously. As they contract they thicken and in constant ratio. Aside from the thickening of the muscularis the lumen as well as the whole diameter of the tube de- creases, due to the shortening of the circular fibers, and the length otf the segment decreases, due to the shortening of the longitudinal fibers. In the carotid of ox during contraction there is thickening of the coats accompanied by a decrease in diameter, both of the lumen and the whole tube. There is little change in length, for there are few longitudinal muscle fibers. 2. Two types of contraction have been described: peristaltic, where a series of wave-like thickenings cross the fiber; total, where the entire fiber shortens and thickens. In the material studied all the muscle seems to belong to the first type, although in arterial muscle there is in places a near approach to total contraction. 3. In peristaltic contraction in the digestive tract contraction Waves pass over the muscularis. These are irregular anastomosing areas of contracted tissue between which there are areas of resting muscle. The contraction waves vary much, both in length and width as well as in number. In length they may cross only a few muscle fibers or they may include the entire muscularis. In width they may be only narrow bands or they may be so wide as to include most of the length of a muscle fiber. Where narrow and close together they give the tissue a distinctly striated appearance. In the contraction waves the muscle fibers are shorter, thicker and more deeply stain- ing than in the uncontracted areas; the nuclei are crowded closely together, the connective tissue is much condensed. From the mate- rial studied there is no evidence that the propagation of contraction waves is due to nerve control. They are probably conducted from fiber to fiber by some protoplasmic connection between the cells themselves. In syncytial muscle the protoplasmic anastomoses probably serve this purpose. In arterial and urino-genital muscle there are no distinct contraction waves. Here there are merely scattered contracted fibers or groups of fibers among uncontracted tissue. 4. The portion of the muscle fiber in the contraction wave is the contraction node, that in the uncontracted area is the internodal Structure of Smooth Muscle. 541 segment. The contraction nodes, both in living and in fixed material, show as conspicuous thickenings of the fiber. In arterial muscle they are long, so that a single one may include most of the length of the musele fiber. In the muscle of the digestive tract they are usually narrower and a number may be present in each fiber. The nodes stain more distinetly with eosin and iron-heematoxylin than do the internodal segments. With Mallory’s anilin-blue connective- tissue stain the nodes are colored orange, the internodal segments red. With ordinary differentiation the nodes appear homogeneous, the internodal segments are distinctly fibrillated. Although the con- traction nodes of smooth muscle have always heretofore been de- scribed as homogeneous, when the material is carefully stained in ivon-hematoxylin the myofibrille may be traced through the nodes and be shown to be continuous with the myofibrille of the inter- nodal segments. As the myofibrille enter the nodes they show a distinct inerease in caliber. Where the contraction nodes are nar- row and close together the myofibrille appear segmented. That the contraction nodes represent contracted portions of the muscle fiber is shown by the following: In both fresh and fixed material the smooth muscle fibers are thicker at the contraction node than else- where. The myofibrille run straight through the nodes and as mentioned above apparently thicker as they pass. In the nodes the nuclei are shorter and thicker than in the internodal segments. In the region of the contraction node the collagenous fibers are condensed and the elastic fibers take a wavy course. 5. During contraction the nuclei of smooth musele undergo an active shortening and thickening. This change in shape ean be seen in living muscle. Typically, with the possible exception of some arterial nuclei, and the nuclei of passively shortened muscle fibers, there is no folding or spiral winding of muscle nuclei during econ- traction. 6. In general, as the nucleus contracts there is a rearrangement of the chromatin. The fine strands collect into coarser fibers, which arrange themselves in loops or festoons at the two ends of the nucleus. During the process there is an apparent streaming of the ehromatin toward the poles. 542 Caroline MeGill. 7. There appears to be no change in the volume of smooth muscle nuclei during contraction. 8. Fatigue was shown to cause no apparent change in the structure of the nuclei. 9. Nuclei in muscle contracted by pilocarpine, adrenalin, atropine, ete., have the same structure as nuclei contracted by electrical or mechanical stimuli. 10. During contraction more changes take place in smooth muscle than can be attributed to morphological causes, such as thickening of the myofibrille, ete. At the contraction nodes the staining reac- tions would indicate that there is a marked chemical reaction taking place also. 11. The development of contraction was studied m chick and pig embryos. The muscle cells become contractile soon after the myo- fibrillee appear. Thus in the chick, where the myofibrillz arise on the fifth day, distinct contraction was observed on the seventh day. This, together with the fact already mentioned that the myofibrillee become shorter and thicker during contraction, weuld seem to indi- eate that the myofibrille are the contractile elements. Contraction waves do not appear until comparatively late in development. LITERATURE LIST. (Papers not accessible in the original marked *.) 1. ApatTHy, 1890. Nach welcher Richtung hin soll die Nervenlehre refor- miert werden? Biol. Centralbl., Bd. 9. 2. ARNOLD, 1871. Strickers Handbuch der Gewebelehre. 3. BARFURTH, 1891. Ueber Zellbriicken glatter Muskelfasern. Arch. f. mikr. Anat., Bd. 38. 4. Benpa, 1902. Ueber den feineren Bau der glatten Muskelfasern des Menschen. Anat. Anz., Bd. 21. BoHEMANN, 1895. Intercellularbriicken und Saftrviiume der glatten Mus- kulatur. Anat. Anz., Bd. 10. OO 6. BBuyNE pE, 1895. Berichtigung zu Hl. Bohemanns vorliiufiger Mit- teilung tiber Intercellularbriicken und Saftriiume der glatten Mus- kulatur. Anat. Anz., Bd. 10. =I DrascH, 1895. Der Bau der Giftdriisen des gefleckten Salamanders. Arch. f. Anat. u. Physiol., Anat. Abth., 1895. 8. FLEMMING, 1878. Ueber Formen und Bedeutung der organischee Mus- kelzellen. Zeit. f. Wiss. Zool... Bd. 30. Structure of Smooth Muscle. 543 Forster, 1904. Die Kontraltion der glatten Muskelzellen und der Herz- muskelzellen. Anat. Anz., 3d. 25. GILMAN, 1903. The Effect of Fatigue on the Nuclei of Voluntary Muscle Cells. Amer. Jour. of Anat., Vol. 2. HemENHAIN, 1861. Zur Frage in Form der kontraktilen Faserzellen wiihrend ihrer Thiitigkeit. Studien d. Physiol. Instituts zu Breslau. Heft 1. HEIDENHAIN, 1900. Struktur der kontraktilen Materie. Ergebn. d. Anat. u. Entwick., Bd. 10. Ilerericn, 19602. Glatte Muskelfasern im ruhenden und tiitigen Zustande. Anat. Hefte, Bd. 19. HENNEBERG, 1901. Ruhende und thiitige Muskelzellen in der Arterien- wand. Anat. Hefte, Bd. 15. Hertwic, 1898. Zelle und Gewebe, Bd. 2. Keck, 1891. Experimentelle Untersuchungen tiber die Zellbrticken in dem Darme der Raubtiere. Diss. med. Dorpat, 1891. KOLLIKER, 1849. Beitrige zur Kenntnis der glatten Muskeln. Zeit. f. Wiss. Zool., Bd. 1. IXULTSCHITZKY, 1888. Ueber die Art der Verbindung der glatten Mus- kelfasern untereinander. Biol. Centralblatt, Bd. 7. LautH, 1834. Recherches sur Vorganisation intime de nos tissus. L’In- stitut, No. 70. LENHOSSER, 1899. Das Mikrocentrum der glatten Muskelzellen. Anat. Anz., Vol. 16: LeEypiG, 1857. Histologie. Marao, 1862. Neue Untersuchungen tiber die Entwickelung, den Bau, etc., der Muskelfasern. Wiener Denkschr., Bd. 20. MARSHALL, 1887. Observation on the distribution of striped and unstriped muscle in the animal kingdom. Quart. Jour. Micr. Science, Vol. 37. MAZONNE, 1851. Untersuchungen tiber die Gewebselemente glatter Muskeln and tiber die Existenz dieser Muskeln in der menschlichen Milz. Wiener Sitzungsberichte, Math. Natkl., 1851. McGinty (1), 1907. The Histogenesis of Smooth Muscle in the Alimentary Canal and Respiratory Tract of the Pig. Internat. Monatschr. fiir Anat. u. Physiol., Bd. 24. McGiLut (2), 1907. The Syncytial Structure of Smooth Muscle. Anat. Record, 1907. McGiiyt (3), 1907. The Structure of Smooth Muscle of the Intestine in the Contracted Condition (preliminary paper). Anat. Anz., Bd. 30. MEISSNER, 1878. Ueber das Verhalten der muskel6sen Faserzellen im kontraktierten Zustande. Zeit. f. rationelle Medizin, Bd. 2. 544 Caroline MeGill. 29. Mtwnwen, 1903. Ueber Nuclein-spiralen im Kern der glatten Muskelzellen. Arch. f mikr. Anat., Bd. 60. *30. Prevost and Dumas. 1823. Sur les phénoménes qui accompagnent la contraction de la fibre musculaire. Magendie, Jour. de Physiol., T. 3. 31. RANviER, 1889. Les muscles de la vie animale a contraction brusque et contraction lente chez le liévres. Comptes rendus Soc. de biol. de RaAGISse 10s 33. REMAK, 1861. Ueber den Bau und die Zusammensetzung der Muskel- fasern. Wiener Sitzungsber., Bd. 44. 34. RonpeE, 1905. Untersuchungen tiber den Bau der Zelle. Zeit. f. Wiss. Zool., Bd. 78. 35. RouGeT, 1881. Phénomeénes microscopiques de la contraction muscu- laire et striation transversale des fibres lisses. Gaz. Med. de Paris, 1881. 36. RouLb, 1890, 1891. Etude sur la structure et le développement du tissu musculaire. Diss. med., Paris, 1890, 1891. 37. ScHAFFER, 1899. Zur WKenntnis der glatten Muskulatur, inbesondere ihrer Verbindung. Zeit. f. Wiss. Zool., Bd. 66. 38. ScHAPER, 1892. Ueber kontraktile Fibrillen in den glatten Muskelfasern des Mesenteriums der Urodelen. Anat. Anz., Bd. 22. 39. SCHLATER, 1905. Zur Frage der sogenannten “Spiralwindung der Mus- kelzellenkerne.” Anat. Anz., Bd. 27. 40. SCHWALBE, 1868. Beitriige zur WKenntnis der glatten Muskelfasern, Arch. f. mikr. Anat., Bd. 4. 41. ScuHuttz, 1895. Die glatten Muskelzellen der Wirbeltiere. Arch. f. Anat. u. Physiol., Physiol. Abth., 1895. 42. Scuutrz, 1897. Quergestreifte und lingsgestreifte Muskeln. Arch. f. Anat. u. Physiol., Physiol. Abth., 1897. 43. Sort, 1906. Sulla struttura della fibre muscolari liscie dello stomaco degli uccilli. Anat. Anz., Bd. 29. 44. Sort, 1907. Sulla struttura della fibre muscolari liscie dello stomaco degli uccilli. Bibliographia Anatomique, T. 17. 45. UnNGer, 1907. Beitriige zur Kenntnis der Wirkungsweise des Atropins und Physostigmins auf den Diinndarm von Katzen. Arch. f. d. gesammte Physiol., Bd. 119. 46. VAN GEHUCHTEN, 1889. Des noyaux des cellules musculaires striées de la grenouille adulte. Anat. Anz., Bd. 4. 47. VreRzAR, 1907. Ueber die Anordung der glatten Muskelzellen im Amnion des Hiihnchens. Internat. Monatsschr. f. Anat. u. Physiol., Bd. 24. 48. WATNEY, 1876. The Minute Anatomy of the Alimentary Canal. Philos. Trans., London, Vol. 166. 49. WeRNER, 1891. Zur Histologie der glatten Muskulatur. Diss. med. Dorpat, 1891. Structure of Smooth Muscle. 545 ABBREVIATIONS. l. m., longitudinal muscle coat. e. m., circular muscle coat. e. w., contraction wave. ¢ n., contraction node. i. S., internodal segment. i. c., interstitial connective tissue. c. f., collagenous fibers. * e. f., elastic fibers. g. p., granular protoplasm. p. S., protoplasmic syncytium. ce. mnf., coarse myofibrillee. f. mf., fine myofibrillee. t. nf., thickened myofibrille. nu., nucleus. r. nu., nucleus of resting muscle. ch. f., chromatin festoon in contracted nucleus. All figures were drawn by means of a camera lucida. The following microscopic lenses were used. Zeiss achromatic D and apochromatic 2 mm. 1:30 objectives with compensating oculars 4, 8 and 12; Leitz */,. in. oil immersion and Bausch and Lomb 1/,, in. oil immersion objectives with 1 in. ocular. EXPLANATION OF FIGURES. Fic. 1. Cross-section of the entire muscularis of the contracted large intes- tine of dog—l. m., longitudinal muscle; ¢. m., circular muscle; ¢. w., contraction wave; i. s., internodal segments of muscle fibers; ¢., an oblique contraction wave. Zenker’s fluid. Iron-hematoxylin. x 3590. Fic. 2. Longitudinal section of a group of muscle fibers from the muscular stomach of chicken showing a portion of a contraction wave (c. w.) with uncontracted areas on either side. The enlargement of the muscle fibers in the contraction wave is marked. In the contraction wave (c. w.) the con- traction nodes (c. n.) appear homogeneous and are stained very deeply. In the uncontracted areas the internodal segments (i. s.) are fibrillated and lightly staining—i. ¢c., interstitial connective tissue; f. mf., fine myofibrillz ; r. nu. an elongated resting nucleus. Zenker’s fluid. lTron-hzematoxylin. << 1275. Fic. 3. Cross-section of a portion of the circular muscle coat of the con- tracted large intestine of dog. This section shows plainly the enlargement and deep staining of the muscle fibers at the contraction nodes (¢. n.) as compared with the uncontracted internodal segments (i. s.). The myofibrillee in the internodal segments are only indistinctly shown—i. ¢., interstitial con- nective tissue. Zenker’s fluid. Iron-hzematoxylin. 2250. Ic. 4. Cross-section of a portion of the media of the contracted carotid of ox. ¢ n., contraction node. The contraction nodes in this section are distinctly fibrillated. In them the cut ends of the coarse myofibrille are shown, ¢ mf. In some of the fibers not firmly contracted (d.) both coarse and fine myo- fibrille (f. mf.) appear—i. s., either an entirely relaxed fiber or an inter- nodal segment of a contracted fiber; i. ¢., interstitial connective tissue; r. nu., resting nucleus; ¢. nu., contracted nucleus. Zenker’s fluid. Tron-hzematoxylin. < 1650. Fie. 5. Longitudinal section of a portion of a contracted muscle fiber from the media of the carotid of ox, showing two contraction nodes (¢. n.)—i. s., internodal segment; r. nu., resting nucleus; c, mf., coarse myofibrille. Zenker’s fluid. Iron-hematoxylin. >» 1650. Fie. 6. Section of a portion of a partially contracted muscle fiber from the intestine of Necturus, showing the continuity of the myofibrille through the contraction nodes (c. na. and ¢. nb.). The enlargement of the myofibrillze is shown in contraction node c. nb. This cell has only fine myofibrillze—g. p., granular protoplasm; f. mf., fine myofibrillz; ¢. f., collagenous fibers; ¢. n. b., a contraction node just beginning to form; ¢. n. a., a more firmly contracted node. Zenker’s fluid. Iron-hzematoxylin. 2250. Fie. 7. Cross-section of a small portion of the circlar muscle coat of Necturus taken near the submucosa, showing the continuity of both granular protoplasm and myofibrille from cell to cell. The tissue is a complete syneytium—g. p., granular protoplasm; f. mf., fine myofibrillze. Zenker’s fluid. Tron-hematoxylin. > 2250. (Continued on next page.) (Continued from preceding page. ) Fic. 8. Longitudinal section of a portion of a muscle fiber from the carotid of ox—just beginning to contract, showing both fine and coarse myofibrillaee— g. p., granular protoplasm; ¢. mf., coarse myofibrille; c. f., collagenous fibers ; e. f., an elastic fiber showing the wavy course due to contraction of adjacent muscle fibers. Zenker’s fluid. Iron-hzematoxylin. 1275. Fic. 9. Cross-section of three uncontracted muscle fibers from just outside the elastica interna of the carotid of ox, showing two types of fibers, one with a large amount of granular protoplasm (g. p.) and only a peripheral layer of myofibrillz, the other with no granular protoplasm and closely packed myofibrillee—i. s., internodal segment; c. f., collagenous fibers. Zenker’s fluid. Tron-hreematoxylin. > 1375. Fic. 10. Section of a portion of a muscle fiber from the internal iliac artery of man, showing two nuclei in one mass of granular protoplasm (g. p.). This fiber has only fine myofibrillz (f. mf.). Formalin. Hensen’s hematoxylin. s< 1275. Fie. 11. Longitudinal section of muscle fibers from the circular layer of the cecum of chicken, showing regular contraction waves. The number of contraction nodes in each fiber gives the tissue a cross-striated appearance. The sharp contrast between deeply staining homogeneous contraction node and lightly staining internodal segment is well shown;. (a) shows end to end union of the muscle fibers. The shortening of the nuclei in contraction nodes is indicated—ec. n., contraction node; i. s., internodal segment; c¢. f., col- lagenous fibers. Opposite the contraction nodes the collagenous tissue is condensed. Zenker’s fluid. Iron hematoxylin. x 1275. FI . Cross-section of a portion of the circular coat of the large intes- tine of a dog to show the straight course of the elastic fibers (e. f.) in the uncontracted muscle and the wavy course through the contraction waves. The enlargement of the muscle fibers in the contraction waves is marked. The union of muscle fibers end to end may also be noted—i. s., internodal segment; ¢. n., contraction node. Zenker’s fluid, Weigert’s resorcin, elastic tissue stain. x 925. STRUCTURE OF SMOOTH MUSCLE. CAROLINE M’GILL. PLATE I. THE AMERICAN JOURNAL OF ANATOMY.—Vot. IX, No. 4. Fig. 15. A portion of the uncontracted muscularis mucosze of the small intestine of pig to show the continuity from cell to cell of both granular protoplasm and myofibrille, through end anastomoses. The large amount of granular protoplasm and the few peripheral myofibrillz give the tissue the appearance of primitive muscle—a., protoplasmic anastomosis; i. ¢., inter- stitial connective tissue; g. p., granular protoplasm; c. mf., coarse myofibrillie. Gilson’s fluid. Iron-hematoxylin-eosin. >< 1760. Fic. 14. From the same region as Fig. 13, but showing in addition anas- tomoses between granular protoplasm of muscle cells and that of connective tissue cells (b.). The whole tissue is distinctly synceytial—g. p., granular protoplasm; p. s., protoplasmic syncytium; c. mf., coarse myofibrille; ¢. f., collagenous fibers. Gilson’s fluid. Iron-hzematoxylin-eosin. > 1760. Fic. 15. Section through the circular muscle coat of the small intestine of adult pig, showing the syncytial structure of the tissue. This material was slightly contracted, probably by the fixative, so the nuclei are oval instead of rod shaped, although no distinct contraction nodes are present—a., myo- fibrillee, continuous from cell to cell; g. p., granular protoplasm; e. f., elastic fibers; f. mf., fine myofibrillze. Gilson’s fluid. Tron-hzematoxylin. > 1760. lia. 16. Cross-section of muscle from the same region as is shown in Fig. 15. Each muscle cell contains a large number of fine myofibrillz, which in the cross-section appear as fine dots—mf., myofibrille; i. ¢., interstitial connective tissue; e. f., elastic fibers. Gilson’s fluid. Iron-hiematoxylin-eosin. >< 1380. Fic. 17. Cross-section through muscle similar to that shown in Fig, 13. Figs. 13-17 are all drawn from the same section. The peripheral arrangement of fibrillz as “Grenzfibrillen” is shown in Fig. 17—i. ¢c., interstitial connective tissue; g. p., granular protoplasm; ¢. mf., coarse myofibrillze. Gilson’s fluid. Iron-hematoxylin-eosin. 1760. Fie. 18. Portion of a partially contracted muscle fiber from the carotid of ox, showing the varicosity of the myofibrille in beginning contraction. The coarser myofibrillze are in the main black, the finer, red—t. mf., thickening of a coarse myofibrilla. Zenker’s fluid. Iron-hzematoxylin-eosin. x 13880. Fie. 19. A portion of a fiber from the large intestine of dog to show the peripheral position sometimes taken by the nucleus—c. nu., contracted nucleus ; c. n., homogeneous appearing contraction node; i. s., fibrillated internodal segment. Zenker’s fluid. Hansen’s hematoxylin-eosin. x 520. Fics. 20-22. Portions of muscle from the circular layer of the contracted large intestine of dog. The intestine was contracted from exposure to the air. The contraction nodes (¢. n.) in all three sections are apparently homogeneous and are stained very intensely in eosin. The internodal segments are lightly stained and show the myofibrille distinctly. The connective tissue (c. f.) is condensed at the contraction waves. The nuclei in the internodal segments are (Continued on next page.) (Continued from preceding page.) long, rodlike structures with delicate chromatin reticulum (2. nu.). In the contraction nodes they are shorter and thicker with the deeply staining chromatin massed at the ends (¢c. nu.). Zenker’s fluid. Hansen’s hzematoxylin- eosin. < 1760. Fic. 23. A contracted portion of the circular coat of the small intestine of Necturus, contracted by electrical stimulation. In the contraction wave the fibers stain deeply with the hzematoxylin, the nuclei are short and thick with the chromatin massed at the ends, the connective tissue is condensed—e. n., contraction node; i. ¢, interstitial connective tissue; f. mf., fine myofibrillee ; e nu., contracted nucleus with coarse chromatin reticulum. Zenker’s fluid. Tron-hematoxylin-eosin. 880. Iie, 24. An uncontracted portion of the circular muscle coat taken from the same section and magnified the same amount as Fig. 23, showing the structure of the relaxed muscle. r. nu., resting nucleus with fine chromatin reticulum ; fmf., fine myofibrillz; cf., fine collagenous fibers. Zenker’s fluid. Tron-hematoxylin-eosin. > 880. Fic. 25. Cross-section of a portion of the longitudinal muscle coat of intestine of Necturus, muscle contracted—e. n., contraction node; i. s., inter- nodal segment showing myofibrille; p. nu., a muscle fiber with the nucleus at the periphery; ¢. nu., contracted nucleus: i. ¢., interstitial connective tissue. Zenker’s fluid. Iron-hzematoxylin-eosin. > 880. Fic. 26. Cross-section of uncontracted muscle taken from the same section as Fig. 25 and drawn to the same scale. When Figs. 25 and 26 are compared the marked thickening of both fibers and nuclei during contraction is very apparent—r. nu., resting nucleus. Zenker’s fluid. Iron-hzematoxylin-eosin. < 880. i, The Structure of Smooth Muscle. Caroline McGill. : Pn sow Pe) A The American Journal of Anatomy: Vol. IX. Fics. 27-29. These drawings were all taken from the same region of the intestine of Necturus. One side of the intestine was firmly contracted, the other was relaxed. Fig. 27 is from resting muscle—r. nu., resting nucleus; f. mf., fine myofibrille; i. ¢, loose interstitial connective tissue. Fig. 28 is from muscle beginning to contract. Two contraction waves cross the section. The portions of the nuclei in the contraction nodes are contracted, showing distinct shortening, thickening and massing of the chromatin. The portions in the internodal segments are still relaxed. The rearrangement of the chromatin meshes during contraction is shown well in this preparation. Each end of nucleus (a) is in a contraction node, so shows contraction phenomena, the center lies in an internodal segment, so has the structure of a resting nucleus. The condensation of the connective tissue at the contraction node is shown—ec. n., contraction node; i. s., internodal segment; i. ¢., interstitial connective tissue. Fig. 29 is from a firm contraction wave. The nucleus (ec, nu.) is short and thick. The chromatin is arranged in loops or festoons (ch, f.) at the two ends—i. ¢., interstitial connective tissue. Ammonia alcohol, Van Gieson’s stain. x 15V0. Fig. 30. A section of a portion of the longitudinal muscle coat of the cesophagus of pig stained in Mallory’s anilin-blue connective tissue stain. The contraction nodes (c. n.) are stained orange and appear for the most part homogeneous. The myofibrille in the internodal segments are stained bright red (f. mf.). The massing of the blue stained collagenous fibers in the contraction waves is well demonstrated (c. f.). In the region of the internodal segments the collagenous fibers are in a loose meshwork. The enlargement of the muscle fibers in the contraction wave is distinctly shown. In this preparation the nuclei are not well differentiated. Zenker’s fluid. Mallory’s anilin-blue connective tissue stain. > 1100. Fig. 31. Cross-section of a portion of a contraction wave and uncontracted area from the cesophagus of pig. In the contraction waves the contraction nodes (c. n.) appear homogeneous and are stained orange. The internodal segments (i. s.) are of less diameter and show the cut ends of the myo- fibrille stained red. The nuclei in contraction nodes are larger in cross- section than they are in the internodal segments. In the contraction wave the connective tissue (c. f.) is much condensed. Zenker’s fluid. Mallory’s anilin-blue connective tissue stain. > 1100. Fic. 32. Cross-section of muscle fibers from the contracted carotid of ox— ¢ n., a contraction node. It is stained orange, but at the same time shows the cut ends of myofibrille—i. s., either internodal segments of a contracted fiber or an uncontracted fiber. The red myofibrille are seen throughout the entire sarcoplasm—e. f., elastic fiber; c. f., collagenous fibers. Zenker’s fluid. Mallory’s anilin-blue connective tissue stain. > 1700. Fic. 33. Portion of a contracted muscle fiber from the carotid of ox. Around the nucleus is a wide contraction node (c. n.). The end of the fiber is uncontracted (i. s.). The contraction node is stained orange. The Continued on next page. ] (Continued from preceding. page. ) myofibrille may be seen indistinctly here. The internodal segment is dis- tinctly fibrillated and the myofibrillze are stained red. The connective tissue is condensed in the region of the contraction node (c. f.). Zenker’s fluid. Mallory’s anilin-blue connective tissue stain. x 1700. Fic. 34. Portion of the media of contracted carotid of ox, showing a contracted fiber and a portion of an uncontracted fiber (a)—ec. n., contraction node; i. s., internodal segment; f. mf., fine myofibrille; ¢. f., collagenous fibers; e. f.. elastic fibers. Zenker’s fluid. Mallory’s anilin-blue connective tissue stain. x 650. The Structure of Smooth Muscle. Caroline Me. Gull. The American Journal of Anatomy VolIX. ac p AY ; 50 ~~ ad « - Fic. 35. Portion of a muscle fiber from the contracted large intestine of dog, showing two small contraction nodes (c. n.). The myofibrille run con- tinuously from internodal segment to internodal segment through the con- traction nodes, apparently thickening in the region of the nodes. (t. mf.) shows such a thickening—r. nu., a resting nucleus; ¢c. f., collagenous fibers. Zenker’s fluid. Iron-hsmatoxylin-eosin. x 3000. Fic. 36. Is taken from the same section as Fig. 35 and shows similar structure. The contraction nodes are wider than those shown in Fig. 35. Zenker’s fluid. Iron hzmatoxylin-eosin. < 38000. Fic. 37. Cross-section of a portion of the circular muscle coat of the intestine of dog. In the contraction wave (c. w.) the thickening of the fibers, the change in staining reaction, the clumping together of the nuclei, along with the shortening and thickening of each nucleus, are clearly shown— r. nu., resting nucleus; ¢c. nu., contracted nucleus; ¢ n., contraction node. Zenker’s fluid. Delafield’s hematoxylin-eosin. x 880. Fic. 38. Is taken from the same section as Fig. 35—ec. n., contraction node deeply stained so that no myofibrillee show; i. s., internodal segment; ¢. mnf., coarse myofibrilla thickening as it enters the contraction node. Zenker’s fluid. Tron-hematoxylin-eosin. >< 3000. Fics. 39-40. Drawn from the same section as Fig. 35. Portions of muscle fibers showing beginning contraction nodes (c. n.) with the myofibrillz enlarged in the contraction nodes (a, b)—t. mf., thickened myofibrillz. Zenker’s fluid. Iron-hematoxylin-eosin. < 3000. Fie. 41. A group of myofibrille from a partially contracted muscle fiber in the large intestine of dog, showing numerous enlargements of the myo- fibrille—t. mf., thickened myofibrille. Zenker’s fluid. Iron-hzematoxylin. << 38000. The Structure of Smooth Muscle. Caroline Me Cull. ” ae : : : a a Diwan wie PLN EY b = os Oy AO cm rn ru tmt The American Journal of Anatomy: Vol Ix Fies. 42-53. 1700. STRUCTURE OF SMOOTH MUSCLE. PLATE V. CAROLINE M’GILL. Fig. 51 THB AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. Tics. 65-67. Three nuclei from the bladder of cat, showing degrees of shortening of the contracted nucleus. Zenker’s fluid. Jron-hzmatoxylin. < 3000. Fics. 68-72. Nuclear contraction in the muscular stomach of chickens. Tig. 6S shows a resting nucleus. The remaining figures show the changes in shape and the arrangement of the chromatin at various phases of contrac- tion. Zenker’s fluid. Iron-hzematoxylin. x 3000. Fics. 73-76. Stages in the contraction of the nucleus in the muscular stomach of a 14-day chick embryo. There is during contraction a decrease in length. There are few chromatin changes. Zenker’s fluid. Iron-hgma- toxylin. x 2200. 5 Fics. 77-84. Nuclear contraction phases in the carotid of ox. Fig. 77 shows a resting nucleus. Figs. 78-80 show shortening and thickening of the nuclei. Figs. 81-84 show folding or spiral winding of the nucleus which may possibly be typical at times for arterial nuclei. Figs. 81, 82, along with coiling, have also undergone shortening and thickening. Zenker’s fluid. Iron- hematoxylin. x 8000. STRUCTURE OF SMOOTH MUSCLE. PLATE V? CAROLINE M'GILL. TuHB AMERICAN JOURNAL OF ANATOMY.—VOL. IX, No. 4. Fies. 85-89. Nuclei from the small intestine of Necturus, contracted by electrical stimulation. Fig. 85 is a completely relaxed nucleus. There is a fine chromatin reticulum and a single plasmasome (pl.). In Fig. 86 a nucleus is shown, one end of which is just beginning to contract. The chromatin has changed from a granular reticulum to fine longitudinal strands (a). Fig. 87 shows the chromatin arranged in loops at one end of the nucleus. At the same end the nuclear wall is serrated. Fig. 88 shows a festoon of thick chromatin strands. In Fig. 89 is shown a completely contracted nucleus. The heavy chromatin strands are arranged in festoons at either end (ch. f.). The ends of the nucleus are serrated. Zenker’s fluid. Iron-hzematoxylin. x 1700. Fics. 90-92. Resting nuclei from muscle of intestine of Necturus kept for 20 minutes in 1 per cent cocaine solution. Zenker’s fluid. JTron-hematoxylin. x 1100. Fics. 93, 95. Contracted nuclei from muscle of the small intestine of Necturus. The tissue was contracted by placing it in pilocarpine solution. Zenker’s fluid. Iron-hzematoxylin. 1100. Fics. 96-99. Nuclei from the same source as those shown in Figs. 93-95. Instead of fixing the tissue frozen sections were made and stained in methylene-blue. Figs. 96-98 are partially contracted nuclei. The chromatic changes are marked. Fig. 99 is a completely contracted nucleus. Its struc- ture is almost identical with that shown in Fig. 89—ch. f., chromatin festoon. Frozen sections. Methylene-blue. x 2200. LS ee LU eee Ee ee eee a STRUCTURE OF SMOOTH MUSCLE. PLATE VII. CAROLIND M’GILL. THb AMERICAN JOURNAL OF ANATOMY.—VoL. IX, No. 4. bs bakit i Ni i i de y Mt > te x YY th) HOI Library - Serials ri PAL’. 5 WHSE i) ‘ Ih Gear ye wf, Vag g. 4 | , tag eerhitetey Mi tiaey ia if PEL CRT TUNE tire COETORLQE AMC epeedgrapge eeceere ‘ PP ey eee seUPe hae 1 qertete ta t Hes a