# Talk:Anatomical Record 15 (1918-19)

THE

ANATOMICAL RECORD

EDITORIAL BOARD

Irving Hardesty

Tulane UnlversUy

Clarence M. Jackson

Unlvcralty of MlDnesota

Thomas G. Lee

Uclverslty of MiDQesota

Frederic T. Lewis

Harvard Unlvenlty

Warren H. Lewis

John! Hopkins Unlvenlty

Charles F. W. McClure

Pllnceton University

Wiluau S. Milixr

University of Wisconsin

Florence R. Sarin

Johns Hopkins University

George L. Streeter

University of Michigan G. Carl Huber, Managing Editor

1330 Hill Sueet. Ann Arbor, Michigan

VOLUME 15 AUGUST, 1918-FEBRUARY, 1919,

PHILADELPHIA THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY

St!

CONTENTS

1918-1919 XO. 1. AUGUST

Kaethe \V. Dewev. a contribution to the study of the pathways of the cerebrospinnl

fluid and the choroid plexus. Nine figures 1

II. E. Jordan. A description of a case of false hermaphroditism. One figure... 27

II. E. Jordan. The histology of lymph, with special reference to platelets 37

William Snow Miller. A differential injection massfor use with stercoroentgenograms . 47

William F. Allen. Surface view of.injected intestinal villi. One figure 49

William F. Allen. An inexpensive .microscopic projection and drawing apparatus.

One figure 53

NO. 2. SEPTEMBER

Henry Erdmaxn Radasch. A duplication or branching of the neural canal. Twelve figures 57

Wilhtjr C. Smith. On the process of disappearance of the conua arteriosus in Teleosts.

Sixteen figures 65

Wayne Jason .\tw'ell. The development of the hypophysis of the Anura. Eighteen

figures 73

HoRTON R. Casparis Lymphatics of the omentum. One figure 93

Matthew Marshall. An unusual right lung 101

NO. 3. OCTOBER

Hal Downey. Further studies on the reactions of blood- and tissue-cells to acid collodial dyes. Six figures (two plates) 103

K. D. Conodon. The embryonic structure of avian heart muscle with some considerations regarding its earliest contraction. Ten figures 135

Eliot R. Clark and Eleanor Linton Clark. Phagocytosis of carbon and carmine

granules in the transparent tails of tadpoles. One plate 151

NO. 4. NOVEMBER

Richard E. Scammon and Edoar H. Xorris. On the time of the post-natal obliteration of the fetal blood-passages (foramen ovale, ductus arteriosus, ductus venosus) . Three figures 166

Wayne J. .\twell and Ida Sitler. The early appearance of the aniagcn of the pars tubcralis in the hypophysis of the chick. Five figures 181

F. A. McJuNKi.v. The identification of endothelial leurncytcs in human tissue. Third

report of studies on the mononuclear cells of the blood. Two figures 189

Frank Blair Hanson. The scapula of Tragulua. Nino figures . 197

iii

iv CONTENTS

NO. 5. DICCKMHKU

Wamu NaKaiiara. Some obsorviitions on the KrowiiiK oncytes of the stoncfly, Pcrla immnrRirinlA Sny, with R|ivoinl regarii to the origin iind function of the nucleolar

•trurlurt's. Nino figuroa 203

ll\Hiii-.>s U Hu.sT. Variability in the common carotid arteries of the domestic cat.

\ IV. limm-s 217

Makuison It IIVNT. AbBciirc of one kidney in llie domestic cat. Two figures 221

AtH)Lr H. S< iirLTX. Ob.ierval ions on the cnnalis basilaris chordae. Three figures 225

Mleanoh I.intdn Ci.akk and Eliot R. Clakk. On the reaction of certain cells in the

tad|Kile'8 tail toward vital dyes. Four figures 231

Kdward I'iiclps Allih, Jr. On the origin of the hyomandibula of the Teleostomi. One figure '. 257

NO. 6. JANUARY

RiruARD K. PcAMUoN. On the development and finer structure of the corpus adiposum biircao Nine figures 267

Frank Ulaih IIan.hon. Nerve foramina in the pig scapula. A peeuliar relation existing b«'iworn the dorsali.s branch of several spinal 'nerves and the suprascapula in the pig Twenty-one figures 289

Florence May Aij*op. The effect of abnormal temperatures upon the developing nervou-t system in the chick embryos. Thirteen figures 307

I'rofccdings of the American Society of Zoologists. Sixteenth Annual Meeting 333

Proceedings of the American Society of Zoologists. Abstracts 341

NO. 7. TEBRUARY

JoMKi-ii M. Tni'KiN<iER. The anatomy of a dicephalie pig. Monosomus diprosopus.

Ten tigure.i 359

\. (!. I'oHLUAN. Double ureters in human and pig embryos. Three figures 369

A. <i. roHL.MA.v. The Urtc of a simple graphic method of recording the relations in serial

sections, particularly for use in teaching embryology. Four charts 375

\. Ct. I'oiiLMAN. The use of bayberry wax in hardening paraffin blocks. One chart.... 385

.\. O. Poiii.Mw. .\ modiHration of the Born pa|)er-wax reconstruction i)late 389

II. K. JoRDA.s. The histogenesis of blood-platelets in the yolk-sac of the pig embryo... 391

actuok'0 abstract or this papcr issued

BT THE BIBUOORAPHIC ABRVICB, iUlVB 34

A CONTRIBUTION' TO THE STUDY OF THE PATHWAYS OF THE CERERROSPINAL FLUID AND THE CHOROID PLEXUS

KAETHE W. DEWEY The Research hnhnralory of the College of Dentistry, University of Illinois

NINE nouRES

In oxperiniental work for demonstrating the presence of Ij'mphvessels in the dental pulp,' use was made of vital staining as a supplementary attempt to obtain some histological information regarding endothelial lined perivascular or other lymph-channels. There are observations recorded in the literature which are vaguel\- suggestive of such a possibility. Of particular interest from this standpoint are statements made by Brass* and by Evans,' that the endothelial cells lining the sinuses and h-mphchannels of lymph-glamls are always intensely stained, and by Kiyono* that vitaliy stained endothelial cells are found "in the lymph spaces of the interstitial tissue of the lung."

A large number of rabbits and a few dogs and cats were injected intraperitoneally and chiefly intravenously with trypan blue and lithium carmine, and microscopic preparations made from practically all organs. Chief attention was paid to those vitally stained cells, which occur in the connective tis.sue of the various organs and which have been classified as endothelial cells, reticuhmi cells, resting wandering cells, clasmatocytes, rhagiocrines, histiocytes, pyrrol cells. The impression witli me from

' Dewey, Kaethe, and 1". B. Noyes, "A Study of the lymphatic vessels of the dentiil pulp." Dental Cosmos, 1917, 58, 43C.

' Brass, "Cber physioloRisohe PiKmentatilafcerunR in den Kapillarcndothelien des Knochenmarks." .Vrch. f. mikrosk. .•Vnat.,'1913, 81, 61.

'Evans, "The macrophages of mammals," Amer. Journ. of Phvs., 1915, 37, 242.

• Kiyono, "Die vitale Karminspeicherung. Jena, 1914.

1

THE ANATOMICAL RKCORD, VOL. 15, NO. 1. AUOUST, 11)18

2 KAETHK W. DKWEY

thos«» studies wjus that tlu\se colls arc intimately related to the iymphatir system. The regularity with which they occur in the same regions within the organs, the apparently systematic arrangement in wliich they present themselves, the definite course wliich so oft^'ii tlu-y seem to jmrsue, an; striking features and tend to «lispel the impression that they are cells indiscriminately dispersed within the connective tissue, resting wandering ceils waiting for a chance stimulus to arouse them to functional activity and l<K-omotion. When the uijections have been continued for some time the cells are large and crowded with stained granules in their cytoplasm and their long processes: the latter not infre<iu«'ntly almost touch one another so that nearly continuous cell tracts are formed, running along the blood-vessels and independently. From these observations, in conjunction with studies of the literature on the lympiiatic distribution throughout the body, I have been led to suggest the possibility that these vitally .stained cells are for the most part endothelial cells lining the l>nnph-channels within organs and that \ital .staining may furnish us the means of demonstrating lymph-channels in organs and regions where they are not yet definitely known or are difficult to demonstrate by the usual methods of injections.

.\s to the central nervous .system it has been and still is a much debated (jue.stion whether the perivascular spaces should be regarde<i as lymph-vessels, the decision hinging on this point whether they have an endothelial lining. It is a general observation that the favorite location of vitally .stained cells within the connective tissue in all organs is the immediate neighborhood of the blood-\e.ssels. It was therefore to be ascertained whether vitally staineil cells are also found about the cerebral ve.s.sels. With this point in view, the brain with its membranes and the choroid plexus were studied. The dura and pia, and the choroid plexus of rabbits were carefully removed and sjiread on glass slides; the choroid plexus of dogs and cats was reduced in thickness by the freezing microtome. Of the membranes the dura always pres<-nts the greatest abundance of vitally .stained cells; it is also one of the tissues in which a ilefiniteness in the arrangement of these cells is pronounced. They form long continuous

CKaKBRO.SPIXAL FLUID AN'D CHOROID M.EXL'.S 3

tracts, niiminK aloiin the l^lood-vo.ssols, but luurc often apparently in entirely independent directions. Two-, three-, and four-cell tracts may run parallel to one vessel, from which they may he entirely separated. Their independent course is also very conspicuous in regions wher<> tlie blood-vessels form Ktops; here the blue- or red-cell tracts sweep across them in continuous straight lines. Compared with the enormous supply of these cells in the dura, those of the pia arachnoid are .scanty and more slender. There are always areas where there are no cells or only a few here and there, and the.se are slender and barely- visible with the low-power lens. But there are always patches where they are in rather dense collections, are larger and more deeply stained, and from these cell groups lines of cells run in all direcJtions. .\fter lithium cannine injections the number of vitally stained cells always seem increased and the individual cells are larger. Frequently they have very long processes which aUnost touch one another, ami thus very definite tracts are formed which ari)i1ranly, it seems, follow some of the blood-ves.sels, while others and especially all tlie larger vessels are unaccompanied by such cells. The irregular distribution and the presence of cell patches is a prominent feature in all preparations of pia. In the choroid plexus the ependymal cells covering the vascular convolutions are all densely tilled with a finelj' granular .stain and form a continuous blue or red layer around the tufts. In the i)ial connective ti.ssue of these and the velum interpositum there are always numerous vividly stained cells like those in the dura and pia. They do not form absolutely definite cell tracts, nor do they follow the blood-vessels closeh'; in fact, they are generally at some distance from them. They are quite evenly distributed throughout and anything resembling the cellular patches of the pia does not occur here. In the intensity of the staining, the large number and the distribution of these cells, the velum interpositum and the pial tissue about the tufts n*semble more the dura than the pia.

Being inclined to consider the vitally .stained cells as endothelial cells lining the lyjuph-ehannels and lymph-spaces I suggest that the cell complexes in the pia arachnoid might be looked

4 K \KT1IK W. DKWKY

upon as thn stnu'turf> ilcsc-ribcd hy W coil :is urachiinid \illi, strurturps normally present in the anic-hnoid of anbniils arul man, and through which, according to hlin, the cerebrospinal fluid is filtered from the arachnoid spaces into the venous sinuses. In view of the presence of such vitally stained cells in the membranes of tlie brain and the choroid plexus, clearly indicating the passjige through and into them of fluid containing the staining solution, it is the more striking that the brain tissue fails to show any vitally stained cells whatsoever. The only other exception from the general observation is the fet-us which also remains unstamed. CJoldmann' explains this fact b}' the staten\ent that the dye derived from the plasma is filtered off by the cubical epithelial cells of the j^lexus and the .syncytial cells of the placenta and thus kept off from the cerebrospinal and the anmiotic fluid. Those two tis.sues form a protecting limiting membrane to the brain and fetus. TrjTian blue, according to him, is a substance extremely toxic to the nervous tissue. He claims that while 50 cc. of a 1 per cent st)lution may be injected in a single do.se into a rabbit intravenously without any unt-oward effect, J cc. of a J per cent solution of the stain injected into the spinal or cerebral subarachnoid space produces tonic and clonic convulsions within ten to twenty minutes and death in coma within one or two hours. In these cases he found that the ganglif)n cells had a dilTu.sely stained cytoplasm and a .stained nucleus, indicating that death of the cells had taken place. But he also (U'.>icribed gemiine vital staining of cells within the connective ti.ssue of the pia and along the vessels of the pial septa. (These cell.s are called in all his publications 'pyrrol cells,' because he first observer! typical granular vital staining of these cells after injections of pyrrol blu*-. ) "The supposition." he writes, "that

• CuiihiiiK. Wrijd iiiul \VL'({i'f«rth, "Studies on the cerebrospinal fluid." Journ. of Med llpurnrrh. 19I4, 2('., .il.

• fioldmann. 1. Die fiusscre und innuro .Sckrction des gcsuiiden und krankcn Organiamua in Lichte vitalcr I'lirbung. Bcitr. z. klin. Chir., 1909, 54, 192, iind 1012, 78, 1.

2 Kxpvrimcntcllc Untersuchungen iiber die iMinktion der Plexus choroidcii und der llirnhnut Arch, f kliii Chir . 1913. 101. l.ir,.

3 Die Vitnlfarlmnn des Centriilnirvcnsystems Berlin, 1913.

Cf:REBR08PINAL FLUID AXI) CHOROID PLEXUS O

the poriviiscuhir prolongations of tho pial funnels functionate as 'lyinj)lispH('es' finds a further support in the fact that the cells which line these spaces, as well as the fine connective-tissue trabecuia trn versing these spaces, store up our vital stains in fine granules in exactly the same way as the reticulum cells in the lymph glands." These statements by Golilmann induced me to also use subarachnoid injections of trypan blue solutions not only for the demonstration of endothelial lined perivascular lymph-channels in the brain, but as well for some further infonnation about the choroid plexus. However plausible his explanation of its function as a protecting memlirane, some questions evolving from such a conception remain still obscure; for example, how is it possible that the filtering jjrocess bj' the cuboidal epithelial cells of the choroid plexus can protect the entire brain against injurious substances from the blood plasma when other vessels beside the choroidal arteries, in fact, the larger number of the cerebral vessels, pass directly into the brain substance? In what manner is the spinal cord protected which has no choroid plexuses?

The clinical results in twenty-four rabbits which I injected intraspinally and intracranially with tr>'pan blue, did not present the grave symptomatic picture which (Joldmann describes as characteristic for the rabbit. Sjinptoms occurred in a number of these anunals: but there were no fatal cases, and in several rabbits even repeated injections failed to elicit any sj'mptoms. In the absence of all grave conditions of convulsions and of coma, the results res(>mble(l much more tho.se observed in dogs. Concerning the striking dissimilarity of results observeti byGoldmann in these two animals, this author remarks hunself: "It seems to me very remarkable that in the dog the symptoms of motor irritation elicited by the dye cannot be compared in any way with those in the rabbit above all, those grave conditions

of convulsions may be lacking in the dog which are so (>X('(>edingly characteristic of the toxic effects of the dye in the ral)bit." I am inclined to believe that the sjiiiptoms do not arise so much from any toxicity of the staining substance, as that they are due to mechanical and physical factors and direct injuries of the

G KAKTilK W. tlKWKY

ncrvdus tissue in the process (if the injections, which jire not so eiisily coiitrollei! in the rabbit. The most severe paralysis oceurntl in n control rabbit after the first injection of distilled wiitor: the pjiralysis persistetl, i)ut the anunal recovered otherwise and remained well in spite of a second injection of distilled watiT and two injections of trypan blue.

The injections were made, as a rule, in the lumbar and lower thoracic region: eight rabbits were injected into the cerebral subarachnoid space. After having anaesthetized the aniinal with ether, the needle was jiasseil through the skin and juuscles into the spinal cavity by way of the interspinous foramina, which are rather large in young rabbits. The cranial injections were nuide through the occipito-atlantoid ligament. I found soon that it was best to use a very fine needle and pass it quickly through the cord in an oblique direction and inject the fluid into the .space opposite the point of entrance. The needle's striking the bony wall makes it sure that the Huid is not forced into the nervous tissue, an accident which is not so easy to avoid if the fluid is injected wliere the needle passes through the interspinous ligament. In injections into the cerebral subarachnoid space some of the fluid was always withdrawn before injecting the staining solution. In larger anunals the drops would flow out spontaneously through the medium-sized needle; in very young rabbits a very fine needle was u.sed and here the fluid was aspirated with a medicine dropper. As to the .spinal cord, attempts to let some of the fluid escape before injecting the solution were given up as rather difficult. For this reason I cannot claim, in .spite of all practical considerations anil careful .study of the anatomic conditions of the spinal cord in the rabbit, to furnish a positive proof that the staining solution actually passed into the subarachnoid space and not into the epidural space. But the clinical and histological observations in these injections being largely the same as in intracranial injections, the controlling tests of the latter should be sufficient to justify my general statements.

In the following table the distinguishing feature for the results, niarke cells indicates a grave impainnent of their vitality, and since I claim that this is not the result of any toxic action of trvf)aii blue, but arises from other causes incidental to the injections, the presence of dilTusely stained cells is applied to represent 'negative results,' and the presence of vitally stained cells 'positive results.' There is evidence that the cells may recover from the condition in which they arc susceptible to dift'use staining. Those rabbits which were killed when serious sjanptoms arose and persisted for several hours were nearly all 'negative' cases; on the other hand, in instances where the animals presenting grave symptoms were allowed to li\o and to recover and sub.sequently were again injected with trjpan blue without any untoward effects, the results were generally 'positive.'

TABLE 1

Inlroipinous injections Nervous symptoms of a more or less serious nature with negative results

a <

a o

M

Si

X

b. O

z a

ill

Z M

8TMPTOUS

REacLTa

11

X

1?

trtmi

ce.

p9rc«nl

ifayf

1

700

1

1

1

Paralysis of hind legs; spnsms of muscles of mastication, excitement

2

650

1

1.0

i

Severe spinal symptoms; paralysis, motion in a circle from

3

1020

4

15

1

5

right to left Severe symptoms after the fourth injection due to injury

4

810

3

10

1

3

in injecting Paralysis of hind legs and slight

_

5

590

2

5

1

2

spinal symptoms Transitory slight symptoms

^^

6

1130

1

1.0

i

Remark : injection was made when the animal was sucoumhing to ether anaesthesia

Tota

I

A

KAETHE W. DEWKV

TABLE 2

Intraspinal injrcltonK

Grave nervous symptoms with positive results

r

■»

o

is§

«s

a j

E

a

5 »

• 2

J:

'; l^

1

a

HYUPTOUS

n

1'^

|l

fram

te.

7

1487

1

1.5

»

20 hours

Paralysis, excitement, spasms of muscles of mastication

+

8

1015

•i

5-15

J

5 days

Paralysis and spinal symptoms after the 4th injection

+

9

1372

4

15

i

5 days

Paralysis and spinal symptoms after the 4th injection

+

Tola

1 .. .

3

TABLE 3 Absence of symptoms with positive results

u

Si

S

4

.S

BBSULn

li

X M

IS

5 5

IH

srslPTOMS

n

1^

»

K

4

S

a.

A

trams

ec.

fv ant

Jair<

10

1380

2

1

\

3

Membranes light

+

11

1265

5

1

J

9

Membranes very light

+

12

1070

13

1 fr-1.5

ItoJ

16

Meinbrances very light. Grave paralysi.s followed first unsuccessful attempt to inject, without any fluid entering the spinal canal. Complete recovery from this in course of following 16 days of experiment

+

13

018

4

06-1.8

1

4

Membranes light

+ 4

Toll

tl

Injeclions

1 AHI.K 4

into the cinlerua magna

li

i

S

Si Si

z

in

i°2

gS3

STUPTOUa

■ncLra

11

i

i^

gram*

«.

p<r Mill

ifan

14

640

1

5

1

Grave symptoms due to impeded outflow from eisternu

15 16

550 620

2 2

5 0.5

1 1

3 4

mngnti

Grave symptoms after the second injection

Disturbance of equilibrium, transitory

+

+

17 18

730 950

1 1

5 75

i

3 3

Slight transitory symptoms NystuKmus; motions of the head

+ +

19 20 21

1060 1530 1500

3 5 1

5

1 5

i

, i

i

5 8 4

S'o symptoms

Xo symjjtoms.

Direct injury of medulla oblongata; lastinR disturbance of equilibrium

+

-f+

Tota

il .

8

TABLE 5 Tests for the comparative offeel o( distilled water and trypan blue solutions in the

same animal

5

a 2

5

NI'UBER or INJBCT10N8

1

Ik o

s

o o

a <

z o

5

a:

H u Z

8

a

.SYMPTOMS

■ BSrLTS

X

5

1

s

1

grama

cc.

ptr ctnt

da,.

22

1-280

2 aq. dost

10

Serious symptoms and

+

3 trypan

1.0

i

9

complete and pcrsisting paralysis of hind loRs after first injection of distillod water. Otherwise (gradual recovery

28

1250

2 aq. dest.

1.0

Paralysis of hind legs af

1 trypan

10

i

7

ter the 3d injection. Xo other symptoms

24

1460

2 aq. dest.

10

Xo symptoms except dul

+

2 trypan

0.5

J

7

nesa

Totf

il

3

10

K \KTIIi: W. DKWKV

lAHii:

Kiiwinnrul' (.. ,i.^r.rl,uu tchfthtT 'KdrtichetUfllcn' in lesioHS such as may occur

, ,. < lake up tri/pan blue when this is injected intravenously

r

a

H

H

I

KCMBaa or

s

1

o

3

i I

8

a

■ TilPTOUS

RCStTLn

s

t IIUBCTIOKa

s

a

«M.

ee.

Sli

in.

25

1005

6 intrav.

8

No nervous man

No vitally stained cells

7 intraa.

10

1

8

ifestations

in nervous tissue at

aq. deal.

site of injection or otherwise. Blue Kornchenzellen in membranes of brain.

26

980

6 intrav.

10

8

No symptoms

No vitally stained cells

12 introap.

1

1

17

in nervous tissue ex

aq. deat.

cept a few along vessels in one restricted region of brain.

n

1240

3 intrav.

1

Complete persist

No vitally stained

4 intraap.

10

1

9

ing paralysis of hind legs after 1st injection of distilled water apparently due to direct injury

cells or Kornchenzellen in region where needle had been passed, but enormous numbers of such in the membranes of the region.

In taliulating my rosvilts I may say: IT) rabbits gave 'positive' results, 8 rabbits 'negative' results. That the greater number of these were obtained during the latter part of the experunent is very probably due to the greater skill in injecting acquired by practice. Three rabbits were killed after one injection because grave symptoms dc\el(tped; all gave negative results. One rabbit was injected in a dying condition of the effect of ether; the autop.sy was made half an hour after death. Microscopically all the cells and libers of the membranes were diffusel}' stained. Control rabbits were among the last in the series and had the benefit of the better techni(|ue. One of the.se was paralyzed in both hind legs by the first injection of distilled water. The

CEREBROSPINAL FLUID ANU CHOROID PLEXUS 1 1

paralysis rcjiiitincd, l)Ut the animal recovered ()ther\vis(* and remained well in spite of another injection of distilled water and throe injections of trypan blue. It was killed nine daj's after the first injection, Microsc()|)ically the results were positive. Two oth(T control rabbits had each two injections of distilled water and only one injection of trypan blue. There were no unu.sual symptoms from either of the injections; both ^ave negative re.sults. In two rabbits the deep cervical glands grossly showed bluish areas and microscopically revealed rather large regions with vitally stained cells. In two f)ther rabbits the kidneys revealed a distinct bluish tint throughout the cortex: microscopically the epithelial cells of the contorted tubules in ilisseminate areas contained extremely fine bluish granules; only by most careful study with the oil inmiersion lens could the granular nature of the stained protopla.sm be detected. One of these two cases was a rabbit which suffered from an experimentally produced nephritis. Only in two of the negative cases was intense staining of nerve cells of the cord observed comparable to that described by (loldmaim. It was due to the direct injection of the staining solution into the nervous tissue. In remoter regions of the cord there was no such intense staining and it was absent in the brain.

The gross appearance of the brain and cord is characteristic and practically the same when the injections have been succe.ssful. The membranes on the convex surface of the brain are always light and in the best cases appear almost entirely colorless. The coloration begins at the olfactory prolongation. On the base there is always more or less intense staining, most markedly over the olf.ictory lobe, the optic and acoustic nerve, the pons and the medulla oblongata. The membranes of the cord are uniformly tinged. After intracranial injections the coloring gradualh' decrea.ses downwards.

The histological lindings reveal peculiarities. If we take those in intracranial injections as a standard, we hnd, on the average, that the dura is less supplied with vitally stained cells than the pia. Towards the olfactory lobe the lunnber incre^uses enormously; at the same tijne the cells are larger. In the pia there

12 K\KTIIK W. DKWKV

may Ix- an ovorwIifliiiiiiK aimuint of cells in this roRion. Generally thoro are also throunliout more cells in this membrane than in the dura. In case of marked liypercmia or pial hemorrhage there is sometinu's an amazing number of granular cells and 'Kornclienzellen' in the convex portion of the pia. The thin membrane over the cisterna magna is well suj)plied with vitally stained cells. The more surprising is the observation that the tissue about the tufts of the chorioid plexus is totalh' devoid of \it:illy stained cells. There is also no trypan blue in the epithelial c»^lls them.selves. As to the cord, diffu.sely stained cells are absent, unless the staining solution has been accidentally injected directly into the nervous tis.sue. There are no vitally stained cells along the intracerebral or intra.spinal vessels in nobu;dt,ger they may occur in regions here and there. Such foci are, as a rule, small in luunber: sometimes careful search for them will fail to revejd any.

In a few rabbits lesions had been produced in the spinal cord which were followe<l by destruction of the nervous tissue and immigration of 'Kornchenzellen' in enormous numbers: these cells are all vitally stained. Those which occupy the region of necrosis and necrobiosis lie close together, are large, more or less rounded and besides blue granules contain diffusely stained cell inclusions. The chemotactic influences emanating from this focus have been juade visible, so to speak. Throughout the .sections cells marked by blue granules swarm from all directions towards this center: they are slender, increasing in size as they apjiroach the field of phagocytic action; they are all in the immediate neighborhood of the ves.sels and follow the course of their divisions so that through them the distribution of the vessels is well illu.strated. There cannot be any doul)t in this case but that these "Kornchenzellen' are derived from endothelial cells lining the perivascular spaces. A singular observation, however, is that as we get away from this region of necrosis, blue stained cells along the vessels are no longer .seen, (iokhnann also found such phagoc.vtic vitally stained cells in inflanunations of the meninges at the place of the injury. They were not only in the subarachnoid meshwork, but could be followed from the pial

CEREBROSPINAL FLUID A.VI) CHOROID PLEXl'S 1 iJ

funnels fur int(j tho cortex. Ho fonsidcrs thorn us normal constituents of the meninges and their intracerebral, intraspinal, and intraneural prolongations.

.Some i)nj)()rtaiit observations from intraspinal injections of trypan l)luo are then Ijriofiy these: \ita.lly stained i)erivascular cells, endowed with the phagocytic properties of 'Kornchenzellen,' are abundant in a wide range about encephalitic foci. Vitally stained endothelial cells about ves.sels in the cord and brain are not generally in e\iilence; they may be present about a scanty number of vessels in the cord and are scarcely, if at all, seen in the brain: apparently there is less tendency in such cells to take the stain as we get away from the region of the injection. The staining .solution, however, is early carried with the cerebrosinal fluid to the optic, olfactory, and all peripheral nerves demonstrated by the early staining of their dural sheaths; it is conveyed to the deep cervical glands. May we not conclude from these observations that the endotheUal cells of the perivascular spaces in the brain and spinal cord have less affinity for the stain than similar cells in the meninges and in other tissues of the body? that some stimulus is needed to arouse them, so to speak, to reaction to the stain? and that the stimulus in these cases come from the local effects of the injections, not from the stain itself as a 'nerve toxin,' but from direct and indirect mechanical injuries to the nervous tissue, from pressure, from disturbances in the circulation? Such an hj'pothesis is not without a parallel. Taking it for granted that these particular cells are endothelial in nature or origin as admitted by various investigators, it is a well-known fact that this property of vital staining is peculiar only to a certain })r()p()rtion of this class of cells. The endothelial cells lining the blood-vessels as a whole do not take the stain. \n exception to this rule are those of the capillaries and venules of the spleen, of the blood-vessels of the bone-marrow and the hemal glands, juid the Kupfifer cells, supposed to belong to the capillaries of the liver. .Vs to the Ipnphatic system vital staining has been ob.served in the endothelial cells lining the lyjnphatic sinuses of the lymph glands. .Vn interesting observation in vital staining concerning these organs has been emphasized bj'

14 KAKTIIK \\ . I) i:\VKV

Kvans,' who writes: '"This purticipation on the part of thopndofhehujii. however, is sharply Hmitod to those definite tracts of it well within the heiiial and lymphatic glands. The entering or draining trnnks. in the ca.se of the hanph ghmd do not show any |x»culiarity on the part of their lining cells, whereas, in the case of the heniolyniph glands, nothing is more striking than the abrupt assiunption of brilliant dye granules by the entlotheliiun of a venule just as it enters and resolves itself within the gland." I have ventured to suggest' that this difference in the behavior (tf the «'ndothelial cells towards the stain may correspond to a diflference of function on the part of the Ijinph-channels. Those within the organs are collecting vessels, those outside conducting ve.s.sels. .Vs to the Kupfler cells, it cannot be said that a decision as to whether thej' belong to the blood capillaries or to perivascular hinph-spaces has been absolutely agreed upon. There are a number of writers who assign them to the latter. The other organs in which vitally stained endotheUal cells of the blood-vessels occur, namely, the spleen and bone-marrow, are curiously enough those in which blood and l>^^^ph are in most intimate relationship to each other: the spleen and the bonemarrow are the birthplace of both elements. The hemal glands may be of a similar nature; they are essentially Ijinphatic structures.

From the differences in the staining phenomena observed in the central nervous system, we may perhaps draw some conclusions regarding the flow of the cerebrospinal fluid and the relatitmship of the general lymphatic circulation to the central nervous .sy.stem. It is certainly ver\' singular that the specific connectiveti.ssue cells about the epithelial tufts of the choroid plexus, which invariably are intensely stained after intravenous injections, are ab>o!utely free from any stain after subarachnoid injections. Since this tissue is a continuation of the pia, we should expect to find vitally stained cells in this location, when tJiey ar(> present in abundatice within the pia. It wovdd seem from this that the cerel)rospinal fluid containing the staining solution does not

' I.e. "The iiiHcrophuicrs of mnnimiils." .\m. Journ. of Plus., 1915, 37, 242. •I.e.

CEREBROSPINAL FLUID AND CHOROID PLEXUS 15

enter chauiiels witliiii the connective tissue of the plexus, but that sucli channels receive the dye with a fluid from another source, i.e., the plasma or lymph.

It is stated that diflferenoes exist in the character of the ventricular and the subarachnoid fluid. Investigations by several authorities also seem to show that there are lymph systems within the cerebral and spinal h-mph system as a whole, independent of one another. D'Abundo,» (niillain,'" Marie and (luillain," Orr.'aud Homen," from the n^sults of injections by various methods, came to practically the same conclusions, namely, that Ij-mph from the general circulation, wliich passes along the perineural Kanphspaces of the periplieral nerves and enters the nervous system, follows paths within the limits of definite territories. Thus bacteria or their toxins, experimentally or pathologically inoculated into peripheral nerves, are carried along the IjTnph-stream to the sj)ine ami brain in an ascending direction, capable of producing lesions in regions of the central nervous system quite remote from the seat of the original infection. All these writers agree iu the statement that the cun-ent of Ijanph ascending the posterior roots and columns is independent from that of the anterolateral columns, (luillain and Marie showed that the lesions in tabes, always limited to the posterior roots and columns, are the result of tlie direct propagation of the syphilitic virus along the perineural hinph-vessels. They go so far as to call tabes a l>^nphangitis of the posterior Ijiiiphatic system of the spine.

• D'.Vbundo, Sullc vie linfatiche del Sistema Nervosa Centrale. .A.nnali di

Nevrologia, 1S90, 14, -'M.

" Guilliiiii. Ln circulation dc la lyniphe dans la moelle (?pinierc. Revue Neurologiquo, 1899, N». 2.'}. 796.

" Marie et Uuillain, Ia's li^sions du systdme lymphatique postc^rieur de la moelle sont I'oriKine du processus anatomo-pathologique du tabes. Revuo NeuroloRiquc, 190:j, U. t'l, 103 and 106.

" Orr, .\ contribution to our knowlcdpoof the course of thelyinph-strcaniin the spinal roots and cord Ucv. Neurol, and I'sychiatr.. Kdinburgh, VMS, 1, tj39.

" Homcn, Die Wirkuntc riniRcr anaoroben Haklericn narnontlich bei Synibiose und aeroben H.aklcrion, sowic ihrer Toxinc auf poriphcre Nerven, Spiualnanglien und da.i lUickoninark. .Vrbeiten aus dem pathologischen Institute der Universitat zu llt-lsingfurs. 190.'>, 1.1.

Ili KAETIIK W. DKWKY

Accept iiiR such division of l>in|jh systems, we do not necessarily d*>dun' the cxistonro in tl>(« brain and spinal cord of conditiniis rntin'ly ('xccptional from those in tht> rest of the body. Apparent deviations from the general laws of anatomy and physiol'>j5>- are not fundamental differences, but variations conditioned by the structural and functional nature of the organs involved. The meningeal spaces arc equivalents of l3Tnph-vessels adopting the fomi most suitably adjusted to the particular requirements of such an organ with such surroundings. Perivascular lymph-sheaths do not only exist in the brain tissue, which is excee<lingly sensitive to variations in the blood-pressure, but are also found in such an unyielding tissue as bone. The cerebrospinal fluid differs from ordinary Ijanph to the same extent that lymph in other parts of the body is modified by the nature of the tissues which it drains. .\s to tlie independence of restricted lymph systems within the central nerv'ous system, parallels can be found in other anatomic units. Sicard, in discussing this question, refers to the independence of the subserous lymphatic network of the stomach from such a subserous network of the duodenum.

Following these lines, we may very well assume that after intravenous and intraperitoneal injections the dye enters the cerebral and spinal membranes anrl the choroidal connective tissue along re^il l>inph-channels, and the endothelial cells lining these channels take up the vital stain as the fluid containing it reaches them, .\fter intracranial and intraspinal injections, the .staining solution follows the paths of th(> ventricular and sul)arachnoid fluid, and in leaving the cranial and spinal cavities is not only taken up by the venous drainage, but is conveyed along true lymph-channels.

The pronounced affinity for vital stains exhibited by the specific connective-tissue cells of the meml)ranes over the olfactory tract and at the exits of the cranial and .spinal nerves probably ci»incides with a difference in the nature and function of these cells. .\t the olfactory lolx-, where this change is especially striking, we have a transition from the subarachnoid spaces to the IjTnph-channels of the na.sal apparatus. Sunilar transitions

CEREBROSPINAL FLUID ANV CHOROID PLEXUS 1 /

we nmy :issuine to exist at the seat of the arachnoid villi, where the piii-aruohnoid channels more or less directly conununicate with the IjTiiph-channels of the dura.

The choroid plexus is, accordiiiK to (loldjnann, a protective limiting nK'jnl)riine filtoring off from the blood-plasma a substance which acts as a violent toxin to the nervous tissue. He also suRRoststhat there may be protective substances inherent in the granules of the ganghon cells in the dog, which he observed to be sometimes vitally stained, while in the rabbit he only found diffuse staining of the protoplasm and the nucleus of such cells. Providing the necessity f)f protection to the nucleus of these nervecells, we should reasonably assume that the ganglion cells throughout would possess and manifest such protective powers, which, however, was not observed. It would be ec|uall}' reasonable to suspect that this is a property common to all cells staining vitally, for it is a characteristic of the phenomenon of vital staining that only the granules take up the dye, while the nucleus remains unstained. On the other hand, we know that diffuse .staining of the nucleus and protoplasm, the indubital^le sign of injury or death of the cell, takes place not only in the cells of the central nervous system, but in those of all organs of the body. It would rather seem that the ability to take up the stain is dependent on definite chemical or physical properties of the constituents of the granules in certain types of cells. These cells have welldefined characteristics in common, whicii they do not share with other cells provided with granules which are refractive to vital stains. Investigations of the morphological and histological occurrence of lipoids tend to show that the cells capable of storing cholesterol are the same which react to vital staining, and in eitliercasetheability is confined to their protoplasmic granules. In my own experiments with cholesterolized rabbits,'* I have observed that cholesterol is stored not oidy in the more or less coarse granules of interstitial cells classified as macrophages, histiocytes, endothelial cells, etc., but also in the exceedingly fine granules of certain epitheUai cells, notably those of the kidney.

'* Dewey, Experimental hypercholesterolemia. Arch. Int. Med., 1916, 17, 757. TUB ANATUUICAL RKCORD, VOL. 15, NO. 1.

IS KAETHE W. DEWEY

In tlit« clioroid plexus tli<^ lipoid .sul)st;inc('.sare perhaps best demi>ii.strate«l by tlic pctlariziiiK Jiiicroscopo. The vessels appear as if invested in an outer silver lining; the doubly refractive substances occur in excet>dingly fine divisions; they are distributed all through the tissue.

The presi'iice of granules within the protoplasm of certain types t)f cells has been associated by some writers with secretory functions. It has als<j been claimed that these granules are surrounded by a lipoid membrane. In the choroid plexus the extracellular droplets of secretion, which are plainly visible in fre.sh or fonnal in-fixed specimens, are manifestly enveloped in such a membrane. Nothing is more striking than Sudan III stain(Ml preparations of choroid from cholesterolized rabbits. The tissue is riddled with orange-red spheres and circles. Also in rabbits not injected with cholesterol, these yellow and orange colored tiroplets jnay be verj' conspicuous Evidently the lipoid of this membrane is present in a form other than that within the cells; it does not appear as doubly refractive or anisotropic substances under the micropolarisoope.

In a recent puI)lieation, Sunclwall describes granular interstitial cells in the choroid plexus of the ox, which because of their morphological and staining characteristics he places in the category of mast cells and which he believes may be concerned in some type of secretion. But for his statements that he did not observe such cells in the choroid plexois of other animals — rabbits, guinea pig. dog, hmnan — I should be inclined to consider them as identical with the vitally staining granular cells which I found, without exception, in the choroid plexus of the rabbit after intravenous and intraperitoneal injections of trypan blue. I also observed them in the few dogs which I examined. Sundwall mentions variations in the number of these cells with different anunals. I have occasionally noticed slight difTercnces, the letwencd number coinciding with the diminished intensity of staining, especially in the study of the dental pulp; it was generally found in aged animals, but the observations are not num " Sumlwnll, Tho choroid plexus with speciul reference to interstitial granular eelli. Anttt. Rec. 1917, 12. 221.

CEREBROSPINAL FLUID AND CHOROID PLEXUS 19

erous enough to warrant the statement that age is responsible for the decrease in the number of the cells.

An assiunod function of whatever nature, inherent in the specifically staining granular cells of the connective tissue within the various organs of the body, gives a peculiar interest to this particular tissue; far from being merely a supporting stroma, this tissue would have all the signilicance of a vital organ. .Such a view is expressed by Renaut"' in the statement that the connective tissue is ' ' like the largest of the glands with an internal secretion which exists in the body of the vertebras, because it keeps the elements ready for inmicdiate action or susceptible to such action at any time, wherever blood-vessels course through the connective tissue." I am inclined to consider these cells as endothelial cells, being in the most intimate relationship to the Ipuphatic ajjparatus and plaj'ing a more important r61e in this system than that of merely lining the lymph-channels.

SUMMARY

Concerning the results of these experinients the following observations are to be particularly eniphasized :

An apparently exceptional behavior towards vital stains is exhibited by the endothelial cells lining the perivascular spaces, or, if the existence of such cells be denied, by specific cells of the perivascular connective tissue within the brain and spinal cord.

Unlike such cells in the perivascular connective tissue in other organs and tissues, they do not habitually take up the vital stain, but do so only under the influence of stimuli from pathological conditions. This lack of affinity for \'ital stains may be due to a (liff(^ronce in specific functions inherent in these cells. DilT(>rences in the behavior of endothelial cells towards the stain are observed with constancy as follows:

1. With reference to blood-vessels, affinity for the vital stain is absent, in general, in the endotheUal cells of the inner lining

'• Reimut, "Les cellules connectives rhagiocrines." Arch, d'aniit. niicroacop., 1907, 9. 495.

20 KAETHE W. DEWEY

of artorios, veins, and capillarios; prosont o) in the rapillarios and vonuK's of thf spleen, the capillaries of the bono-niarrow and the blood sinuses of the hemal glands, i.e., in tissues the source of blood and lymph elements, b) in the Kupffcr cells of the liver, i.e.. in cells of which it is not yet absolutely certain whether they belong to the capillar}' wall proper or to the perivascular lymphspaces.

2. With reference to hinph-channcls, affinity for the vital stain is absent in general in the endothelial cells lining the inner wall of lymph-vessels outside the organs; present in lymph-channels within the organs except the brain and spinal cord.

3. With reference to the central nervous system, affinity for the vital stain is absent in general, in the perivascular spaces within the brain and spinal cord; present in these conditionally and in focalized distribution in the presence of pathological stimuli, in general, within the membranes along channels conveying l}7nph or cerebrospinal fluid.

CKHKHHOSIMNAI- II, I II) AM) CUOUOIU PLEXUS

i;.\l'I,\N A TKiN 1)1 1 KilKKS

FiR. I Dura. (IntruvennuB injcctioim of trypiin l>Iui'; lilood-vcsscis lillcil with cariiiiiu- nfliiliiil Tracts of vitally stainrd rclls niiiniiiK aloiiu thi' lilooilvcssols and also in iiidc|>ciid(>iit directions. MaKniticatiim KKIdiani.

Kin. - I'ia-araclinoid. (Intravenous injections of lithium earniine; hloodvcssols filled \vill\ Herlin lilue gelatin.) Two complexes of vitally stained cells: more or less distinct lines of Krunular colls radiating from those centers. Magnification 100 dtam.

Fig. 3 Choroid plexus. (Intravenous injections of trypan blue; hlood-vossels filled with carmine gelatin.) The niuiiorous vitally stained interstitial cells are <|uitc everdy distrihiitecl; there is no distinct perivascular arrangement Magnification KM) diam.

Fig. 4 Dura. (Injections of trypan Mue into the cislerna magna Kogion of trnnsilion from the hraiii to the olfactory tract. Tracts of vitally stained granular cells closely following the hlood-ve.s.sels. There was a striking lack of vitally stained cells in the dura over the hemispheres. Magnification 1(X) diam.

Fig. 5 Intracerehral lilood-vossol. ( Injection of trypan hlue into the cisterna nuigna.i Perivascular vitally stained granular colls. Magnification iOO diam.

Fig. I) Spinal cord. (Intraspinal injecticui of trypan Idno.) ICnormous accumulation of 'Iviriicheh/.ellen' in a region near the central canal Tracts of slender granular cells converging towards the focus; they follow the course of the lilood-vessels and increase in size as they approach this center. Photomicrograph. Magnification 18.') diam.

Fig. 7 Choroid plexus. Stained with Sudan III and hematoxylin. I^xtracellidar droplets of secretion with a clear center and a lijioid memlirane. Fron\ an apparently nornuil ralil>it. Magnification 100 diam.

Fig. 8 Choroid plexus. From a ralibit injected with cholesterol. Olohular and ring-shaped extracellular droplets of secretion; stained with Sudan III ; the tissue is very faintly stained with hetnatoxylin. Photon icrograph. .Magnification '275 (liatn.

Fig. !) Pia-arachiioid. i Intraspinal injection of try p in Mue. i Diffuse staining (if cellular elements and all the fillers of the cmineclive tissue demonstrating the complex structure of this memlirane.

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A I)i:S('I!IIl"l()\ OF \ CASI': OF FALSK lli:i{.MAlMlliol)l TIS.M

II. K. JORDAN Dcparlment of Anatomy, University of Virginia

ONE FICJURK

.My iiitcrc.'^t in tlii.s (■use wu.s tirsi arimscd Ix'ciiu.so tlic autopsy materials included, besides two abdominal testes, a bilateral pair 111' syninictriciilly placed smnll alxinminal l)otlies suK^e-stive of ii\arian icmnants. These ixxlies i)ro\-ed on microscopical exaniinatimi to 1)(> altered lynii)li-notles, such as are typical for certain inlianiniatory conditions. The nodes are chanvcterized by fil)rosis, hyalinization, and arteriosclerosis. The case is tli<>refore one of masculine pseudohermaphroditisinus, and is oonse(|U<'n11y of relatively lesser importance. However, since tlic inicrosco|)ical data in refjard to the testes have a bearin}? <in I lie alleiied causal relationship l)etw(>en the interstitial cells and the development and maintenance of the male secondary .sexual chnracters (Bouin et .\ncel'), as also on the hypothesis that ' heat ' is dejiendent upon an internal .secretion on the part of the.se cells, a detailed descrijition of these organs seems warranted. In view of the wide ffcncrjili/at ions current . as comjjareil with the relatively meager body of definite data, concerning the function of the interstitial cells, it would seem that any additional relevant microscopical ol)servations should be carefully recordeii.

The subject was a patient of Dr. ('. S. I'nderhill, of Mc( iahe.\ s\ ille. \'a.. who first directed my attention totheca.se. The aiilo])s\- was performed on October .'^l by the coroner of Hockiiridge ('ounty. Dr. .1. .M. Hiedl(>r, of Ilai'i'isonbui'g, \ a. The

' Hoiiiii el .\iiitI. I'.MU. lici'hcri'ln'.s siir Wi .•iiuniliciiioii |>hy.iici|ci)(i(|iii> ilo la kIhiuIc intor.sliticllc lUi tcslii-uU- dcs iiijimtiiif^rcs. .lour. <ir plivsiol. ct do piith. Kon., T. 0, i>. lOIJ.

•js

II. !■:. .IDKD.W

iiitrriial and rxtcriiul R«>iiitiilia were pn'sci\cil in 10 per ci-iit fi)riiialiii. I am ^;|•(•ally iii(l('l)t<'il to Unth <>( tlicsc gent l( mm for their interest in this case and fur kindly sending me wellfixed tissues and full ailtojjsy notes.

Dr. I'liderliill's notes ijielii<le the fnllowinjj pertinent data: 'I'he siihjoet was thirty years of as*', white, of p)od i)hysiop;nomy. with narrow forehead, hi^h eheek-l)ones, and oidy a thin growth of hair on top of head. He had supernumerary fingers and toes, the foiH' additional digits being accessory to eacii i>l' llie fiftli

Kin 1 I'liiit<iKra|>h of iiui.'triiliiic fiilsc hrrmaphroditc, witli |>olydiictylisin and tali|i08 varus ( Kiiidiics.s of Or. (' I) riidcrliill.)

nioinhers. Hoih fe<'t were chibbed (taHpes varus). The mammary ghmds were well developed, the penis was rudimentary, the .scrotum was vestigial, and the hands, hips, thighs, and legs were of female conformation. 'I'here wa.s much fat everywhere; all organs were covered and infiltrated with fat; the subcutaneous fat was 4 inches thick. Opposite the internal ring on each side was a ma.ss having the 'feel of ovary or testicle." Two wnde.sceiide<l te.sticles were removed from a .sort of caii.il cxtern.il to the internal ring, from which the finger could i)e pushed down into a 'pouch of skin where the scrotum should have been."

CASE OF FAI.8E HERMAPHRODITISM 29

On subsequent incjuiry tlio following additional facts were ascertained: The subject was 'very feeble-minded,' left-handed, unmarried, with no tendency to 'venery.' His parents were 'about third cousins.' His mother had a severe case of heat prostration at the age of 15 to 17; she was in poor health when married at 22. She has been married twice; her one child, a daughter, by her first hust):ind is 'alive and well.' She had three chiklrcn by her second husband; one died in infancy, and a feeble-minded daughter, sister of the subject, is still living; 'she is able to work about the house.' The parents deny any abnormalities; they do not have supernumerary digits.

The specimen of the external genitalia shows only a .short penis, of 2.5 cm. length. The internal, or liidden, portion of the penis has a length f)f 9.5 cm. Taken as a whole, this organ is therefore about two-thirds average length, and not actually, as apparently on external view, 'rudimentary.' The scrotal region is marked by a small area of pigmented, loose, greatly wrinkled skin.

The bodies originally suspected of being ovaries were enca.sed in an envelope of fat, about 6 mm. thick. The larger measured 3.5 X 2 X 0.5 cm. ; the smaller, 2 x 1.25 x 0.5. The general shape was flattened reni^)rm. The central core of the larger body, apparently of connective-tissue structure, was triangular in transverse section, measuring 8 mm. from base to apex and 5 mm. along the base; the latter was indente<l by a hilus.

The central cores onlj' of both of the.se bodies were embedded in paraffin, sectioned at 5 microns, and stained with iron-hematoxylin followed by van Gieson's stain. The preservation was superb. The bodies are unmistakably lymph-nodes, in which tile reticular tissue has increased enormously. .Approximately half the volume of these nodes consists of dense hyalinized reticulum. .\t first sight this appears homogeneous, but closer inspection reveals a fine fibrillar constitution. The larger denser areas occur in the cortex, apparently at the centers of the original nodules, and along the medullary cords. The reticulum of the denser masses shades o(T into the loo.se reticulum of the medullary siiuises. The lymphocytes are well preserved and appear normal.

go <"ASK OK FA USE HEUMAPHRODITISM

The tosticlcs have a flattniod oval form, roundly triangular in transection. Thoy differ Rreatly in size: the larger measures 5 X 2.5 X 1 cm.; the smaller, 2 x 1.5 x 1 cm. Microscopical examination shows that they ar(> atrophic, similar in appearance to undescended testes, but with the usual degenerative phenomena greatly accentuated, the degree of abnormality corresponding with the size difference. The sections were stained with the iron-hematoxylin van Cieson's combination. The tunica albuginea is greatly thickened. The testes are characterized in general also b.y fibrosis and arteriosclerosis.

Seminiferous tubules are still distinguishable in certain regions of th«' larger testis, but their tunica propria is greatly thickened and converted into a fibrous hyaline wall. Throughout this wall nuclei of variable form, size, and staining capacity are sparsely scattered. The nuclei are relatively small; they have an oval, bilobetl or irregularly lobed form, suggestive of amitotic division. The larger oval nuclei are generally vesicular; the smaller and more irregular fonns are usually pyknotic. The perinuclear region, corresponding to the cj'toplasmic area, appears clear and homogeneous, the tissue as a whole thus resembling somewhat fibrocartilage. The surfaces of the wall have become sinuous or corrugated through contraction. The thickness of the wall varies as the diameter of the cross-sections, the smaller tubules generally having the more robust w-alls. In certain regions of the larger testis the tubules have collapsed and become solidified into cords of hyaline material.

In the smaller testis practically all of the tubules have become solidifietl. These denser, tubular, areas are separated by narrow areas of hyalinized fibrous tissue with few cells containing mostly pyknotic irregular nuclei, and an occasional small degenerating interstitial cell. All of the intratubular lining cells have disappeared in the smaller testis.

The p.'itent tubules of the larger testis contain a peripheral layer of irregularly c<ilumnar or polyhedral cells, one to three layers thick. It seems most probable that the lining epithelium wjus originally single-layere<l and that this became 'stratified' thnjugli a dislocation of certain cells following the shrinkage of

CASE OF FALSE HERMAPHRODITISM 31

the tubules during the process of hyalinization of their walls. Certain of the smaller tubules are filled with cells arranged in the form of a reticulum. The reticulated appearance is due largely to a vacuolization of the cytoplasm. Certain cells contain a single large spherical vacuole, the nucleus having been pushed to one pol(>, giving these cells the appearance of fat cells.

The nuclei of the less degenerate cells are relatively large, oval, and vesicular, and contain a delicate reticulum; those of the more extensivelj' vacuolated and irregular cells appear collapsed and pyknotic. In the larger tubules an occasional cell may be found of spheroidal shape with large vesicular imcleus suggesting a spermogonium. Besides these very infrequent cells, the lining cells include no elements which resemble typical spermog<jnia or spermocytes; they are most probabh' largely remnants of Sertoli cells and possibly in very small part of prunordial germ cells. Between the tubules occurs a coarsened fii)rous connective tissue with numerous small, irregular, more generally p\knotic, nuclei. Relatively few scattered interstitial cells are discernible.

The chief point of interest in the case centers on these interstitial cells, considered in relation to the mixture of secondary sexual characters ami the absence of sexual desire in the subject. The interstitial cells were identified by comparison with those of a normal testis, the spermogenesis of which was previously described.- Compared with those of the normal testis, the interstitial cells of this hermaphrodite are very few, much smaller, approximately half the nonnal size, and the cytoplasmic content of lipoid granules, instead of being scattertnl. is grouped into one or several large clunips. The cells could be studied best in unstained preparations. Thanks to their brownish-j-ellow granules they could be easily detected. They are widely scattered; they ha\e a spheroitlal or polyhedral shape; certain have a large, oval, vesicular nucleus, but the majority contain only a small, frequently irregular and pyknotic, nucleus.

• Jordan, H. E., 1014. The spermatoKcncsis of the mongoose; and a further

comparative study of mammalian spcrmatof^enosis. with special reference to sex chromosoines. I'lil). 182, Curnonio Institution of Washington, p. 16.'J.

82 H- E- JORDAN

T1h« niicrospiipical evidence shows, then, that these testes contauiotl oriKinnlly l><>th the intra- and intertubular cellular elements characteristic of normal fetal male gonads, but that both liad sufTcnKl a gradual dcficnoration, probably in part patiiological, until only modified intratubular, chiefly sustentacular, cells and degenerate interstitial cells remained throughout the hyaiinizcil stroma. The persistence of even a few recognizable interstitial cells under the conditions which obtained in these testes, even in the more atrophic of the two, indicates a most remarkable vitality and suggests an important r61e on the part of these cells.

The case under consideration appears to be one of arrested male development. All things considered this pseudohermaphrodite is very decidedly more male than female. The female secondary sexual characters are superimposed on a male substructure. And it may be emphasized that the 'female' characters are not infantile; witness the well-developed mammary glands. Nor are they wholly dependent upon the considerable adiposis. For some reason certain female secondary sexual characters have gained formal expressicm in this essentially male individual. The underlying proximate cause is most probably the atrophy of the testes. The ultimate cause would seem to have been one conmion to the hernuiphroditism and the concomitant physical (supernumerary digits, club-foot) and functional (left -handedness; feeble-mindtHlnessj anomalies. In respect to this congeries of anomalies, presumably dependent upon a common germinal factor or disturljance. the ca.se is practically a duplicate of one previously described,* where a very similar pseudohermaphrodite was one of nine children, all of whom showed various anomalies, including polydactylism and gigantism, and adds another to a large and varia!)le group of pseudohermaphroditic malformations coincident with other minor anomalies.

Thus the 'rudimentary' penis, vestigial scrotum, and cryptorchism appear to be different aspects of a general anomalous condition. The case may be conceived to have developed as

' Jordan, H. E., 1912. Studies in human heredity. Bull. Philosophical Society, Univ. of Vir((inia, Scientific Series, vol. 1, no. 12, p. 293.

CASE OF FALSE HERMAPHRODITISM 33

follows: The atrophy of the testes, and the suppression of the scrotum, supervened upon the inguinal retention of the testes; the development of certain female secondary sexual characters resulted from the atrophy. The effect of the original cryptorchism may have been modified by pathologic factors, for undescended testes commonly have an increased number of interstitial cells coincident with, and reciprocal to, a decrease in the number and activity of the intratubular .spermogenic cells. It would be of interest to know in this connection also the condition of the hypoi)h3'sis. but this organ was not removed at autopsy.

The well-attested lack of .sexual desire in connection with the paucity and abnormality of the interstitial cells is perhaps the most important phenomenon pertaining to this case. That the control of the male secondary sexual characters is not dependent upon the interstitial cells of the testis seems proved by this case. where a mixture of secondary sexual characters is maintained in the substantial absence of healthy interstitial cells. Such conclusion must be drawn also from the various castration and spaying experiments where certain secondary sexual characters, generalh' reciprocal, originate and persist in the absence of interstitial cells. The same conclusion is indicated also by conditions in certain turtles which lack secondary sexual characters and where the male nevertheless possesses abundant interstitial cells during the spring, for a period probably corresponding with the breeding season.

The hypothesis that 'heat' is dependent upon the integrity of the interstitial cells seems to rest upon a tinner basis of observational data. The case describetl by \Miitehead,^ of a stallion with a third abdominal testis, seems conclusive on this point. The whole ma.ss of data relating to infertile hybrids (e.g., mules) and cryptorchid individuals supports the .same conclusion. For here heat remains in the absence of potencj', correlattMl with a numerical increase of interstitial cells and an ab ' Whitehead. 1{. II , lilOS. .\ |HTuIi:ir case of cryptorchism. and its hearing upon tln' prohliMM of Ihi- fuiictiun of the interstitial cells of the testes. Anat. Rec, vol. 2. p 177.

THE ANATOMICAL RBTORD. VOL. IS. NO. I

34 H. E. JORDAN

sence of complete spennogenic activity. The hermaphrodite here descriho«i adds similar e\'idonce, though of negative character. Here heat was absent, a condition correlated with few interstitial cells and these of degenerate character.

The sufficiency of this latter hypothesis, as well as the now more generally disearded earlier one of Bouin and Ancel, is controvert etl by Boring and Pearl' on the basis of their results from a microscopic study of the testes of chickens, in individuals of which of over six months they claim interstitial cells are whollj' lacking though of 'full sexual norraalitj- both in respect of primary and secondarj' characters' (p. 205), These results, however, are at variance with those obtained bj' the recent careful work of Reeves,* who reports interstitial cells in the testes of chickens at all ages up to eighteen months.

The coincidence of left-handedness in this case with various anomalies (polydactylism, rudimentary and atrophic genitalia) supplies additional interest. In my studies of hereditary lefthanilednes" I have been led to adopfthe hypothesis that the condition follows a fetal structural variation in the cerebral bloodsupply, of a nature to effect a better nutrition of the right cerebral hemisphere in contrast to the usual condition where the left hemisphere is better supplied. An examination of the fetal vascular system suggests that the arrangement of the common carotids, iimominate and subclavian arteries is such as to favor the po.ssibility of a larger blood supply to the left cerebral hemisphere and the right arm, thus presumably determining a bias towartls right-handedness. If this morphologic explanation of right-handedness and left-handedness is correct, then it is significant that also left-handedness should occur in this case where various other anomalies are associated with hermaphroditism.

In view of these data the conclusion is suggested that a condition of pseudohennaphroditism is of the same category as the

• Boring, Alice M., and Pesirl, Raymond. 1917. Sex Studies. IX. Interstitial cells in the reproductive organs of the chicken. Anat. Rec, vol. 13, p. 253.

• Reeves. T, P., 191.5. On the presence of interstitial cells in the chickens'

teates. Anat Ric , vol. 9. p. 3.S3.

'Jordan, H E, 1914. Hereditary left-handedness, with a note on twinning. Journal of Genetics, vol. 4, p. 68.

CASE OF FALSE HERMAPHRODITISM 35

common anonialies of other systems. The accompanying functional (listurl^ances in this case, expressed in loss of sex potency and suhsidcnre of 'desire,' resulted from the induced atrophy foIl(»sving the inguinal retention and proljablj' later disease of the testes. Whether subsidence or accentuation of heat follows cryptorf'hisiii seems to depend upon whether the resulting testicular atrophy involves also the interstitial glandular cells or only the seminal epithelium.

AtTTHon n ADATRArr nr tmipi PAreR irhitkd

BT TIIK UlllLIOUItiPHlC AKItVlCe. JULT 19

THE HISTOLOGY OF LYMPH, WITH SPECIAL REFERENCE TO PLATELETS

H. E. JORDAN Department of Anatomy, University of Virginia

The primary object of this investigation is to determine whether blood-platelets occur in lymph. The material employed consists of stained smear preparations of lymph from the thoracic duct of the dog, treated according to Wright's method for blood smears. The desirability for further studies on the structure of Ij'mph arises by reason of the discrepancies in the results reported by recent investigators. Schafer ('12) makes the unqualified statement that "both lymph and chyle contain thrombocytes" (p. 394). Davis and Carlson ('09) in their study of the cell-content of lymph, make no mention of platelets, permitting the inference that they did not see these elements in lymph. Vinci and Chistoni ('10), on the basis of careful microscopic studies of the lymph, of dog, cat, and rabbit, conclude that lymph lacks platelets (p. 209). Howell ('14) comes to the conclusion that "platelets do not occur in lymph from the thoracic duct of the dog."

The following general facts would lead one to expect to find platelets in lymph: 1. Platelets play an important part in the clotting of blood "by liberating an additional supply of thromboplastic substance and of prothrombin " (Howell, '14). Lymph, in common with blood, contains fibrinogen and thrombin, and under similarly favorable conditions clots promptly. Since thrombus formation in blood is apparently lurgcly dcpoiulent upon the presence of platelets, the latter would be exin-cted to occur also in lymph, which has a similar physical and chemical constitution and clots like blood under similar conditions. 2. With the possible exception of platelets, lymph contains the same cellular elements, in vastly ditTerent proportions, as blood. If any bar 37

38 H. E. JORDAN

ritT l)ot\v(»on the hlood-vusruhir ;iiul tlu- lymph-vascular systems can bo passed by all the other cellular elements of the blood, even the erytliropiastids, it seems reasonable to suppose that platelets also would not be excluded from lymph. 3. The origin of blood-plat elels from segmenting pseudopods, and from fragmenting larger areas of cytoplasm, of a certain type of megakaryocyte in the red bone-marrow is well established nVright, '10; Bunting, '09; Downey, '13; Jordan, '18). .Since the platelets pass readily into blood-capillaries of the marrow, no plausible explanation presents itself as to why they are excluded from the IjTnphatics of the marrow.

The recent work of Howell ('14) is the most thoroughgoing that has yet been devoted to the question of platelets in lymph. It is requirefl, therefore, that this work be first carefully analyzed, with a v\ow to determining whether any possible contingency remains which has not been fully met in this search for the possibly very elusive platelets in lymph. Such a contingency would serve as the only excuse for any addition to an otherwise apparently conclusive research.

Howell's investigation of platelets in lymph is part of a larger research relating to the mechanism of coagulation in blood and l>anph. He obtainofl the lymph of his experiments from dogs anesthetized with morphia and ether, by placing a vaselined cannula into the thoracic duct. The rate of flow was variable, but averaged about 0..') cc. per minute. The lymph from dogs starved for forty-eight hours clotted in from ten to twenty minutes. The more milky lymph from fed animals clotted in from thirty to sixty minutes in the case of earlier specimens; Ij^mph collected after an hour or two only clotted after from one to tliree hours. Howell searched for platelets both in the fresh and in oxalate<l Ij'mph of these experiments. He examined also the corpuscular sediment obtained by centrifugalization of oxalated lymi)li, but neither in the deposit from a first nor in that of a second centrifugalization could he discover platelets, though the same Tnethod applied to blood-plasma revealed them in great abundance. Howell C14) concludes that "platelets do not constitute a normal element in the lymph."

HISTOLOGY OF LYMPH-PLATELETS 39

Acoordinp; to Howoll, clottinp; involves the cr)6poration of four constituents: 1, fibrinogen, in solution in plasma and lymph; 2, prothrombin, liberated by platelets and by lymphocytes; 3, antithrombin, present in lymph and in bl()()d-i)lasina in substantially equal amounts, and 4, thromljoplastin, liberated by platelets and lymphocytes and tissue cells in general, and operating to neutralize the antithrombin. Pure lymph uncontaminated with tissu(> juices is said to coagulate more or less imperfectly compared with manmialian blood, l)Ut it coagulated promptly and (irmly if tissue extract is added to it. Howell explains the inferior clotting of lymph as due to the comparative paucity of thromboplastic material, the result of the absence of the less stable platelets and the consequent restriction of its source to the relatively more stable lymphocytes. This explanation is in accord with the earlier cimclusion of ^'inei and Chistnni f'lO) that coagulation can occur in the complete absence of platelets. These authors concluded, moreover, that "the principal morphological blood element intimatelj' related to the phenomenon of coagulation is the white corpuscle" (p. 212).

In view of the facts that general considerations relating to the comparative microscopic structure of IjTuph and blood, and to the mechanism of clot formation as fornmlated by Howell, seemed to favor the probability that lymph contains platelets as expressly claimed bj' Schiifer ('12), it occurred to me that failure on the part of Howell, and of Vinci and Chistoni, to discover t"he alleged elements might be due to some unfavorable factor in their technical i^rocedure with reference to platelets. It is well known that the leucocytes in the blood-vessels tend to collect peripherally. .Vlso the extreme adhesiveness of the platelets and leucocytes in general is obvious. It seemed possible that the slow flow of the lymph (0.5 cc. per minute, Howell) might permit the elimination of platelets from specimens collected by caiinula from the thoracic duct, by reason of their adhesion to the wall of the duct. A final link in the chain of apparently complete evidence respecting the absence of platelets in lymph as given by Howell, seems to demand an ex:miination of lymph in the form of specially preparetl smears made directly from the thoracic duct.

40 H. K. JORDAN

Such hypothotical doinaiul fiirnishod tho roasnn for the present investiRntioii and suggested the special technic employed.

This investigation includes lymph smears from two different dogs prepared at an interval of several weeks. Control specimens of blood were made from these same animals. The technical procedure, for assistance in which I am greatly indebted to Dr. J. A. Waddel, professor of Pharmacology, at the University of \'irginia, was as follows: The first dog was quickly killed with ether. The thoracic and abdominal cavities were immediately opened, and a segment of the thoracic duct tied off and excised. The segment was thoroughly rinsed in physiologic salt solution at body temperature, to cleanse it of any possibly adherent blood. \ cut was promptly made into this .segment of thoracic duct and the lymph content allowed to flow onto slides. The lymph had a clear, watery appearance. Subsequently the segment was cut along the entire length and slides were smeared from the inner surface of the duct. The slides were at once treated according to Wright's staining technic. Control blood smears, stained according to the same method at the same time, showed abundant platelets. But the lymph smears apparently lacked platelets completely.

I was at first surprised to find erythroplastids among the cellular content of the lymph. I suspected that their presence was the result of the death struggle, they having possibly been sucked back from the subclavian vein. However, if their presence was tf) be accounted for on this basis, typical platelets should occur for the same reason. 1 fiiul that Howell, and Davis and Carlson, also record the presence of a certain small number of red corpuscles in lymph. In the second experiment almost immediate death was caused by administration of chloroform by mouth, with the idea of obviating any possible reverse flow from the subclavian vein at death. But the preparations from this specimen also showed the presence of a few red corpuscles. Accordingly, we may conclude, I believe, that erythroplastids are a normal constituent of lymph. Nor did any of this second series of slides reveal typical platelets.

HISTOLOGY OF LYMPH-PLATELETS 41

The ((uestioii arises as to how the presence of the erythroplastids in lymph is to be interpreted. Davis and Carlson ('09) only regard the presence of crythroplastids as due to blood admixture (p. 16). In accord with the numophyletic theory of bloodcell origin, such red corpuscles might represent differentiations of certain lymphocytes, serving as hemoblasts, in their slow passage through the lymphatic tree. However, if this conclusion were correct, one should probablj* expect to find also erythroblast stages; but such could not be detected in my slides. It seems perhaps more reasonable to conclude that the few erythroplastids present in lymph have their origin in the red marrow drained by certain perivascular lymphatic terminals of the system. This conculsion, however, requires that a consistent answer be given to the question as to whv platelets, which theoretically could enter the system in the same way, cannot be found in lymph from the thoracic duct. It seems altogether probable that platelets do actually enter the marrow lymphatics in small numbers. The lymph of the thoracic duct contains elements generally liberated by platelets, namely, prothrombin and the thromboplastin (Howell), but in relatively smaller amounts than found in blood-plasma. Platelets are notoriously unstable structures. In their relativeh* slow passage (compared with the blood circulation) through the lymphatics to the thoracic duct, the relatively small number that may have entered the system may have entirely disintegrated, meanwhile contriliuting the relatively small amount of prothrombin and thromboplastin characteristic of lymph; to which the lymphocytes under favorable conditions may contribute a certain additional amount, as suggested by Howell. Considering the origin of the hulk of the lymph from the plasma via the tissue spaces, and the continual disintegration of blood-platelets, the presence of these elements (thrombin and thromboplastin) in lymph can be accounted for at least in part on the basis of this relationship between blood plasma and l\'mph. Similarly with respect to the thromboplastin of the ti.ssue cells.

Starling ('15) inclines to regard blood-platelets as precipitation products in plasma, following contact with foreign bodies or the lowering of its temjjerature from 37°C. to 18° (p. 837). He

42 H. E. JORDAN

baws his opinion on llie observations, following Huckmaster: 1. When a film of blood, held in a platinum loop and kept at body tenip<'raturo, is examined under the microscope, no platelets can l>e seen: on cooling, platelets make their appearance. 2. AMien noncoagulablf plasma, e.g., peptone plasma and oxalate plasma, is kept for twenty-four hours at 0°C., after removal of all formed elements by centrifugalization, a precipitate forms 'indistinguishal)le from blood-platelets.'

A\'ith regard to the first observation it may be justly urged that the fact that the platelets are not microscopically visible in the jilatinum-loop preparations at body teniperatur(> is not conclusive proof that such are not actually present. The re^ fractive index of the platelet protoplasm at body temperature may be so close to that of blood-plasma as to render the platelets practically invisible; on cooling, the refractive index of the platelets may change to a point where they become visible against the background of the fluid plasma.

In opposition to the deductions from the second set of observations, stand the results of the present investigation, namely, that in the smear preparations (essentially of lymph which was allowed to cool and thus suffered coagulation and precipitation of certain proteids) certain platelet-like bodies formetl which at first appeared indistinguishable from genuine blood-platelets* However, a careful study, as described in detail below, showed that these bodies are quite different structures, essentially compound precipitation products. Moreover, considering the chemical and physical similarity between blood-plasma and lymph, and in large part their conmion origin, it .seems strange that genuine platelets should precipitate only from the former and not from the latter. And in addition, the fact should again be empha.sized that the original results of Wright ('10) regarding the megakaryocyte origin of platelets have been confirmed by at least three subsetiuent independent workers (Bunting, Downey, Jordan) and his conclusions may therefore confidently be regarded as firmly established. Continued skepticism regarding the genesis of platelets as first described by ^\'right can only rest, it seems to me, on a failure to repeat in detail Wright's technic in the study of red marrow.

HISTOLOGY OF LY^fPH-PLATELETS 43

In view of Starling's interpretation of platelets as precipitation products, it becomes important to give in detail the steps by which I r«>:ichcd the conclusion, on the basis of my microscopic preparations, that the lymph from the thoracic duct of the dog lacks platelets completely. The slides were made a year ago and were at once carefully studied. Numerous structures were seen which sinudated platelets. The possibility seemed to present itiself tiiut these botlies were modified or disintegrating platelets. The investigation was temporarily abandoned with the idea that the material at hand did not permit of definite conchisions. I subsequently again studied the slides, and again arri\-ed at a point of uncertaintj'. A third, more intensive effort, has brought me to certain and definite conclusions: Platelets do not occur in lymph of the thoracic duct; the half-dozen bodies in my preixirations regarding which some douljt may remain do not materially alter the precision and generality of this conclusion; various and numerous precipitation products appear which simulate platelets, but which are clearly of different origin and of compound structure. This material accordingly jields additional ami significant evidence for the disproof of the conclusion that platelet-like bodies in peptone and oxalate plasmas are genuine platelets.

The stained smear preparations of lymph have a background of a homogeneous or ver\- finely granular vacuolat^'d substratum, ranging in color, depending upon a varying density, from a faint pinkish-blue to a light lilac color. Among the spherical vacuoles may occasionally be seen small spheroidal or oval areas of more condensed substratum. Throughout this substratum are scattered the lyinph cells. These are mucii more abundant in the specimens from the first dog, a difference depending, as Howell has shown, upon nutritive conditions, that is, whether the animal was st-arved or recently fetl. The small l>Tnphocytes predominate, forming at least 9() per cent of the entire cell-content. Large lymphocytes are also relatively abundant; similarly large mononuclear leukocytes with reiiiform lilac-colored nucleus, ami a wider shell of faintlj- pink cytoplasm. Occasional neutrophils, eosinophils, and erythroplastids also occur, probablj- of marrow

44 H. E. JOHDAN

uiiil tissue-space origin. Occasional endothelial cells, detached from the duct wall in the process of making the smears, also occur. The large mononuclear leukocytes are in the vast majority of cases distorte<l or fragmented. The.se cells are eviilently much more adhesive and fragile, both in respect to cytoplasm and nucleus, than the small lymphocytes. In the close vicinity of such broken leucocytes clumps of filirin-fibrils occur, suggesting that the.se cells are more favorable regions for thrombus formation.

Scattered throughout the substratum are also innumerable spheroidal and bacillary lilac-colored metachromatic granules, most probably proteid in chemical nature. Such frequently collect in small groups and may thus simulate platelets. The resemblance is especially close where several such granules have collected within one of the minute oval coagula above described. It is only when constant reference is made to the platelets of the control blood-preparations that precise dififerences are established. In this work, whenever such a platelet-like body was encountered, reference was immediately made to the blood smears. The following are the essential criteria finally established for definitely distinguishing between such platelet-simulacra in lymph and genuine platelets of blood: The blood-platelets, like their sinmhicra in lymph, vary greatly in size; the size extremes of platelets are in the proportion of at least 1 to 10 volumes; but true platelets are very frequently collected into groups; their Ij-mph simulacra are generally scattered and never in large groups. The peripheral hyaline layer of blood-platelets is very pale blue, the central granules violet, after Wright's stain; in the lymph simulacra the correspcjnding parts are pinkish-blue or light lilac, with deep lilac or violet granules. Moreover, the granules in the case of the lymph structures are coarser and less regular (spheroidal and bacillary) than those of true platelets.

The inference ma\' perhaps be justly drawn on the basis of these comparative observations on Ijiuph and blood, with respect to platelets, that the so-called 'platelets' of peptone and oxalate plasmas, on which is founded the precipitate-explanation of platelets (Huckmaster; Starling), are homologous with the platelet-sinmlacra of lymph. The evidence seems conclusive that

HISTOLOGY OF LYMPH-PLATELETS 45

platelets are preformed structural elements of blood — not precipitated products — and that they do not form a normal constituent of lynipli of the thoracic duct. This may mean only that the possiblj' small quota contributed to the peripheral portion of the lymphatic tree in relation to red bone-marrow, from which the relatively smaller amount of prothrombin and thromboplasfic substance of lymph may have a partial source, suffered disinte^riition during their relatively slow progress towards the thoracic duct, and accordingly do consequently not appear as structural elements among the blood-cell content of thoracicduct lymph.

LITERATURE CITED

Bdnting, C. II. 1909 Blood platelet and megakaryocyte reactions in the rabbit. Jour. Exp. Med., vol. II, p. 541.

Davis, B. F., and Carlson, A. J. 1909 Contributions to the physiologj- of lymph. IX. Notes on the leucocytes in the neck lymph, thoracic lymph, and Mood of normal dogs. .Am. Jour. Physiol., vol. 25, p. 173.

Downey, H. 1913 The origin of blood-platelets. Folia Haemotologica. Bd. 15, p. 25.

Howell, W. H. 1914 The coagulation of lymph. .\m. .lour. Physiol., vol. 35, p. 483.

JoKUA.N, 11. E. 1918 A contribution to the problems concerning the origin, structure, genetic relationships, and function of the giant cells of hemopoietic and osteolytic foci. .\m. Jour. Anat., vol. 24, p. 225.

Sfn.\FEH, E. A. 1912 Text-book of microscopic anatomy. (Quain's Anatomy, vol. 2, pt. I, p. 394. 1 Longmans, (irecn & Co.. London.

.SxAHLlNfi, E. II. 1915 Principles of human physiology. Lea & Febiger, Phila., p. 838.

ViNi'i, Ci., ET Chistoni, .\. 1910 Plaquettes et coagulation (Contribution k I'l'tude do la coagulation du sang). .\rch. ital. dc biol., vol. 53. p. 206.

Wrioiit, J. II. 1910 The histogenesis of bluod-platelcts. Jour. Morph, vol. 21, p. 263.

ADTBOn B AOaTRACr OP TUlfl PAPER USOBO BT TUE illULIoaitAPIllCflBRVlCE, AUOtJBT 15

A DIFFERENTIAL INJECTION MASS FOR USE WITH STEREOROENTGENOGRAMS

WILLIAM SNOW MILLER Anatomical Laboratory University of Wisconsin

In studying the distribution of the blood-vessels within the lung and their relation to the bronchial tree, there are certain disadvantages connected with wax corrosions, celloidin corrosions and the clearing method of Spalteholz which can be overcome by special injection masses used in connection with stereoroentgenograms.

Wax con-osions are very brittle and easily broken; moreover, they do not show clearly the relation of the blood-vessels and bronchi to any variation in the lobation of the lung.

Celloidin corrosions are less brittle than wax corrosions and, when once they are mounted in their proper preservative, they are more permanent; but they also fail to retain the relationship of blood-vessels and bronchi to variations in lobation.

Spaltoholz's method shows the lobation and its variations perfectly, but the blood-vessels and bronchi often fonn a confusing mass.

To offset these disadvantages, I have made use of the following method which shows variations in lobation, the distribution of the main arterial and venous trunks, and their relation to the bronchial tree.

The pulmonary artery is first injected with a starch mass in which vermilion gramiles are held in suspensibn; the puhnonary veins are next iujocteil with a starch niass in which ultramarineblue granules are held in suspension, ^\^len the injection masses have set the lungs are distended with air through a cannula and tube which is tightlj' tied in the trachea and a stereoroontgenogram taken. WTien this is viewed in the stereoscope it will be

47

48 WILLIAM K. ALLEN

fourul tliat the mass in the arti-ry gives a uniform dense shadow ; the mass in the vein gives a finely granular and less dense shadow ; the hroiu'hi distended with air have their walls well defined.

The use of starch masses for x-ray photographs is not new; but used in the manner I have indicated, by which a differential density is obtained, it is, so far as I know, new. The injection mass is |)repared l)v mixing cornstarch and the pigment with 70 per cent alcohol until the re<iuired consistancj' is obtained. If only the large vessels be desired, a mass that will flow with a pressure of 100 mm. of Hg. will give the desired results; but if the finer vessels are to be injected, the mass nmst be diluted with 70 per cent alcohol and the pressure increased.

No set rule can be given for the preparation of the injection ma.s.ses, for I ha\e found that no two lots give just the same results. Each new purchase requires one or two experimental trials before the best results are obtained. This is especially true of ultnunarine blue, and in preparing an injection mass with this pigment a smaller f|uantity should be used than is the case with vermilion.

AUTHOR 8 AUSTHACT OF TniS PAPKR IBSUBDDTTUB DIDUOQRAPUIC aiBVICI

SURFACE VIEW OF INJECTED IXTE.STIXAL VILLI

WILLIAM F. ALLEN

Department of Anatomy of lh<: University of Oregon Medical School, Portland,

Oregon

ONE FIUURE

The figure is from a sjnall portion of a class microscopical demonstration which the writer prepared to illustrate the bloodvascular supply of a rabbit's intestine. It also shows much more conclusively than microscopical sections how the absorption surface of the intestine is enomiously increased through the mucosa's being thrown up into numerous villi. When this preparation is examined with a binocular microscope the villi appear like numerous mountain peaks arising from the floor of the intestine.

This demonstration was prepared by first removing the blood from the intestine of a living rabbit by forcing a normal salt solution through the blood-vessels from the mesenteric vein. This was followed by an injection of chrome-yellow gelatin injecting mass, prepared after a fonnula given in a previous paper fProc. Wash. Acad. Sci., 1905), which easily passes through the most minute capillaries. This injection was followed by a second injection of a cannine injection mass (formula given in above-mentioned pajjor) thickened sufficiently with cornstarch to prevent its passing through the capillaries of the villi. When this injection was completed the various mesenteric vessels were iigatetl and a j)ortion of the intestine was removed and placed in cold water to solidify the injectictn masses. From whence it was transferred to a 10 per cent .solution of formalin to fix. Small pieces of the intestine were then dehydrated, cle.ared in cedar oil, and mounted on a slide with djvnunar so that their inner walls and villi faced the cover-glass.

TU ANATOSIICAL RBCORD, VOL. \i, NO. 1

50 WILLIAM F. ALLEN

The writer takes pleasure in expressing his acknowledgments to one of his students, Mr. R. G. Young, for his skill in reproducing a small portion of this preparation in a drawing for the accompanying illustration ffig. 1).

It will be seen from figure 1 that two moderately large submucosa vessels (S.A. and S.V.) sujiply the immediate neighborhood with numerous branches {s.a. and s.v.). These vessels are Ukewise confined to the submucosa, but supply each villus with an arterial and a venous branch {V.A. and V.V.), which follow up opposite sides of a villus to its apex, within the connective-tissue layer close to the epithelium. In this preparation the viUi arteries are fully as superficial as the corresponding veins. Each villus artery and vein sends off numerous branches to either side, which break up umnediatcly into a capillary network in the tunica propria close to the basement membrane. This capillary network is considerably finer at the apex of a villus than at its base. Sometimes a number of these capillaries form a continuous vessel (v.a.) which follows the long axis of a villus close to the basement membrane. It should not be confused with the main villus artery or vein.

IXJECTKI) INTKSTINAI, VIM.I

'A

5-^ s.v.

EXI'LAXATIOX OK KHITRK

The figure is from a drawiup of a cleared luicniscopiciil preparation of a small portion of a rabbit's intestine, which had its arteries and capillaries filled with a yellow injecting mass and its veins with a red mass, looking at the inner surface. If any criticism is to be made of the drawing it is that the villi are a little too far apart. The arteries are colored red and the veins blue.

M.S'il., mucosa capillary network .i.e., branch of submucosa vein

S.A., submucosa artery I'. .4., Villus artery

s.a., branch of subnuicosa artery I'.a., small villus vessel

■S.V'., submucosa vein V. V., villus vein

At'TIIOR M AIWTR^CT OK THIS PAPEK l«BVKU IIY Tin: IIIKI.IUfJKAPillC HEItVKX

AN IXEXPENSIVP: .MlCltOSCOPIC" PROJECTION' ANO

hi; \\\ INC Ai'i'Ai; A rus

WILLIAM l'. \I.M;.\

Deparlmenl of Anatomy <>/ (Ac Cniversilij of Oregon Meilirnl Sclinnl, Purtlttinl,

Oregon

ONE FIOURK

This appjiratus, as sliow n hy tli(> fip;ure, consists of tho Bausch it Loinh ■■Siiiiiilc (Irawiiifz: (Miuipinciit" supported l>y a stami and a soeoiul stand lioldiiifj an ad.iusial)l(' dra\vin}i;-i)i)ard. The outfit serves every iiMiuii'cnicnt lor rapid and offifiont work, notwithstanding its total cost, cxclusiNc of the niici'os('o|)<>. was alxMit forty dollars.

'i'ho stand lor supi)ortiny: 15ausch iV Loinli's "Simple drawing ciiuipnicnl" has a wooden top (.1) '2'2 \ 14 inches, ahout 4 feet 3 inclies from tiie floor and is supported hy four legs constructed out of old gas pipe. 'I'hese were hent outward at the ba.se and strengthened Ki inches up by cro.ss pieces. On top of the .stand will be .seen the Bausch it Lomb "Simph' (h-awing e(iuipment," which, including i)risni in front of th(> (\v(>)iiece for the microscope, is listed in catalogue at 83"J.o(l. As shown l)y tlie figure, the rheostat (R) is placed at the extreme left, next, in center, is a .small arc light and conden.ser (L), elevated 4 inches by a wooden stool. At the extreme right a microscope is clami)ed on the adjustai)le microscopical stage and bent at right angles so the prism (/•*) extends over the center of the drawing-board. It is an easy matter to ele\ate or lower the microscope by the adjustable micro.scopical .stand .so that the conilen.ser of the microscope (D is in liirect line with the light condenser (L.).

The framework for the adjustable drawing stand was al.so constructed out of old gas jiipe. Its four legs w(>re bent outward at ba.se and aliout ti inch(>s from the floor were l)ound together

53

WII.I.IAM K. \i,i,i;.\

\t\ cross j)ioc('s of the saiin' iiiiitcrial. As will Ix' seen fmiii tlic (ifjiin'. uprJKlits '.\ feel Id inclics were screwed iiitn the re;ir ami (wo siile U'Rs und were cnniieclcd ;il liic tup hy cioss jjieces to ni\(' solidity. Tn proxidc ;iiii[)lc rnimi t'oi- drawing ami (■(Mitciiiiii the iiiianc oil the draw inn-l)oanl the two side lejjs and their uprights were placed 2 inches nearer- I lie hind le^ than the front lep. 'I'lie drawing hoard iD) is of 1 incli eedar, 2() x 21 inches. and is attached to <'ach of the three uprights by a metal daiui) (N), which allows for clainiiinfi; the drawinf^-hoard firmly at any tiesired distance frmn the eyepiece |)risiii iPw so that tli<> size of the imafje c;m be regulated from an inch in diameter to the total width of the draw iiifj-board. The total amount of moxcnient n|)ward or dowiiwai'd of the di'aw in^;-board is 'A feet 10 inches and the adjustable ]nicroscoi)ical stantl cm l)e raised about (i inches, j^ivin^ a total distance of al)out 4 feet 4 inches for projection of the imaKc.

If a greater diameter of the ima^e is de.sired, the adjustal)le ilrawinn stand and tlie prism (/') may lie discardetl and the ima^e can be |)rojected on a \(!rtical surface for da.ss u.se or drawing a chart. I'or class demonstration the imago can ho projected on frround ghi'^'* to advantage.

For low magiiifieations it is best to remove the microscope condenser ((') i'lui de|)end entirely on the lantern condenser, or a third ct.ndenser placed before the mieroscop.' condenser will bring .'dxiut the s.imc results.

I'in- I i" friiiii 11 |ili(>ti>Kr:i|ili iif the iiiii'rd.scopic'il priijnctiii)! .iml drawitin apparntii.s. .4. staiiil fur liiiliiiiiK HaiHch ,V l.iinih's simple (lr!i« inn cmiipiiKMil , (', iiiirr<>srii|H- rotiilcnscr, It. :iiljii--litlil(' ili:i«iii)j-l"'ar(l . /,. small aic lidlil ami cimiIiioit: /'. prism iivrr i-vcpiiTc; If, rliiMislal; .V. i-laiiip for Imliliri); ailjiislalilr >lrauiiiK-l">ar<l.

I'HO.IKCTION AM) DKWVIM; A I'l' ARATCJS

.JO

AUTIIOltM MlMTHAl.'r urTlltl PAPftllt INHI^tH liV TlIK UlKt IDimil-lIK: nKRVirK, HKI^KUUCn Iti

A i)riM.i(\'i'i()\ ()i: liHAXCHixc OF Tin: xkiijai.

CAXAL

IIKXKV KUD.MANX UAUASCII

LaboratiiricH of the Daniel liniigh Inxtilulc iif Anatniiiy of tin- JcjTcrson Metlirnl

TWELVE FIfiCRES

111 ;t provinus article' concrrninpc the lesions pnxlufed hy (^ieetricity as ()l)serve(l aft<'r le}>;al electnteutioii. the writer called attention to a diiplicatiou (tf the neural canal in one of the cases studied (Case 111, C. (!.).

This structural peculiarity was first noted in examininjr sections at the level of the motor decussation, and upon careful study it was found at the lev<(l of the .sensor decussation also. The remaininfj; unstained sections also were examined, and tho.se that would fiive any assistance in the interjjretation of this condition were .stained and mounted. Of these the best and most characteristic were photographed. The accompanying illustrations represent only the canal area, and they have been so arranfi;ed that the top represents dorsal and the bottom ventral directions. Hitiht and left represent the san\e re.spective directions as far as can be determined.

Tlu^ order in which the illustrations ha\"e been placed .s<>ems to be the proper one according to the characteristics of the rest of each section. The first ei<iht illustrations represent this peculiarity at the motor decussation level and the last four illustrations represent the condition at the sensor decussation level. The order of the se(|Uence here may s(>em odd, perhai)s. but ;in exanunation of the superficial arcuate nucleus and fibers m.aterially assists in the arranjjement , as progressive enlargement of these structures is readily noted. Throughout all of the seclions the canal is much larger than s;>(>nis usual .'itid the direction of the long iixis varies at the two levels here described.

' Aiiiriioan .loiirn.nl of tho Mcilical .'^ricnces. Sciiti-iiil)rr. 1!UJ.

THE ANATOMICAL RECORn. Vol.. l.V NO 2. MErTKMHIlR, I91S

58

The first (Mght s<'ctitms \v(m-«' i)li()t()frr;ipli<'<l witli a Ki-mm. ()hjtH-tiv<« uiid SI 10 X ocular, inakiiiji; a luaj^iiilicutioii of al)oiit 12'> times. Ill the romainiiif; sections the canal was larger so that the 7.5 X ocular was used with the Iti-iiiiii. nhjective giving a nuiRiiification of about !K) tunes.

Figure 1. In this section the canal shows a |»'culiar Ixilge, but the ependyiiKi! liniiiy; is distincl and iinl)rnki'ii i linniiiliinil . though cilia are not dcinoiistrable. The long axis of tiu> canal is transversely jilaced and the iiiuuediate neuroglia is rather dense and devoid of nuclei. This seems to be the lowest section of the series.

Figure 2. In this section the canal is more extensive, laterally, than in figure 1, but less so dorso vent rally. Tlu' ependyiiial cells are clear and distinct, but cilia are absent. The neuroglia in the ijmn(Mliate area of the canal is dense and contains very few nuclei.

It is difficult to say which section comes next. If it is figure 3, then the canal has branched and the branches are at about their gT-eat<'st distance apart and are about to approach each other. If figure 4 comes fii-st, then the left canal (/) represents the direct contiimation of the canal of tiic spinal cmd wliil(> the right one (r) represents a blind branch.

P'igure 3. This is the section in which the double condition was first noticed. Here are two distinct canals, the larger of which measui-es about 112^ by (5.'^ and the .smaller 63^ liy 43^. They are about 224 microns apart. The ependymal cells are clear and distinct, but devoid of cilia. In the neuroglia imjuediately surrounding each canal there are many nuclei, whil<! in the central part of the area between them the neuroglia seems ([uite dense.

Kig. 1 level. FiK ■-' Kin -i I'iK I Vig 't

Thp ranal of tho oblonuata :it lower portion of the motor decussation

Tho cuiial of thi' oMonnatii iit n sliuhtly InKli'^r level. The (liiplicntion of the canal. L. left; U. rijsht. the two ennalH closer to each other, rhe curved canal. L, left limb; ){, rinlit linil). Fid. ft The canal at a hinher level (reverse picture) L, left limb; H, riRht limb; a, branch from left limb.

UHAXCHINC OF NEtrtAL CANAL

59

-A.- *

\.

4

X

,

'^l-

V ■■

a

4

FlKUro 1. In llii> section the twn canals arc rather lariie and closo logctluM'. TIk^ cpondyinul colls aic clcai', ilistinct. ami complete «>\(>i\\\\ hci<'. 'rii(> luiclcaf zon<' aroiiiul cacli canal is not so well inaikcd. If this section really precedes (ifiure 'i. then the caiuils are really diverginfi and one will end iilindly. The writer, however, believes that they are conxcriiinfj ;ind t'orni the ptnniliflr canal .seen in fifjure .5.

Iij>;ure .">. In this section the canal i- licnl ;ind the eix'ndym.al is clear except at the toji of the left linii): here it shows a tendency to send cells into the filial tissu<> as thoiifiii an extension of the canal were indicated. This Ix-comes delinite in higher sections.

Figure (i. This illustration is a reverse picture, so that the parts have Ixmmi labeled as they should be and not as they ap])ear. At this level the left limb (/) shows a tendency to extend the canal dor.salh' (as this liml) points dne dorsally). i'lie right linib is unclianged. From the left limb, howev<>r, a branch (a) has de\'eloped and is quite cxtensi\-e. The ei)eudymal cells are dear and distinct throughout except in /. where an extension of the canal is forniing.

Figure 7. In this section the right limb of the canal is still unchanged. The branch («) is less distinct and it appears as though it will soon be closed i>f!. The left limb shows a welldeveloped extension of cells at h; this outgrowth is ahuo.st as extensive as the canal. These cells seem to r(>present a bud that will become hollow and constitute another branch.

Fig. 7 The canal near the upper part of the motor ileciissation level. L, left liiiil>; H. ri({hl limb; n. branch; h. cell cord extension from left limb.

FiH- s The canal at the upper part of the motor deciissalion level. I/, left limb; It, rijchl limb; «, branch tending to occlusion; h. accessory canal.

Fin. i) Till- oblonxatal canal at the lower pari of the sensor decussation level. I), dorsal; \', ventral; a, dorsal extension of ependymal cells.

Fig. 10 The canal at a slightly higher level. D, dorsal; V, ventral n, accessory canal; b, f-shaped group of connecting cells.

Fig. U The canal al a slightly higher level. I), dorsal; \'. veiitrul; «, accessory cnnal; h, f-shaped group of conned ing cells.

Fig. 12 The oblongata! canal at the upper part of the sensor decu.ssation level. D, dorsal; V, ventral; n, accessory canal.

UI{A\(HI\<; OK NKfHAI. CANAI,

I'.I

^'•*«.^« .« BtfA. •

■P'TJ'^

r '^'^ '

II '

^^KS^I ^^^B

M

_b

V

n

l'"inun« S. Ill this section tlic ]\ix}\\ limb is as usual. The hraiicli a swms to lie closing. Tho loft limb (/) has afiain distinct bouii(lari(>s, but a part has bo(Mi soparafcd at I). This rc|)rcsciits the biidileil cells of tli(> ])rece(linji illustration, and fhrso have now formed a little canal: in all i)i(il)ability this is a little blind diverticulum. Wheth(>r the iiranch b is a i)liiid canal or joiiLs the main canal is imi)ossil)l(> (o saj', ;us no other .sections at higher levels show it at all.

The succeeding sections are all at the sensor decussation le\-el. and althoiitih the arrantrement may seem odd it coiifoi-ms to the internal structure of the (il)!on^ata at this ]e\'el.

Figure !). In tliis section tiie neural canal is ajjain single. It is much larger than in the preceding sections even though the magnification is only 90 times. Its long axis is dorsoventrally directed and is peculiarly bent. The ependymal cells are clear and distinct except at the dorsal tip of the canal wli(^r(» they seem to be fonning a dorsally directed bud.

Figiu-e 10. In this section the canal is .seen to be douljle. It seems as though the cells noted in the preceding section had formed (|uite a dorsal extension. These when hollowed out fonn another canal shown here at n. This canal is not coml)letely free from the main canal, as at !> there is the conn<>cting group of cells. The ependymal cells of the main c.in.il arc distinct and clear except at the dor.sal area where the acces.sorv canal is connected.

Figure 11. This .section shows a main canal -till extensive; its cpend^nnal is distinct except at the dorsal area. The accessory canal at a is much smaller than in the preceding illustration and its epeiidyma is not distinct. It would seem .as though there was a tendencj' to occlusion. At /' tliere is a ["-shaped cord of cells that extends from the left side of the dorsal boundary of the main canal over and then down to the dorsal bouiuhuy of tlie accessory canal. These cells probablj^ represent some of the general mass of buckled cells from which the acces.sory canal is deriv(Ml.

Figure 1'-'. In this section the main canal is large and shows a tendency to branch at the lower right-hand corner. The

HHANCHIN(i OK XKIHAL ( A.NAI. (W

('I>rMi(lym;il cells ('\-<'ry\vh<'r(> arc clciir and ilistitict. The accfssdiy canal is larscr llian in the prcccdiiif^ sc'ctii)n and it is well sepamtcd from the main canal. This accessory curia! soems to he a hiind tlivcrticulum that arises at the lower part of the sensor ilecussation le\<>I and extends toward the mid-olivary level. It seeii\s to !)(> constricted and pn>l)al)ly does not reunite with tile main ( aii.il at all. Imi < nds either blindly or l»y becoming |)art of I lie liiiirili ventricle as it (the branch) extend^ to liinher levels.

AUTlltJilH Ah«TltA<T UK THIS TAt'EH IWtTCIi lir TIIK IIIIII.IOfJKAI'HIC HKHVICK, AVOVn 7

()\ Tin; i'i{()ri:ss OF TMSAiMMiAirwcK or III).;

COM S AIMlilJlOSlS IX TKLEOSTS'

UlLULU V. .SMITH Thi- Dipfirliiirnt of Anatomy, Tulane Univerxitij <»/ Loiti.iiann

SIXTEEN KKiUKES

C'onsidoralilo ilifUculty lias hcfii ciicountcrod in tlio classificatioii <if (ishos and their ditToront genera, and especially in drawintl a distincf line between the (l.-uioids and Teleosts.

( i<>'!;enl)aur ('(ili) found in the ( ianoids aiul in a limited number ot Teleo.sts, interralated between the ventricle of the heart and the truncus arteriosus, two segments clearly distinguishable from one another. Of these segments, the caudal is calleil the conns arteriosus or bulbus cordis, while the cranial is called the l)ull)us arteriosus.

The cunus arteriosus is a well-marked muscular structure, furnislied with numerous valves, and is one of tlu; characteristics uf ih(> Elasmobranchs and (ianoids. Teleosts, with the excej)linn of a very limitiMl nujuber, have no conus.

It was onee thought that the rudimentary conus, or its total ab.sence. with l)ut one tier of valves, was characteristic of the Teleost heart. Stannius in liis researches ('54) found that Albula vulpes possessed two tiers ()f valves instc^ad of one. Likewise .S'liior ('()7) found the sanu^ true of Megaloi)s cyprinoides. Pterothri.ssus gissu, and Tarpon atlanticus. .Vmia calva has the shortest conus and a fewer number of valves than any other (Janoid. Some of lh(> Teleosts seem to have descended from a stem somewhat akin to Amia, notably the iierring group. .Sime of I hi fnmihcs of this group, for instance, Albula vulpes, Pterothrissus gi.ssu, .Magaloi)s cvi)rinoides. and Tarpon atlanticus have ahiKist as hirge a comis as .Vmia itself, but with two rows

' .\I:il«'ri!il ilsrd iliicfly finiii the I'nivcrsily and Ili'Ili'Viir lli>s|iil:il Mi'ilicjil Srliool oiilli'ctiiiii.

GG wii.m u t . sMiiii

i)f vnlvos instojid of thnH\ Other Hourly iclaiccl ffcncra luivo a distinct oomis with niily (Hic row of valves.

Hoas ("SO), in his diagram of tho Teleost hoart, shows the valves to arise from fibrous tissue. This is (Troneous, since it has .since been found that all conns valves of Teleosts have tlwir ba.ses attached to nui.scle with some mu-scle fibers probably extending into the cusps. Boas' diagram for the Teleost repro•scnts the condition found in both Teleosts and Ganoids.

Favaro ('10) states that, from the point of view nf compaj-ative anatomy and embryologj', we are not obliged to recognize in the conus and bulbus arteriosus autonomous and distinct organs, such as are observed in single orders and species, but rather structures which in part correspond to one another. Thereby he contradicts Clegenbaur's statement that the conus and bulbus arteriosus are separate ami distinct .segments. However, when (me considers that Favaro had in mind only the part derived from vascular endothelium and ( iegenbaur that derived from splanchnic mesoderm, l)()th are correct. The intima and media of tlH> bulbus are derived from embryonic vascular endotheliujn and are homologous with the intuna of th(! coiuis and general vascular apparatus, (iegenbaur said that th(> conus difi'<'i-('<l fri.ni the luillnis in thai the greater ])art of its thickness consists of heart nuiscle. This is (juite true.

Hcjyer ('00) advanced the theory that in Teleosts the conus had disappeared by intussusception into the ventricle.

I have examined serial sections of the hearts of the following fishes: Etnuiieus teres, ("oilia nasus, Clupea harengus, ("tengraulis my.sticetes, Engraulis mordax, Clupanodon coeruleus, Stolephorus compressus, Harengula macropthalma, Chanos chano.s, Opisthonema thrissa, Poniolol)Us i)seu<lnhareugus, Doro.soma cepcdiaiuun, Clupanodon lacepede, Xotopti^rus, Pantodon, MormjTUs caballus, Alepocephalus agassizii, Osteoglossuni bicin-hosujn, Mesopus pretiosus, Thpnallus signifer, Salmo irideus. ('luj)ea alosji, Oncorhynchus chouicha, Osmerus mordax, Balh\lagus benedict i, Nolemigonus chrysoleucus, I"]lojis .sjiiu-us, C'hrosomus erythrogaster, Chirocentrus dorab. Tarpon atlanticus, Hiodon tergisus, C'ampostoma anomalum, Albula

DISAl'I-KAKANCK OK TIIK CD.VrS AKTKKIOStS (17

vulix's, I'tciois vdhiiis, I'lotosus anguillaris, Cantheriiies sjiiulw icliriisis, li:ili>t;ii)iis iiinliilut lis. Oslnicioii fornutuiii, ChiK"todmi uctifer, ( 'lijictodoii trifiiscijita, Hctcrotis iiiloticus, Opliioc'('i)liiilus strialiis, Ilippocaiiipus atterimus, SyiiKathus p<'l('aKi<"Us (iastrotokciis l)iaciiloalus, Aiiiia calva. PdIvimIkh spatliula. ami L(>pi( lost CMS plastnstdJIUIs.

'rii(> specimen of Ileterotis iiilotirus and OsteoKlossuiu hicirHiosuiii jn(>asiire(l 4(1 cin. in leiijitli. Tlie others ranged from () to 20 cm. The ilhistratioiis as here re|)ro(hiced arc not all of the sjune magnification. It was con.sidereil most convenient for comparison to employ un\' magnification necessary to distinctly show the conns, when present, and the attachment of its valves, "^rhe (ip;ur(>s are arranged in the order of what s(H>med to be the projiressiv<' disappearance of the conus arteriosus as found in specimens here studied.

I take this opportunity to thank Professor Senior, of The University and Hellevue Hospital Medical Collef^e, for the privilege of using material in the collection of this institution, and I sincerely ili.iiik Professor iJutli, of the University of Manila. Professor .Ionian, of T.eland Stanford University, and Mr. l!atlit)un, of the United Stales Xalional MuM-um. f()r their kindness in .sending me a numl)er of the sp(>cimens emnnerated.

1 tind tliat tlie hearts of Tarpon atlanticus, Osteoglossum l>icinlio.>um, ( )piiiocephalus striatus, Chaetodon aetifer. Coilia nasus. ("hanos clianos, .\li)ula vulpes. Notopterus, Plotosus anguiliari--. Dorosojiui cepedianum, and Ihierotis niloticus, all show a distinct muscular conus. Figure I illustrates such a conns in Dorosoma cepedianum and figun- 2 the similar condition in Ileterotis niloticus. It was deoned unnecessary to .submit illustrations of the remaining specimens mentioned since the coiuis in th(>m was of similar appearance and structure.

The hearts of Jialistapus undulatus. Pterois volaus. Stoh^ phorus c(inipr<'ssus, Pojnolobus jistnidoharengus, Elops sjiurus. ( lupanodon co(>rul(>u.s. Harengula macrothalma. (^i)isthon«>ma thri.s.sa, Kngraulis mordax. .Mepocephalus aga.ssizii. and Utengraulis mysticetes show a rudimentary conus of elastic tissue including a small amount of .scattered cardiac muscle. Those are shown in figun>s M. 4. .>, (5, 7, and S. respect ivt>Iy.

/^\

f

•^i rt*

^-f m^'

•1 — ■_

r-^A

r__^:.

-^^.-•_.

fri— " ■ ^

/A

-V

KXPLANATIOX <1K TIIK FKJt'KKS

The figures arc all drawn to incliKlc only llu- cephalic ends of the hearts of the specimens named. The sections from which the drawings were made were selected from serial sections of tlie hearts and. as is evident, many of the sections pa.ss ol)li<|nely through the cephalic ends. The figures rcpn'sent hearts of specimens as follows:

I Doro.iotna eepedianiim H Clupanodon coerlueus

'2 Hcterolis nilolicus 4 llariMiKula macropthalma

G.S

.") CtciiKiiiulis inystii'otcs Kii)craiilis iiiiirdax 7 Opisthonoiiia Ihrissii N .Mcpoccplialu.s aKassi/.ii !l Morniyrus cnballtis 10 Cliipoa harrnEiis

B A. bulhiis arteriosus

II KathylaKUs ln'iicdii-ti I'J SyiiKalhus pclcaKifUs \:i lliiiiliin tcTKiiOis II llippix-aiiipiis attcritntis I.) ('hirorcnlrus <|iiral> l(i raiitodoii

RKFERKNCE LETTKKS

C A, conus arteriosus. V. ventricle 69

70 WIIJUH C. SMITH

III llic iciiiaiiiiiiji iiiiiiilicr of 'I'doosts cxjimiiicil, I he (•(Hiiis scH'iMs to have (lisai)i)('arc(l. The Kioator muiiher of tlicin ai)i)(>ar to liav<' lost it l)y intussusception (telescoping) into the ventricle. In (igure 0. Moniiyrus caballus, the valves are seen to be attached Id iiitussusceptetl conus muscle, caudal to the cephalic (Mid of the he;uM, whih' on th(> other hand, in some that have lost their conus, one finds the conus \alves not to be drawn into the ventricle, as in figure 9 but to be attached to its most cephalic end, as in figure 10, Clupoa harengus. In the hearts of Bathylagus benedicti.Syngathus peleagicus, and Hiodon tergisus, figures 11. 12. and 13, resp(>ctiv(;ly, the cusps are solely bulbar in their attachment and show no evidence of being drawn into the ventricle nor any direct strui-tin'al rehition with it. In Hippocampus atterimus antl Ciiirocentrus dorab the vah^es are merely attached to the bulbus and to the aortic end of the \-entricle with al.so no evidence of being drawn into the latter, while in the heart of Pantodon both cusps are attached to the caudal end of the bulbus, which, on the left side, is drawn into the ventricle.

I beliexe that Hoyer's statement that in Teleosts the conus has been lost by intussusception or recession into the ventricle applies to mo.st species of Teleosts, but, on the other hand, it seems to me that in those hearts one .should find the conus valves caudal to the af)rtic end of the heai-t, with their bases attached to the receded conus muscle. In the figiu'es showing no conus and no drawing in of the valves, and in those showing the valves to have only bulbar attachment with no intussusception, it appears to me that in a limited number of Teleosts the conus is not intussuscepted into the ventricle, but is taken up by the caudal elongation of the i)ull)us.

The pheiiomeiirtn of the disappearance of the conus arteriosus without iiitiissuscejjtion of the valve cusps into the ventricle, as nt)ted, may possibly be explained i)hysiologically. One may consider that, on ventricular conti'action when the blood is forced into the conus and bulbus. if during the succ(>e(ling contraction of the bulbus, the greater resistance offered to the passage of the blood through the l)ranchial ves.sels nece.ssitates a

DISAI'PKAHANCK ol THE CONUS ARTKHIOSUS 71

greater pressure ami a hyixTlmpliy <<( tli<- i)iill»ii>. llim there would result a stroiiRer eaiulal reKurgitation of the blood against till' cusps of tlie eoiuis vah'es. Tliis caudal regurgitation might lead to a caudal compression and conseciuent obliteration of the eonus.

I desire to express jny aiijjreciation to Professors H. D. Senior and Irving Hardesty for their aid and >uggestions offered me in the preparation of this paper.

T.ITKHATfRK riTKI")

Rdas, J. v.. \' . IS.SO Cher doii C'oiiims arteriosus l)ci liutirinu.s unci bei andern

Kiicicliciifisfhen. Morph. .lalirlj.. Bil. (i. p. 527. KwAHo. (i. 1011 Cono <■ l)ull)o Artcroso negli anamni. Monitore Zoologico

Italiano. Anno. I'J. p. 121. Gegenb.wr, C. 1866 Zur vcrRl. .Vnatomic lies Hcrzcns. Jenaischc Zcit sciirift, Bd. 2, p. 365. HoYER, H. 1900 Bulletin international de I'acadetiiie des Sciences de Cracovie,

no. 7, p. 263. .I<ini>.\.\ .\.\D KvKH.\i.\.N Bulletin of the United States National Museum no. 47. MiKLLER, J. 1846 (ibcr don Bau unil die Orenzen der Cianoidcn. Berlin.

1846. Senior. II. D. 1907 The conus arteriosus in Tarpon atlanticus. Biological

Bulletin, vol. 12, no. 6.

1907 Teleosts with a conus arteriosus having more than one row of

valves. Anat. Rec. vol. 1, no. 4, pages 83-84.

1907 Note on the conus arteriosus of Megalops cyprinoides. Biological Bulletin, vol. 12. no. 6. Stannu'.s 1846 Bernierkuiigen liber das V'erhiiltnis der Ganoiden zu den Clu pciden, insbcsondere zu Butirinus. Rostock, 1846.

AUTIIOn's AftflTR\CT OF Tlim PAPKR tiflUSD BT THE BIBUOOBjLPBIC BBIIVICI, AVQVWft 7

THE DEVELOPMENT OF THE inTOPHYSTS OF THE

AN UK A

WAYNE JASON ATWELL Department of Anatomy, Univcrtity of Michigan

KIOHTEEN FICUBES

Rocont studies have shown that the epithelial portion of the hypophysis consists of three parts. It has been clearly demonstrated that these three parts are distinct both ontogenetically and histolofiirally. Besides the anterior lobe proper and the pars intermedia previouslj- recognized, Tilney ('13) has shown that a third epithelial part, the 'pars tuberalis' is to be distinguished in mammals and in birds. He gives a brief account of its development in the cat and in the chick. Woerdeman ('14) tre^its of early stages in the development of an homologous part, the 'lobulus bifurcatus,' in reptiles, birds, and mammals. Baumgartner ('16) traces the development of the 'pars tuberalis' in reptiles. Parker ('17) has described its ontogeny in the Marsupialia. In a recent paper ('18) the wTiter has given a detailed account of the development of the hj7)oph}-sis of the rabbit and has followed the differentiation of the three epithelial parts until the tijue of birth.

The most striking feature in the development of the pars tiiberalis is its paired origin. This is noted by all of the authors above enumerated. In the rabbit the pars tulieralis is discernible very early. From the thickened epitheliiun which lies in front of the early Rathke's pocket two thickened ridges are fonned. These are the anlagen which fuse anil form the 'pars tuijcralis' — a thin lamina surrounding the infundibular neck and spreading out under the tuber cinereum.

The pars tuberalis is in many fonns considerably more va.scular than is the pars intennedia. It is further characterizeil by the tubular or acinar arrangenxent of its cells. Tilney

73

ANATOHICAI. RSCOao, VOL. IS, NO. 2

74 WAYNE JASON ATWELL

states that tlio walls of these acini are composed of one or two layers of colls, while Parker and Atwell speak of them as being composed of one layer only.

A\'(H'rdeman ('14) draws an interesting homology between the pars tuberalis (which he terms the 'lobulus bifurcatus' following Bolk) and the inferior sacs of the Elasmobranch fishes, which, as is well known, have a paijed origin. Woerdonian lacked material showing the development of the hypophysis in the higher fishes and in the Amphibia. On this account the WTiter felt that such jm homology must be considered precarious until the ontogeny of the hypophysis had been stutlied for these remaining vertebrate classes (Atwell, '18).

The present study was undertaken for the j^urpose of ascertaining whether a homologuo of the pars tuberalis is to be recognized in the amphibian h3'pophysis. In answering this question it was hoped that light would be shed on the question as to whethir a lobe comparable to the pars tuberalis is constantly present in the hypophysis of all vertebrates.

THE DEVELOPMENT OF THE HYPOPHYSIS IN THE AM KA

The material for this study con.sists of some eighty series of .sections of lanae of Rana pipiens, which range ii\ length from 2 to 25 nun. This wa-s augmented by the preparation of series of hirval Rana clamitans obtained just preceding and during metamorphosis. For the hypophj'sis of the adult frog, specimens of R. pipiens and of R. catesbiana wore u.sed. For purposes of comparison se\eral series of larvae of a toad (Bufo americana) were also prepared.

A series of wax-plate reconstructions was made from typical hirval and adult stages to illustrate the morj^hogenesis of the hypophysis. The hypophysis of the adult frog was first studied in the gross and sketched at a low magnification, using the camera lucida. .\fter sectioning, the same structures were identified and stutlied under higher powers. Oraphic reconstructions were made and compared with the sketches obtained in the gross.

IIVF'OPHYSIS OF THK AiVURA 75

It is not tho purpose of this study to treat at length the early appc.aranco of tho hy|)ophysis. This has boon done for the Amphibia by (ioette ('7')), Orr ('89), Hallcr ('97), and Corning ('99). Of the largo niunbor of series of sections studied only certain t3'pical stages have been selected for description hero.

3-mm. larva of Raria pipicns. At this stage tho anlage of the hypophysis is already well formed. The ectoderm at the anterior end of the embryo is separated into two well-defined layers. It is from the inner of these that the hypoi^hysis arises. ■ In sagittal sections the hypophysial anlage shows us a wetlgeshapcd mass of cells lying between the neural tube and the wall of the foregut. The apex of the wedge is directed caudully. There is usually considerable separation between the two laj-ers of ectoderm at tho base of tho wedge. The appearance given is that an attempt had been made at evaginating to form an hypophysial pocket.

.V transverse .section from near the anterior end of the hypophysis is presented in figure 1. It is seen that this part of the anlage shows a distinct bilaterality. Reconstructions of the entire hypophysis at this stage show that this bilateralitj* is confined to tho anterior portion of the gland. Po.ssibilitios as to the significance of these appearances are discussed on a later page.

7-mm. larva of R. pipieun. \ sagittal section of a 7-mm. larva is shown in figure .3. The oral plate is intact. The hypophysis, hyp., extends caudally from its attachment to the inner layer of tho ectoderm. It does not reach to the caudal end of the thin-walled infundibulum, inf. .V reconstruction, from sagittal sections (fig. 5) shows that the hypophysis maintains its attacluuent to the ectoderm by a relatively broad band. Just caudal to tho stalk the hypopliysis is widened and .shows a p;ur of thinner lateral shelves. It is bolievotl that the.se give ri.se to the more definite lateral lobes of later stages.

S-nnn. larva of R. pipien.'< (fig. ti). The hypophysis has recent Ij' lo.st its attachment to the ectoilerm. It lies flattened out under the floor of the infundibulum juul is now somewhat witler from side to side than nasocaudaliv. Its caudal end is now ahnost

76

WAYNE JASON ATWELL

«'Voii with tho caiKl.'il (>xtroiiuty of the infundihiilimi. Tho rather sudden breaking loose of the hj-popliysis from the eetoderm has resulted in a marked change in the shape of the gland. Before (fig. 5) it was much longer in a nasocaudal direction. Now (fig. 6)

'V?;5

^^

Fig. 1 Transverse section of anterior end of hypophysis of a 3-inm. larva of Rana pipiens, showing bilaterality of hypophysi.s iinlage; hyp., hypophysis; b.w., briiin wall. X 100.

Fig. 2 Transverse section of hypophysis region of a 12-mm. larva of R. pipiens. l.l., lateral lobes; ant. I., anterior lobe proper; inf.. infundibulum. X 100.

it is spread out from side to side. This rearrangement is a very disturbing factor in any attempt to follow the lateral lobes. They cannot be distinguished with certainty at this stage.

12-mm. larva of R. pipicih<;. A transverse section from an embryo of this length is shown in figure 2, a siigittal section in

HYPOPHYSIS OF THE ANURA

77

Fig. 3 Midsagittal section of head end of a 7-mni. larva of R. pipicns. n.c, notochord; inf., infundibulum; f.g., foregut; hyp., hypophysis; or. pi., oral plate. X 75.

Fig. 4 Midsagittal section of hypophysis region of a I2-nun. larva of R. pipicns. ani. I., anterior lobe proper; p. int., pars intermedia; inf., infundibulum; nc, notochord. X 150.

78

WAYNE JASON ATWELL

p. int.

. p. Int

Tif^. 5 Reconstruction of hypophysis of a 7-inm. larva of R. pipona, viewed from the dorsal surface, caudal end at the bottom, l.l., lateral lobe; cut., entoderm; eet., ectoderm; st., epithelial stalk. X 100.

Fig. 6 Reconstruction of the hypophysis of an 8-mm. larva of R. pipiens, viewed from the ventral surface; caudal end is below, hyp., hypophysis; b.w., brain wall. X 100.

HYPOPHYSIS OK THE AVURA 79

figure 4, and a dorsal view of a wax model of the epithelial hypophysis ill fiRiire 7. It will he seen that the hypophysis has its eiiudal end in close proximity to the anterior end of the notochord. The latter, in earlier stages, touchctl the dorsocaudal wall of the infundibulum, causing it to be indented ( fig. '.i). The hypophysis is already differentiated into tiu"ee epithelial portions, one of which is paired. The anterior lobe proper is the thickened central portion of the gland. At its caudal end it is bounded by a bulging transverse ridge, the pars intermeilia. P'rom tlie sides a pair of thinner shelves or ledges (figs. 2 and 7) extend; these are the lateral lobes, the paired anlagen of the pars tuberalis.

18-mm. and 20-mm. larvae of R. pipiens. In these and all subsequent stages the morphological differentiation of the three epithelial portions is very distinct. In figures 8 and 9 models of these stages are \iewetl from the ventral surface, with the caudal end below. The anlagen of the pars tuberaUs are visible as a pair of buds (l.l.) located at each side of the anterior end of the hji^ophysis. At the caudal end of the gland a curving transverse ridge, marked off by a groove, is the pars intermedia. This ridge is considerably longer than the width of the remainder of the gland.

A thickening at the caudal end of the infundibulum, corresponding to the extent of the pars intermedia, is the beginning of the neural lobe. It lies just dorsal to the pars intermedia and so is not visible in a ventral view of the reconstruction.

Fig. 7 Ucconstruction of the epithelial hypophysis of a 12-inm. larva of R. pipiens, viewed from the dorsal surface, l.l., lateral lobe; p. int., pars intermedia; ant. I., anterior lobe proper. X 100.

Fig. 8 Reconstruction of the hypophysis of an l.S-inni. larva of R. pipiena, from ventral surface; caudal end below, ant. I., anterior lobe proper; p. int., pars intermedia; /. /., lateral lobe; b.w., brain wall. X 100.

Fig. 9 Model of hypophysis of a 20-mm. R. pipiens larva, from ventral surface; caudal end below. Abbreviations as for figure 8. X 100.

Fig. 10 Model of the hypophysis of a 22-mni. larva of R. pipiens, from the ventral surface. Caudal end below. .Vbbreviations as for figure 8. X 100.

Fig. 11 Model of the hypophysis of a 24-mm. larva of R. pipiens, from ventral aurface. Abbreviations as for figure 8. X 100.

80 WAYNE JASON ATWELL

Already the two show evidences of coming into intimate relation with ouch other.

iSS-mm. and 24-mm. larvae of R. pipiens. Ventral views of wax-plate reconstructions made from these embryos are shown in fipures 10 and 11. The lateral lobes, l.l., which are the anlagen of the pars tnberalis, are larger and are flattened out under the infundibular floor. The pars intermedia conforms more closely to the shape of the neural lobe (fig. 11).

Larvae of R. clamitans and Bufo americana at metamorphosis. Very similar relations to the last are sho^\^l by a larva of R. clamitans possessing very short hind legs. A model from this embryo is shown in figure 12. That the spreading out of the lateral lobes under the infundibular floor is due to active growth is indicated by the irregularity in outline of the lateral lobes at these stages (figs. 11 and 12). Other larvae of R. clamitans obtained at later stages of metamorphosis show the lateral lobes in the process of separation from the anterior lobe.

A reconstruction from a toad (Bufo americana) which had just completed metamorphosis is presented in figure 13. The lateral lobes, l.l., are seen to be joined. They are united to the anterior lobe proper by a single attenuated epithelial strand. The lateral lobes do not long remain united, however, for another toad of about the same age (not figured) shows one lobe entirely free, while the other is attached to the anterior lobe by a narrow strand.

From what has been observed in R. clamitans and B. americana it seems safe to assume that in general the lateral lobes become separated from the remainder of the epithelial hypophysis at the completion of metamorphosis or very soon thereafter.

A dulls of R. pipiens and R. cateshiana. In the adult frog the pars tuberalis, derived from the two lateral lobes, is seen as a pair of epithelial plaques lying close under the 'infundibular' floor some distance in front of the remainder of the gland (figs. 14 to 18). Each plaque is appro.ximately circular, but not infrequently shows a greater diameter from side to side. Each is 0.3 to 0.4 nun. in diameter and about one-fifth as thick. They

HYPOPHYSIS OK THE ANTRA

81

Fig. 12 Ventral view of a model of the hypophysis of a larva of R. clamitani with small hind legs (legs 6 to 7 nun. long), ani. I., anterior lobe proper; p. int., pars intermedia; l.l., lateral lobe; b.w., brahi wall. X 100.

Fig. 13 Ventral view of a model of the hypophysis of a toad (Bufo americsn*) which had just completed metamorphosis, n.l.. neural lobe; other abbreviations as for figure 12. X 100.

Fig. 14 Model of the hypophysis of an adult of U. pipiens, viewed from Iba ventral surface, p. f., pars tuberalis; other abbreviations as for figure 12. A-B, plane of section shown in figure 18. X 3o.

82 WAYNK JASON ATWELL

lie rioso to the brain wall, indenting but not invading it, figure IS. and are surround(><l I)v tli<' pin mater.

Hy a proper coinl)inatioii of dehydrating and clearing fluids the pars tuberalis may be seen in the gross, preferably with the aiti of a binoeular microscope or a low-power dissecting lens. I'sing a mixture of absolute alcohol, 00 parts, and xylol, 40 parts, it was possible to obtain the camera-Iucida sketch shown in figure 15, which reproduces the ventral surface of the hypophysis of an adult bullfrog (R. catesbiana). Similarly the drawing from an adult specimen of H. pipiens presented in figure 10. V was obtained. To make sure that the parts observed in the gross were the same as those seen in sections, these two brains were later cut into transverse series. A grajihic reconstruction of the caudal half of the so-called infundibulum and adjacent structures of one is shown in figure 16 B, and a single transverse section passing through the center of the pars tuberalis is presented in figure 17.

It will be seen that the two plaques composing the pars tuberalis lie under fairly tliick lateral portions of the 'infundibular' floor. Between these thickenings and caudal to them the floor is very thin, X, Fig. 15. \\']ien observed in the gross this thin floor is almost transparent and this fact, no doubt, has given rise to the rather misleading description of the infundibulum as a "bilobed structure, emarginate posteriori}' and divided by a median longitudinal groove" (Holmes, p. 294). Such relations are not evident from a ventral view of a reconstruction fFig. 14). It seems to me doubtful whether the thickened 'bilobed' portion is properly to be considered as a part of the true infundibulum. 1 would restrict the name infundibulum to that portion possessing the very thin floor, A", figure 15, and exhibiting a single medial thickening at its caudal end, Ih., figure 15 (see also fig. 16). Possibly the bilateral thickenings, from their more anterior position, are to be considered homologous with the tuber cinereum of the higher vertebrates.

Another feature of interest is the presence of small but wellmarked depressions, one above each half of the pars tuberalis, figure 17. Whether these depressions are to be considered

llVrOPHVSIS OK THE ANlUtA

83

Fi(t. 15 Camera-lucitla drawing of the 'infundibulum' and hypophysis of an adult of n. catrsbiana, from the ventral surface. Caudal end is below, anl. I.. anterior lobe: p. int., para intermedia: pi, pars tuberalis; th., thickening of infundibular floor; A', thin portion of infundibidar floor. X 10.

Fig. 10 A Caniera-Uicida drawiiiu showing a portion of the ventral surface of the brain of an adult H. pipicns. Main body of hypophysis had been removed ; its approximate position is indicated in dotted outline, p.t., pars tuberalis: O.I., optic lobe; //, ///, 1', 17, VII, VIII, cranial nerves. X 10.

B Graphic reconstruction, from transverse sections, of portion adjacent in 'A'.

Fig. 17 Trailsvorse section of the frog's brain shown in figure 16 A. p.t., pars tuberalis; ///, oculomotor nerve; o./., optic lobe; inf., infundibulum. X 12.

S4 WAYNE JASON ATWELL

homologous with the 'tuberal recesses' of higher vertebrates is uncertain.

The anterior lobe proper has become the most ventrocaudal portion of the gland. This h>be, then, does not deserv^e the name 'anterior' from its adult position in the frog, but from its very evi<lent relation to the homologous lobe possessed by higher vertebrates. The lobe is oval in outline, being shorter in its nasocaudal dimension.

The pars intermedia is a long transverse ridge with bulging lateral terminations. It conforms in extent to the neural lobe which is definitely constricted from the infundibulum donsally and at the sides. In handling, the hypophysis readily separates from the infundibulum, the break occurring at the narrow attachment of the neural lobe (fig. 18). In such a case the neural lobe, pars intermedia, and anterior lobe proper come loose as a unit. The pars tuberalis remains attached to the floor of the part interpreted as the tuber cinereum (fig. 10 A).

In sagittal sections the neural lobe is cut in a direction transverse to its greatest length (fig. 18). As Stendell ('14) has noted, it presents in this section the form of a right-angled triangle with its hypotenu.se lying against the pars intermedia.

The three epithelial lobes are histologically distinct. The anterior lobe proper, which has been so named from its resemblance to the anterior lobe of higher vertebrates, is very vascular and consists of cords of cells which are chromophilic and granular. The pars intermedia and the pars tuberalis may both be spoken of as chromophobic portions of the gland. There are, however, well-defined differences between the two. The pars intermedia of the frog, in contrast to the same lobe in mammals, for example, is traversed by numerous blood-vessels. The pars tuberalis, on the other hand, and this is also in contrast to the mammalian gland, is not invatied by vessels at all. This may in part be accounted for by its small size in the frog. Many of the cells of the pars intermeilia (adult II. catesbiana) contain rounded hyalin bodies, considerably smaller than the nucleus. These seem to be more numerous in the neighborhood of the blood-vessels. Such liyalin bodies are not to be found in the pars tuberalis.

iivi'Driivsis OK thA anuka

85

DISCUSSION

Entoderm. My observations boar out the assertion <if( 'orning ('99) that entodenn does not enter into the development of the frog's hypophysis after the manner described by Kupffer ( '94) and Valenti ('95). There is indeed a mass of cells, epithelial in appearance, in relation with the anterior end of the notochord in larvae of \{. pii)iens having a length of 2.o to 3 mm. I am not able to find evidence that this mass becomes incorporated into the hypophysial anlage. That this eiiithelium-like mass is comparable with the 'protochordal plate' of other vertebrates, as suggesteti by Corning, ^Irs. Ciage, and more recently by Parker, seems to me not unlikely.

p.lm.

■■■^ifi::^v^^:B^

f. I. I ; , ml. I.

Fig. 18 Parntiiedian sagittal section of hyjiophysis of an adult frog (R. pipiens). Plane of section indicated by line A-B, figure 14. Nasal end is at the left, inf., infundibulurn; p.t., pars tuber.ilis; n.l., neural lobe; p. in*., pars intermedia; ant., I., anterior lobe proper, X 50.

The pam (uhcralis. \ two-fokl origin for the hypoj)hysis of Ajnbylstoma has been recordeil iiy Kingsley and Tiling. These observers believe that the bilaterality involves the entire gland and that the two parts soon fuse. The bilaterality observed in the frog's hypophj-sis seems, in certain stages at least, to be confined to the anterior portion of the anlage and does not extend to its caudal tip. I have never seen convincing evidence that the two parts come together and fus<> in the frog.

It is with some difficulty that the two parts may be traced to the bud-like lateral lobes which later form the pars tuberalis.

86

WAYNE JASON' ATWELL

\\ln'n tho hypophysis anlagp breaks loose from its parent ecto<i«'rrii, there is a very noticeable rearranp;etnent in its shape, as has been notetl. This is a disturbing factor in any attempt to frac<> the history of the lat<>ral lobes. During the stages just preceding and following the separation, however, a somewhat thjjiner shelf is sin-n on each side of the anterior half of the luihige (Hgs. .'> and 7). Tlu>se I have supposed to be the lateral lol)es.

Hy the time the embryo has attained a length of IS mm. the lateral lobes are definite bud-like structvu'es at each side of the nas^d end of the gland (fig. 8). The further development of the pars tuberalis consists in a spreading out of the lateral lobes uinler the brain floor and their subsequent detachment from the anterior lobe proper. This latter occurs, in Rana clanutans and Bufo americana at least, during the latter part of metamorphosis or very .soon after its completion.

In the adult frog the pars tuberalis consists of a pair of epithelial plates, free from each other and from the remainder of the gland. The plates have been described by a few authors, but all have spoken of them as insignificant rudiments and none has considered them homologous with the lateral lobes seen during the development of the hypophysis of higher vertebrates.

The terminology for the various lobes of the hypophysis differs so much among writers that it is customary to gather into a table the names to be found in the literature. In the following table I have included only those authors who speak of three lobes of the .A.nuran hypophysis.

Terminology of the lobes'of the Anuran hypophysis

OBSIKTIR

AXTCRIOR LOBK PHOPCB

rAU INTtRUIDIA

PARS TDOKRAUS

Caiipp ('89)

Para jiostcrior

ParH anterior

Partes lateralcs

U. Haller ('97)

Hypo))hysi8

Infundibulur

Vordcrlappen

drOsc (Saccus

paares

va.sculosii.s)

Stendrll ('14)

Ilaiipt Liippcn

Zwisfhon Lappcn

Pars anterior or Pars chiaaiiiatica

AtwcU ('18)

.■Vnterior Inbn proi>cr

Pare intermedia

Pare tuberalis

JtYPOPHYSIS OF THE AN'fUA 87

GENERAL CRITERIA KoR HOMOLOCWZING THE M)HES OF THE IIVPOPIIVSIS IN THE VARIOfS VERTEBRATE CLASSES

To hoiiK.ldfiizo tlic lobos of the hypophysis throughout tlio vertebrate ehi-sses it is iiocossury to have clearly in iiiiiul both their dovelopmontal and their adult relations. I propose to eiuinierate some of the criteria by which they may be classified in th<^ lifiht of some of our recently acquiretl knowledge. It must be borne in mind that many of the points to be given are as yet supported by relatively few obser\-ations. However, it is hoped that their presentation in concise form may be founfl useful. The adult relations may first be considered.

.Size. In gener.-il the anterior lobe proper is the largest of the three lobes, the jJars intermedia the next smaller and the pars tuberalis the sjiiallest. That these relations may not hold constant in all \ertebrates is quite possible, but they are certainly true for the frog and for some mammals. Woerdeman ('14) believes that the pars tuberalis (his 'lobulus bifurcatus') becomes progressively less important as the vertebrate scale is ascended.

Form and position. The anterior lobe proper tends to maintain an approximately spherical or ovoid shape. It is not mouldeil to any marked degree b\' surrounding structures. It is not intimately attached to anj' tissue of nervous origin and in many forms does not e\en lie in contact with neural tissue. On this account Tilney ('13) has named this lobe the 'pars distalis."

The pars intermedia is always conformed to the shape and extent of the neural lobe. In reptiles, birds, and mammals the pars intermedia is a thin epithelial layer applied to the neural lobe and derived from the superodorsal wall of Rathke's pocket. It more or less completely surrounds the neural lobe, which in these forms has its longest dimension in the sagittal direction. Later in life the pars intenn<Hiia invad«'s the tissue of tlie neural lobe to a considerable extent. In the frog also the pars intermedia corresponds to the neural lobe in shape. Here the latter has its long axis extending from side to side. The par> intermedia has a very similar shape, with the exception that it is

88 •WAYNK JASON ATWELL

rnundod and bulging where it protrudes beyond each side of the anterior lobe (figs. 14. 15, and 1()).

The pars tul)eraUs Hivewise conforms to the shape of that part of the brain wall upon which it lies. It is essentially thin and hunina-like. In nianunals it surrounds the infundibulum and spreads out under the tuber cinereum. Recon.structed and viewetl separately, it may be described as 'saucer-shaped' (Tilney, '13) with a perforation for the infundibular neck. In reptiles thin bands and zones may be formed about the middle of the anterior lobe in addition to the thin plate which is spread out under the brain floor (Bamngartner, '16). In the Anura the pars tuberalis does not extend dorsal or caudal to the attachment of the neural lobe. It consists merely of a pair of small rouniied plafiues, located nasalward from the remainder of the gland.

It is important to note the relations of the pars tuberalis to the membranes of the brain. It lies in the pia mater in close relation to the brain floor, but does not appear to invade the neural tissue after the manner so characteristic for the pars intermedia.

Histology. Much remains to be worked out in regard to the normal histology of the hypophysis. It is my purpose merely to emphasize the fact that the three lobes may be readily distinguished by their histological characters. Tilney ('13) was first in attempting to classify the differences in structure of the three parts. This he did for a number of manunals and for the domestic fowl. According to Tilney, tlu> three parts may be differentiated as to cell structure and vascularity. The pars tuberalis is more vascular than the pars intermedia and less vascular than the anterior lobe proper. Its cellular arrangement is that of cell masses with occasional small, relatively thickwalled acini. The cells are basophilic with rather scanty cytoplasm.

Th«' histological features of the three parts of the reptilian hypophysis an; thus saimnarized b)' Baumgartner ('16):

The pars intermedia has a laminar arrangement of columnar clearstaining cells. The part derived from the lateral lobes are arranged

HYPOPHYSIS OF THE ANURA 89

in columns (or sometimes acini) of clear-stainine polyhodral cells. Tlif anterior lobe proper i.sfornu'd of columns or acini, witli clear-staining ami (larkly-staininR colls which may be acidophilic or b;i.-<ophilic. In Ken('r:il, the pars intermedia and the parts derived from the lateral buds m:iy bi- considered the chromophobic and the anterior lobe the chromophilic part.

Parker ('17) has called attention to the tubular or acinar structure of the pars tuberalis of the hypophysis in marsupials.

In a recent j)aper the writer has ("Uiphasized the differences in structure^ in the several parts of the rabbit's h>'pophysis at birth. Of considerable interest in distinpuishiiiR between the pans intermedia and the pars tuberalis are the peculiar spindleshaped and branching cells which have been observed in the former (Retzius, Hi^rring, Trautmann, Atwell, and others). They ha^e been called ependymal and neurogliar elements (iStendellj. It is important to note tiiat these cells have never been described for the pars tuberalis.

The frog forms an exception to some of the features found in the Amniota. In this form the pars intermedia is fairly vascular while the pars tuberalis is non-vascular. In the specim<>ns studied (which were obtained in October and November) the pars tuberalis does not show a tubular or acinar arrangement of its cells. The pars intermedia i.s clearly in contrast to the pars tuberalis on account of the numerous hyalin bodies to be found in the former, but never in the latter (R. catesbiana).

It will thus be seen that there is very good agreement among the several vertebrate classes, so far as studied, as regards the main features of structure of the three epithelial lobes.

Development. It is by careful ontogenetic studies that the distinction between the three epithelial lobes is most strikingly brought out. Thus the pars intermeilia, in those forms poss<^;sing a hollow hypophysis anlage. develops from the dorsal wall of Ratlike's pouch. In those forms having a solid hypophysis fundament, as the frog, the pars intermedia is <lerivcd from the dorsal or caudal tip of the soliil anlage. During development, in all forms, the pars intermedia becomes intimately united with the neural lobe.

TIIH \X»TOMU-.\L RCCORU, VOU 15, NO 2

90 WAYNK JASON ATWKLL

Tho nnt«^rior lobe propor (iovolops from tho iiiaiu body of the t'pillu'lial anlag<>. In tlio frog tliis is the muklle ami anterior part of the kIuikI with the exception of the hiteral lobes. In vertebrates higher than the Amphibia the anterior lobe is formed mainly from tlie anterior or ventral wall of Rathke's pocket.

'i'lie pars tuberalis has a paired origin, being derived from the lateral lobes, lateral buds, or 'tuberal processes.' It had been lielieved that for niammaR the lateral lobes appear relatively late in development (Tiiney, ^Miller), but the writer ('18) has shown that thej^ are present very early in rabbit embryos. It seems likely that the 'bilateral origin' of the hypophysis observed in Ambylstoma by Kingsley and Thyng is an early appearance of the nnlagen for the pars tuberalis. This, at least, is my interpretation of the bilateral conditions abo\-e recorded for very young embryos of R. pipiens.

The definite lateral lobes are, during development, attached close to the epithelial stalk at the ventral or nasal end of the gland. They may fuse with one another and fonn a single epithelial plate closely applied to the floor of the third ventricle (most .Vmuiota). Thej- maj- maintain their attaclunent to the anterior lobe proper or they may become detached as cell masses (certain reptiles), or as two epithelial plaques (frog). In snakes and in some lizards it is even stated that the lateral lobes disappear entirely (Baumgartner, '16).

SUMMARY

1. The hypophysis of the Anura consists of three epithelial lobes and a neural lobe. The lobes of epithelial origin are the anterior lobe proper, the pars intermedia, and the pars tuberalis.

2. Fn.m their development and their mature structure these lobes may be considered homologous with corresponding lobes of the hypophysis in all higher vertebrates.

3. The anterior lobe proper develops from the main central portion of the solid epithelial anlage. It comes to lie caudal and ventral to the other portions of the gland.

4. The pars intermedia develops from the caudal tip of the hypophysial anlage. It forms a long transverse structure conft)rming to the shape and extent of the neural lobe.

IIVF'OPHYSIS OF TFIE ANIRA 91

"). The pars tuboralis has its origin in the lateral lobes, which appe;ir very early. These two lateral lobes spread out under the brain floor and become detach(!d, forming two discrete, rounded plaques lying close under the brain floor in the pia mater.

6. The pars tuberalis is a constant structure, during development, in the hvfjophysis of amphibia and higher vertebrates. It is characterized by its paired origin (the two lobes appear relatively earh' and have their attachment near that of the epitheUal stalk) by its l;uuin;u- nature and by its adult location in the pia mater covering the tuber cinereum of the brain floor.

BIBLIOGR.^PHY

Atwell, W. J. 1918 The dovolopnifnt of the liypophysis cerebri of the rabbit (Lepus cuniculus L.). Am. Jour. .\ii!it., Sept. 1918.

Baumgartner, K. \. 1916 The development of the hypophysis in reptiles. Jour. Morph., vol. 28.

Corning, H. K. 1899 I'bcr cinigc Kntwickiungsvorgange am Kopfe der Anuren. Morph Jarb., Bd. 27, S. 173.

Gage, Sdsanna Phelps 190C The notochord of the head in human embryos of the third to the twelfth week and comparisons with other vertebrates. Science. N. S., vol. 24, p. 295.

Gaupp, E. 1899 .v. Ecker's und R. Wiedersheim's .\natomie des Frosches. Zwcite .Abtheilung.

GoETTE, .\. lS7.i Entwicklungsgcschirhto dcr Unke. Leipzig.

Haller, B. 1897 I'ntcrsuchungen iibcr die Uypophyse und die Infundibularorgane. Morph. Jahrb., Bd. 25.

Herring, P. T. 1908 The histological appearances of the mammalian pituitary body. Quar. Jour. E.\. Phy.siol., vol. 1, p. 121.

Holmes, S. J. 1906 The Biology of the Frog. Macmillan.

KiNosLEV, J. S., AND Thyng, F. W. 1904 The hypophysis in .\mblystonia. Tufts College Studies. Scientific Series, vol. 1, no. 8, p. 363.

KuPKKER, ('. V. 1894 Die Deutung des Ilirnanhnnges. Sitz. Ber. d. Gesellsch. f. Morph. u. Physiol, zu Miiiichen, S. 59.

LuNDBORG, Herman 1893 Die Entwicklung dcr Hypophj-sis und des Saccus vasculosus bei Knochenfischcn und .\mphibien. Zool. Jarb., .\nat. Abt., Bd. 7, S. 667.

Miller, M. M. 1916 A study of the hypophysis of the pig. Anat Kec . vol. 10, p. 226.

Orr, Henry 1889 Note on the development of the .\mphibians, chiefly concerning the central nervous syHtem: with additional observations on the liypophysis, mouth, and the appendages of the head. Quar. Jour. Micros. Sc., N. S., vol. 24, p. 275.

•»2 WAYNE JASON ATWELL

Parkkr, K. M. 1917 The development of the hypophysis cerebri, prooral (jut,

and related structures in the Marsupiuliu. Jour. Anat., vol. 51, part 3. Retiics, G. 1S94 Die Neuroglia der Neuro-Hj-pophyse dcr Siiugothicrc.

Biol Untcrsuch., N. F., Bd. 6, S. 21. Ste.vdell, Waltkh 1914 Die Hypophysis Cerebri, .\chter Theil in Oppel's

Ix-hrbuch dcr vergleichenden mikroscopischen Anatotnie der VVirbel thiore. Tll.NEY, Fredkrkk 1013 .\n analysis of the juxta-noural epithelial purtioii of

the hypophysis cerebi, with an enibryological and histological account

of an hitherto undescribed part of the organ. . Internal. Monatschr.

f. Anat. u. Physiol., Bd. 30. Traotmann, Alfred 1909 Anatomie und Histologie dcr Hypophysis cerebri

einiger .Siiuger. Arch, f. mikr. .\nat., Bd. 74, S. 311. Valenti, G. 189.5 Sullo sviluppo doll' ipofisi. Anat. Anz., Bd. 10, S. 538. WoERDEMAN, M. \V. 1914 Vcrglcichenden Ontogenie der Hypophysis. Arch.

f. mikr. Anat., Bd. 8G.

AUTHOR H AIIATRACT Of THIS TAPER I4SUED UT TIIK BIBLIOORAPUIC •CRVICE, AVOVVT 7

LYMPHATICS OF THE OMENTUM

HORTOX R. CASPARIS Anatomical Laboratory of the Johns Hopkins Urtiversity, Baltimore

ONE FIGURE

The significance of the omentum has been since the earHest times ;i matter of imirh theory and conjecture. \'arious protective and other functions have been attributetl to it, but these in the main have btH*n basetl on theory. Only recently has there been definite experimental work to prove the accuracy of some of these assumptions. Most recently the interest has been centered on what part the omentum might play in absorption from the peritoneal cavity. Previous work on peritoneal absorption has resulted in emphasis being placed on the diaphragm as the main pathway of absorption, Adler and Meltzer, Muscatello, and others holding that drainage was b}' way of lymphatics, while Heidenhain, Starling and Tubby, Cohnstein, Hamherger, and Dandy and Rowntree hiivc shown that much of the absorption takes place by way of the blood stream. However, asserticins had been made by some that the omentum aided in peritoneal absorption; but not until Rubin in 1911 showed that loss fluid was absorbed from the peritoneal cavities of animals whose omenta had been amputated than from the peritoneal cavities of normal controls, was there any experimental evidence to support the.se assertions.

Knowing the facility with which material passes from the peritoneal cavity through tlie diaphragmatic lymphatics, the question arose as to whether or not the omental lymphatics might play a like rfile, but only a short siu*vey of the hteratiu-e was necessary to cause the matter to resolve itself into whether or not the omentum contained Ijinphatics. Ranvier found them in new-born kittens, but claimed that they disappear by

93

94 HORTON R. CASPARIS

rotrofuossion or by somo (It'Rcucnitivo process by the oiui of the third month. Oth«Ts h;ivo in;iiiit:iincd that there are no lymphatics in the onientiun, and some of the earUer workers who made very ext«>nsive studios of the l>TupIiiifir system do not mention the omentujn at all as one of tiie Ijinphatic-contaiuing organs. And just recently Shipley and Cunningham, who have made a careful study of the omentum, proving definitely that active absorption from the peritoneal cavity takes place -by way of the blood stream of the omentum, were unable to demonstrate the presence of lymphatics. On the other hand, Klein, Norris. and others speak of lymphatics of the omentum, but do not give convincing proof of their existence, and it is this latter fact together with the fact that it is very difficult to adequately demonstrate them in a place where one would think they should be easily demonstrable that is responsible for the uncertainty which those fe<>l who have had occasion to think of or look up the matter.

In my first attempts von Rechliiighausen's silver method was used. Omental sjjroads were made, the surface was rinsed with distilled water, a one-fourth of 1 per cent solution of silver nitrate was applied, and after one or two minutes the silver solution was rinsed off with distilled water. The specunen was then iiiunersed in distilled water and exposed to the sunlight until it assumed a brownish tint. The.se sih-er markings were also brought out by th<> arc light, but loss effectively. Ry this method I found that oulj- the peritoneal lining of the ojnentuni was silvered. Then I followed Klein's technique of penciling off the surface of the omental spread with a camel's-hair brush in order to remove the peritoneal covering before applying the silver nitrate. In this waj^ beautiful rich networks of Ijnnphatics were readily brought out in the centrum tendinum of the diaphragm- just such a picture as that diagrammatically sk<itchod by Klein for the omentum, but in no case were lyznphatics found in the omentum by this method except in the new-born kitten where some of the lymph-ve.ssels were present in the very thin meshes of the omentum. However, since the silver did nf)t with this teclini(}Uo penetrate the perivascular fat and the thicker parts of the omentum around the blood-vessels,

LYMPHATICS OF THE OMENTUM 95

it (lid not exc'luclo tlio possibility of thero being l3^nphatics in those ureas. Attempts were then mjide to inject with rolor inass(>s, but with no success. The extremely fine and delicate structure of the omentum made this method unsatisfactory. It was then decided to return to the silver method and attempt to .silver the perivascular structures by forcing the solution through the blond streuJM under increased pressure. Inunediately after killing the animal a cannula was inserted hi the abdominal aorta, the aorta was then ligated below the cannula and above the coeliac axis. A nick was then made in the portal vein to insure a free outflow, and distilled water was passed through the omental vessels to wash them out. Inunediately following that a onefourth of 1 per cent silver nitrate solution was forced through the omental vessels under rather high pressure. One could readily determine the extent of injection by the immediate milky opacity assujned by the tissues which the silver invaded, and this was the guide in regard to the amount of pressure to use. The omentum was then removed, immersed in distilled water and exposed to the sunUght until the brownish tint appeared. The spt^imen was fixed in alcohol and was cleareil by the Spalteholz method, and it was found that the solution had pa.ssed through the walls of the arteries and had invaded and silvered all structures in the perivascular areas. By this method and with local forced injections lymphatics were foimd in the region of the larger l)lood-vessels of the omentum in the rabbit, cat, dog, and in man. The lymphatic endotheUum is typical and unmistakable and is easily distinguished from that of the veins and arteries by its different jjattern. It must be said here, though, that in no case were the Ijinph-vessels numerous. After finding the lymphatics by the above method, it seemed unn^isonable that one should not be able to inject them with color suspensions, but further attempts along this line were practically without success. Whether the fact that removing the omentum from the peritoneal cavity, exposuig it, and stopping its circulation which gives it a tendency to dry rapidly and contract somewhat, would cause the deUcate-wallcd l>inpliHtics to contract and make injection diflicult is only a possible explanation.

•)t) HORTON R. CASPARIS

Attempts Jit injection did. howevor, bring out one or two inton^stiiiK facts. In the cat und iulult pig, by making shallow injections in the ventral stomach wall along the greater curvature just anterior to the hne of attachment of the omenttim, one was able to get loops of l\inphatics running down from 2 to 4 or 5 cm. into the omentum. From the position of these loop.s it would appear that the lymphatics had been dragged down from the stomach wall in the growth and development of the omentuju. There was no e\ideiice of drainage of lymphatics from the omentum proper into these loops. Also when one injected in the corresponding prepyloric region of the rabbit's stomach, the injection mass would pass from the stomach wall through a single Ijinpli channel which cour.sed down through the ventral leaf of the omentum to its lower splenic attachment. This vessel had no definite relation to any of the large bloodves.sels, but passed across one of the less vascular areas and could not be considered a true omental lymphatic.

Attempts at injection further brought out the peculiar pattern of the blood-A'es.sels of the omentum. In the normal omentimi capillaries are comparativeh' rare, but there is an unusually rich arterial and venous anastomosis.

Reverting to the main subject, at this point it was felt that I had progressed practically no further than others who had clauned positive results. Therefore, in the hope of producing absolutely definite and convincing evidence for the presence of hiiiphatics in the omeiitiuii. it was decided to run some absorption experiments, following the method used by Shipley and Cunningham in their peritoneal absorption experiments. Instead of decerebrating the animal, it was anaesthetized with luminol-sodium (a drug which produces an even quiet anaesthesia which can be made to la-st from one to six or eight hours by regulating the amounts, but from which the anunal never recovers), was then placed in a box in which physiological conditions were maintained, and the omentum was cjirefully drawn out of the animiars body through a midline incision and was kept immersed in a carmine solution. In the .several experiments various lengths of time ranging from one-half to three

LYMPHATICS OF THE OMENTUM

!)7

hours wore allowed for al)sorpti()ii to take placo and then all the possible Innph glands to which drainage might occur (including the mediastinal glands) were removed, sectioned, and were examined for the carmine granules. Also each time a careful

Lymphatic

Artery

Vein

Fig. 1 Specimen of the oiueiituiii of an adult rat in which silver nitrate (0.2."i per cent) was injected into the arteries to show comparison of the size and shape of the endothelial cells of an artery, vein, aixd lymphatic. The transverse lines on the artery and the vein indicate the smooth muscle. The entire width of the artery is not shown, as part of it was covered by the lymphatic.

search was made for an indication of the channels of absorption, but both here and in the case of the glands exjunined the results were negative. Then it occurred to me that possibly I had failed to find the glands or that the drainage might be direct. Additional experiments were made, IjTiiph was withdrawn

us HOKTON H. CASPARIS

from the cysteriii chyli and sjiicars were made, with the result that a few granules were found each time. At other times l>nnph was drawn from the thoracic duct proper and granules wi're found, but in neither case were they numerous. Otlier exp<'riments wore made whicii were sbnihir to the above with the exception that for the absorption material a true solution was use<l (potassiiuu forrocyaiiiile anil iron aimnonium citrate). (Hands were removed as before and were fixed in hydrochloric acid formalin without the formation of Prussian blue, but when the hnuph was drawn from the cysterni chj'li and from the thoracic duct and was placed in hydrochloric acid fonnalin, Prussian blue was fonned.

These experiments then confirm the findings with the pressure injection of silver nitrate into the blood stream — they show deftnitelj- that Ij-mphatics are present in the omentum and furnish a channel for absorptions from the peritoneal cavity. Whether the lymphatics really play a very active role in this latter respect I am not prepared to say, for while absorption was rather meager in the above experunents it might be considerably more pronounced when the omentum is closed u]) in the peritoneal cavity where pressure conditions are necessarily different, or the meager absorption might have Ixx^n due to the small number of IjTiiphatics present. The experinients also show that drainage from the omental Ijanphatics is into the cy.stemi chyli and on through the thoracic duct and not by way of the posterior mediastinal glands as liyjiotliecated by Crouse in his extensive paper on the great omentum.

I wish to take this opportunity to thank Dr. F. II. Sabin, F*rofessor of Histology in the Johns Hopkins IMedical School, also Dr. R. S. Cunningham, Instructor in Anatomy in the same institution, for their helpful suggestions and criticisms and also for placuig e(|uijimcnt at my dispo,sal.

LVMI'IIATICS OF THE OMENTUM 99

BIBLIOGRAPHY

Adi.kh, I , ANU Meltzeb, S. J. 1890 Kxperiiiu'iital contribution to tlie study

of the path by which fluids arc carried from the peritoneal cavity

into the circulation. Jour. Exper. Med . 1. CoHNSTEl.v, W. 1895 Cbcr Resorption aus der I'critoncalhohle. Zentralbl.

f. Physiol., 9. Crocse, H. 1912 The omentum. Bulletin of the Kl Paso Med. Soc, April. Dandy, W. E., and Rowntree, L. G. 1914 Peritoneal and pleural absorption.

with reference to postural treatment. Annals of Surgery, 59. Hambcriikr, H. J. 1895 L'ber Resorption aus der Peritoncalhohle. Zentralbl.

f. Physiol., 9. Heidenhaix, R. 1891 Versuche und FraRen zur Lehre von der Lymphbildung.

Pfluger's Arch. f. d. Kcsammt. Physiol. 49. Klein, E. 1873 Handbook for the physiological laboratory, I. Muscatello, G. 1895 tjber den Bau und das Aufsaugungsvermogen des

Peritoniium. Arch. f. Path. Anat., 142. NoRRis, C. C. 1908 The omentum: its anatomy, histology and physiology in

health and disease. Univ. of Pcnna. Med. Bulletin, 21. Ranvier, L. 1896 Anatomic gc'nt'rale.— Aberration et ri'-gression des lymph atiques en voie de doveloppement. Acad^'mie des sciences. Comptes

rend us, 122. Rubin, I. C. 1911 The functions of the great omentum. Gynecology and

Obstetrics, 12. Shipley, P. G., and Ccnninoham, R. S. 1910 The histology of blood and lymphatic vessels during the passage of foreign fluids through their walls.

II. Studies on absorption from serous cavities. Anat. Rec, 11.

1916 The omentum as a factor in absorption from the peritoneal

cavity. I. Studies on absorption from serous cavities. Am. Jour.

of Physiol, 40. Starling, E. H., and Tdbby, A. H. 1894 On absorption from and secretion

into the serous cavities. Jour, of Physiol., 140.

ACTHOn'll AllATRACT OF THIH PAJ'RR ISaUKD BT THE ItlHLIorjKAi-mc KrRVIf B, At'(H7«T 7

AN r.xrsr.M, i^icii r i.rxc

MATTHEW MAUSllALL Anatomical Laboratories, The Schtiol of Medicine, Univernili/ of Pillnhiirgh

In the course of the regular dissection an unusual right lung was found recently. In brief, its interesting features wore:

1. A rudiiuenturv middle lobe so hidden in the interlobular fissure as t(j make the lung appear two-lobed from a sunple surface inspection, and

2. An unusu£ll distribution of the second lateral bronchus.

The inferior interlobular fissure did not reiich the diaphragmatic surface, but tunu^d around the ventral margin on to the mediastinal surface and thence dorsahvard and slightly upward to the hilus. The superior interlobular fissure separating the middle and superior lobes was not evident from the mediastinal surface; that is, there was no evidence of a middle 1<)Ik> from surface inspection of the mediastinal surface of the lung. On widely divaricating the inferior int(Tlobular fissure, the middle lobe could be seen near the mediastinal surface, Ij'ing in a position ventral to a frontal plane passhig through the hilus. It was shaped as a biconvex disc about 1 cm. thick, elongated in a dorsoventral direction, with its medial edge very close to and parallel with the mediastinal surface of the lung, and its lateral edge roughly parallel to the medial edge and about 4 cm. from the mediastinal surface of the lung. The dorsoventral length was about ."> cm. It was attached dorsomedially to the hilus, the remainder being covered by pleura except the ventral extremity, which was fused with the superior lobe. Thus it was incomplctc^ly separated from tlie upper lobe by the superior interloljular fissure.

The relations of the structures at the hilus were those of a nonnal right lung. When traced (h^'ply into the lung substance the relations of the eparterial bronchus and vessels of the superior

101

102 MATTHEW MARSHALL

lulu- wen- finiiul to lu- iioriiuil. lln' sitoikI hiti-nil bronchus, the nonnal hrouchijil supply of the midillo lobe, was observed, however, to send a large branch to the upper lobe, taking a path through the fusion betwoon the middle and superior lobes, while it supplied oidy a small branch to the middle lobe. The main-stem branches proceeded into ihc inferior lobe in the usual manner.

The tendency of the middle lobe to fuse with the su])eriur lobe is well known. The diversion of the distribution of the larger part of the second lateral bronchus to the upper lobe in this case seems to suggest there is some parallelism between the extent of fusion of these two lobes anil the ilistribution of the sectmd lateral bronchus to the su'perior lobe.

This lung merits recording since: 1) it shows an extreme variation in the relations of the interlobular fissures of the right limg. and, 2) it presents a well-marked case in which an upper lobe of the right lung receives the major part of the bronchial distribution of the second lateral bronchus, and the middle lobe receives the minor part.

autbob's abstkact or thu papbb laat-cD bt

TBB BIBLIOORAPniC SERVICE, SEPTEUBEB ID

FURTHER STUDIES ON THE REACTIONS OF BLOOD AND TISSUE-CELLS TO ACID COLLOIDAL

DYES'

HAL DOWNEY

University of Minnesota, Department of Animal Biology

SIX FIGURES (two PLATES)

It has been cluiiued that IjTiiphocj'tes and leucocytes of the blood will not take up colloidal dyes in animals stained intra vitam. It is also generally believed that the reaction to colloidal dyes is specific and that it serves to distinguish between l\Tiiphoid wandering cells and fixed cells of the connective tissue and those which have migrated into the tissues from the blood.

At the New York meeting (1916) of the American Association of Anatomists the writer showed that blood-cells, both Ijinphocytes and polymorphonuclear leucocytes, will take up the dye and store it in the form of granules if the cells are isolated from the blood stream. It was shown that if the dye is injecteil into a blood-ve.ssel which has been isolated from the general circulatit)n by a double ligature and the vessel is left in situ for twenty-four hours, it will be found that the poh-morphonuclears have stored the dye in large ((uantities. The hiiiphocytes of the vessel have also taken up the dye. .\nother experiment described at that time consisted of the injection of the dye into the intermuscular connective tissue. Sections of the muscle, which wore removed on tlie following day, contained many poljnnorphonuclears with dye granules. These results were described in detail in a paper published in Vol. 12 of The Anatomical Record.

In that pajier observations were described which gave further support to the view of Evans and Schulemann, that the cellular

' Aided by u grant from tlip llcsearch Funds of tho University of .Minnesota.

103

THE ANATOMICAL HECOHD. VOL. I.I, No 3 OCTOBER. 191S

104 HAL DOWNEY

reactions find llioir cxpljuiHtion in the colloidal nature of the dyes. Acc<»rdinK to these authors, the dye particles are taken into the cell by phagocytosis and concentrated in cytoplasmic vacuoles in smaller or larper masses which in the end-stages appear as more or less definite granules. The writer reviewed some of the literature on phagocytosis ^nd showed that the reactions of the cells which are primarily concerned in intra vitam 'staining' with the colloidal dyes are exactly what should be expected if the process is one of ingestion and storage rather than of true staining of preformed cj'loplasmic structures. Phagocytosis on the part of leucocytes is of rare occurrence within tlie general blood stream, but in the tissues and in blood sinuses, where the velocity of the current is greatly reduced, the same leucocytes may be very active phagocj'tes. It, was shown that in their beha\'ior towards the colloidal dyes the leucocytes follow this same general rule. The conclusion was reached that the reaction is no more specific than is general phagocj-losis, and that it does not serve as a means of distinguishing between lymphoid wandering cells of histcv genous origin and those which have wandered into the tissues from the blood.

These studies have been continued during the past year with results which add further support to the conclusions reached in the first paper.

In this later work it has been found that the studj^ of the very early reactions following single intravenous or subcutaneous injections is of verj- great importance. This phase of the work seems to have been neglected by nearly everyone who has experimented with these dyes. The bulk of the present paper is devoted to an account of these early reactions.

It has been found that the polymorphonuclear leucocytes are much more active phagoc.ytes for the dyes than was evident from the first series of experiments. In order to demonstrate polymorphonuclears in the bl<K)d which contained dye it was necessary to inject the dye into a segment of a vessel which had been isolated from the general circulation b}^ means of two ligatures. Since then it has be(>n found that it is not neco.s.sary to ligature the ve.s.sel before the injirtion is given if the blood is removed

EXPEHI.MENTS WITH COLLOIOAL UYES 105

from the vessel within from one-half to two hours after the injoetion, or if a sepment of a large vessel is tied off and fixed within this time limit.

Rabbits were injected with from 20 to 50 cc. of 5 per cent Trypanblau in distilled water or nonnal salt solution through the marginal ear vein and fresh blood smears examined, or a segment of the inferior vena cava removed within the favorable time limit. Spleen, lung, and mesenteric lymph node were also removed at the same time and fixed in Helly's Zenker-formol mixture. Sixteen rabbits were treated in this wa}' and, with two exceptions, polymorphonuclears containing dye granules were found either in the blood or organs, and usually in all of these locations.

The brief period of time between the begimiing of the injection and the removal of the blood and organs probably accounts for the fact that the dye granules in the poljnnorphonurlear leucocytes are usually smaller and frequent h- paler than is the case when the dye is injected between two ligatures and the vessel left in situ for from nineteen to twenty-four hours. In the sections, and especially in the fresh preparations, one frequently gets the impression that the dye has stained or has been concentrated on the surface of the special granules of the leucocytes. In the later stages, in the case of the doubly ligatured vessels, or when the injection has been continued after the death of the animal, the dye granules are much larger and more irrt^gular in shape than the special granules. Th«' appearance of the granules in the early stages, together witli the; fact that it has never been possible to stain the special granules in leucocytes containing dj'e granules, seems to indicate that the dye granule is built up about the leucocyte granule, the latter serving as a nucleus for the larger dye granule.

There is some variation in the results of these experiments which is difficult to explain. In some cases no dye granules could be seen in any of the leucocytes of the blood or sectioned bloodvessel, but examination of sections of the organs would show that the polj'morphonuclears of the capillaries or interstitial tissue of the lung, spleen pulp, or .sinusoids of the liver contained beautiful

lOti HAL DOWNKY

dyo pranulos. In soino other c.isrs the l)hie Kiiinules were f<juiui in the blood, but not in theorgiuis. The usuiil condition, however, wjis to finfl blue pranules in the leucocj'tes of all of these locations.

All of those cells which Evans has grouped under the term 'macrophages' (histiocytes of A.schoff-Ki\'ono) do not take up the dye until later, when most of it has disappeared from the pol>inori)honuclears, which shows t hat the latter are more actively lihagocAtic for the dye than are the former.

\\'hen the blood or organs are examined later than two and onehalf hours after the injection it is difficult to find polymorphonuclears containing dye granules. \n explanation for this is not difficult to find. Immediately after the injection the leucocytes are floating in a .solution of the dye, the concentration of which is relatively high. Consequently the cytoplasm of the leucocytes is in contact with innumerable particles of the dye, and owing to its phagocytic properties is able to take in the dye particles. The dye being very diffusible, its concentration in the plasma is gradually being lowered by diffusion into the tissues.

Evans has shown that the colloidal dyes readily pass from a region of high concentration to one of lower concentration. This probablj' explains the diffusion from the plasma to the tissue fhiids, and also from the pohnnorphonuclears to the plasma, as 80<in as the concentration of the hit ter has been materially reduced. In the circulating blood the poljnnorphonuclears are in contact with the dye during a relatively short period of time, and hence the dye which they have taken up is not so firndy anchored as it is in the case of the doubly ligatured vessel from which diffusion takes place ver>' slowly, resulting in the storage of more dye and the fomiation of more resistant dye granules. In the latter experiment the dye granules of the leucocj'tes are very large and brilliantly colored after twenty-four hours, while the leucocytes of the circulating blood contain no dye granules at this time.

In ca.se the death of the animal occurs during the injection, the condition.s of the doubly ligatured vessel arc duplicated. The circulation is stopped and diffusion from the vessels is very much reduced. The leucocytes, of cour.se, continue to live for .some lime, and being in intimate contact wilii a high concentration of

EXPERIMENTS WITH COLLOIDAL DYES 107

the dye in the quiescent state they are able to phagocytose more of it than would be possible in the circulating blood stream.

The complete protocols of the two animals which died during the injection of Pyrrholblau may be of interest. Accidents of this sort rarely occur when Trj'panblau is u.sed, but with Pyrrholblau one must be prepared to lose some of the animals. In this ease the accidents served a good purpo.se, for they gave further insight into the factors governing the ingestion of the dye.s.

Rabbit 27. 50 cc. of a fresh 1 per cent Pyn-holblau solution in iionnal siilt solution was injected into the ear vein and into both femoral veins. Total time for the injections: one hour and twenty minutes. Animal died one hour and ten minutes after the beginning of the injection. 20 cc. injected after death. Organs removed fifteen minutes postmortem.

Liver: Many polymorphonulears, including those in the larger ves.sels, contain dye granules. No dye in the stellate cells.

Vena cava (section) : Large and brilliant dye granules in nearly all of the pohniiorphonucle^irs.

Spleen: Ver>' hj^ieremic. Some free dye, but none in cells.

Mesenteric Ijmiph node: Negative.

Rabbit 29. 14 cc. of 1 per cent Pyrrholblau in normal salt solution. Animal died fifteen minutes after the beginning of the injection. Injection stopped at time of death. Organs removed two and one-half hours later.

Vena cava : The dye has settled on one side of the vessel. All of the polymorphonuclears which are surrounded by the dye contain largo and brilliant granul(\<.

Lung: Large numbers of ])olym(>rphonuclearsin the capillaries and interstitial tissue. .Ul of them are filled with very conspicuous dye granules.

Liver: Negative.

In the material from both of the.se animals more of the jiolymorphonuclears contain dye and the granules are larger and of greater density than is usually the case when the animals are allowed to live until the organs are removed. In other words, the conditions are more like those obtained by i.solation of a segment of a vessel by double ligature. Reduced diffusibility

lOS HAL DOWNEY

from (ho v<>s«'ls and tlio (|uic>scont state of tlie dye-plasma solution :uv probably the dftcrmininp; factors in both cases.

Although the later series of experiments have shown that absolute isolation from the blood stream is not necessarj' for ingestion of the dye on the part of the blood-cells, it still remains evident that a slowing down of the blood current favors phagocytosis. This probably explains the few cases of early stages in which dy«^ praimles were found in the polymorphonuclears of the lung and spleen, but not in those of the vena cava. However, it does not explain the condition in one animal in w^hich pol>7uorphonuclears containing dye were foimd in the vena cava and not in the organs. The number of polpiiorphonuclear leucocytes appearing in the sections of lung and spleen is extremely variable in the different animals. In this case the organs contained very few of these cells, and it is possible that if a greater number of sections had been .studied some pol3Tiiorphs with dye granules could have been found.

In the first paper of this series (Anat. Rec, vol. 12) the A^Titer reviewed some of the literature dealing with the subject of phagocytosis of Uving bacteria following intravenous injection. There is general agreement among the investigators of this subject that the organisms disappear from the circulation within a few minutes after the injection. The reticular cells of the spleen pulp and bone-marrow, and especially the stellate cells lining the sinusoids of the liver, are the most active agents in the removal of tlie organisms from the blood. Polymorphonuclear leucocytes may take up the organisms, but only in those locations in which the blood current is slowed down (sinusoids of liver and suprarenal body, etc.), or after the organisms have invaded the tissues. They are never able to phagocytose the bacteria in the general circulation in the larger vessels.

In order to gain some personal experience with living organisms the writer injected an emul.sion of staphylococci into the ear vein of two rabbits. The results agree with what has already been reported in the literature.

The first rabbit was killed fifteen minutes after the injection. Hlood smears and sections from the vena cava were negative.

EXPKHIMKNTS WITH COLLOIDAL UVES 109

Thr spleen contained cocci in its reticular cells, but the mesenteric lymph node was negative. In the lung cocci were found in macrophages and in polymorphonuclears. The organisms were more abundant in the liver than in any of the other organs. They were founil in stellate cells and polymorphonuclears of the sinusoids in the neighborhood of the larger bile ducts, i.e., in the peripheral portion of the hepatic lobules.

In the second rabbit the inferior vena cava and portal vein were tied off and the organs removed about thirtj'- minutes after the injection. Results were similar to those of the first rabbit. Blood smears and sections of the vessels were negative. In the liver the stellate cells and polymorjjhonuelears which contained cocci showed the same grouping as in the first animal, i.e., they were in the peripheral portion of the hepatic lobules in the immediate neighborhood of the interlobular vessels and bile ducts. There were not many cocci in the spleen, but a few reticular cells and ])olymorphonuclear leucocytes contained them. In the lung the polymorphonuclears were verj' numerous, and many of them contained cocci.

The results of the experiments of these two rabbits are quite .shnilar to those obtained with the intravenous injection of colloidal dyes. In the circulating blood of the larger vessels, however, an important difference is noted which requires explanation.

Study of the blood smears showed that the cocci are removed from the circulation within a very few minutes after the injection. This fact, together with the mechanical factor of the rapid circulation of the blood through tlie larg(>r ves.sels, probably accounts for the absence of phagocytosis in the general blood stream.

Colloidal dyes are not eliminated .S(j rapidly, and consequently the leucocytes are floating in the dye for a r(>latively long period of time (one-half to two and one-half hours), and they are coming in contact with innumerable particles of the dye during this period, and hence are able to phagoeytoseit. In regioiLs of slow circulation and in the tissues the leucocytes are able to phagocytose both the dye and the cocci. AMien escape of dye and cocci

110 HAL DOWNEY

is pnvriitod by tho double liguturing of a vossel, phaROcytosis on the part of Iho loucocytos is still more active. For the cocci this was checked by the injection of an emulsion of staphylococci into the doubly ligatured jupular vein of a living rabbit. The vein was left in situ for lo minutes and then removed and fixed in lit lly"s Huid. In sections of the vein the polymorphonuclear leucocytes were seen to contain many cocci.

Before leaving the .subect of experiments with intravenous injections of colloidal dyes it may be well to add a few selected protocols of animals kept alive until the organs and blood were removed.

Rabbit 2/f. 20 cc. of 1 per cent Trypanblau. Animal killed one-half hour later. Inferior vena cava tied ofif and fixed in Helly's fluid. Ljanph node and spleen fixed at the same time. Some polymorphonuclears of the vena cava contain dye granules, but none were found in the spleen or lymph node.

Rabhil So. 38 cc. of 5 per cent Trypanblau in normal salt. Organs removed thirty-five minutes after beginning of the injection.

Vena cava: Dye granules in the polymorphonuclears. One large mononuclear with dye granules.

Lung: \'ery few polymorphonuclears. They all contain pale blue granules.

Spleen: Negative.

Liver: A few stellate cells with dye granules.

Rnhbit 36. 58 cc. of .') per cent Tn,'panblau in normal salt. Organs removed one-half hour after the 1) ginning of injection.

Vena cava : Dye granules in pohmiorphonuclears. M iny of the granules are very large.

Lung: Rather pale dye granules in some of the polymorphonucl( ars.

Spleen and liv(r: Negative.

Liver: \'ery little dye in the organ. Some stellate cells have traces of it.

KXPERIMENTS WITH rOLLOIDAL DYES HI

Rabbit 26. 40 cc. of 1 per cent Trypanblau. 20 cc. injected into each ear vein. Animal killed one hour after beginning of injection, thirty-five minutes after completion. Ligatured portal vein and spleen fixed in Helly. Dye granules in the pohiuorphonuclears of the vein, and the spleen contains a few polymorphs with dye granules.

Rabbit, 26. 22 cc. of 1 per cent Pyrrholblau in nonnal salt solution. Animal killed two hours after the completion of the injection.

Spleen: Dye granules in polymorphonuclears, but very little dye in the reticular cells.

T>iver: Dye in many of the stellate cells and in a few pol^^n<J^piionudears, but it is usually not in the form of definite granules.

Mesenteric lymph node: Very little dye in the node. A few polymorphs with dye granules. No dye in the reticular cells. A few histiocytes containing dye in the form of rf)unde(l masses of granules.

Lung: Great numbers of polymorphs with dye granules. A few small medium-sized l\^^^phocytes also contain dye granules, but there are no granules in the larger macrophages.

Vena cava: Some of the polymorphs have pale blue cytoplasm, but in none of them is the dye concentrated in the fonn of granules.

Rabbit 32. 50 cc. of 5 per cent Trypanblau in normal salt. Material removed two hours after the beginning of the injection.

Sjjleen: Dyt' granules in many cells of the reticulum and in many large macrophages.

Vena cava: Very few poljmiorphs in the sections, but most of them contain dy(> granules.

Lung: Not many polj-morphs, but they contain distinct dye granules.

Liver: Dye in stellate cells. No poljnnorphonuclears.

Mesenteric node: No dye granules in reticulum, but some reticular nuclei are l)lue, and some lymphocytes have blue nuclei or diffusely stained cytoplasm.

112 II \ I- DOWNEY

Rdbhil 33. 40 cc. o i)tr cent Tiypnnblau in norninl salt. Killi'd two hours mid twenty niinutos after the beginning of injection.

Spleen: Dye in the c<>Ils of the reticulum, but the total quantity of dye in the organ is .small.

V«'na cava: Dye granules in the polymorphonuclears.

Limg: Some free dye, but none of it in the polymorphonuclears or other cells.

hiver: Manj- dye granules in the stellate cells.

The abtn-e protocols are sufficient in number to give an adequate idea of the results obtained. It will be nof(>d that there is some variat ion in the re.sults even where the time elapsing between the beginning of the injection and the removal of the organs and blood was the same. Difference in the amount of dye injected does not seem to account for these variations.

When the experiments were first begun the dyes were given in aqueous solutions, but the hemolysis resulting seemed to be a disturbing factor. For this reason the dyes were suspended in n«)rmal sjilt .solution for the later experiments. This does not affect the storge of dye in the cells, but the amount of pigment in the organs seemed to be less when the salt solution was used.

At first it was found impossible* to demonstrate dye granules in the polymorphonuclears of blood smears, even though sections of the same veins from which the blood was obtained showed cli'arly that the poljniiorphonuclears contained dye Later it Was found that the technique of staimng {h.ssolved out the dye. WTien fresh preparations were examined without any stain the dye granules could be seen very clearly, especially when the preparation was examined by the light of a small Spencer lamp placed close to the mirror of the microscope. The heat from the lamp was sufficient to permit active ameboid motion. In these active cells t he blue granules could be followed as they circulated t hrough the cytoplasm and into the pseudopodia.

The.se i)repiirat ions can be preserved if they are fixed in heat or in the fumes of full-strength fonnahn and their nuclei can be stained with methyl green, which does not affect the dye granules. Mounting the sme.'irs dry, with just sufficient damar around the

EXPKRIMKN'TS AVITK fOM.OIDAI. DYES 113

edge of tho cover to support it, improves the preservation of the graiuilos.

As has already been stated, the granules in the poijTiiorphonuclears of fresh smears of the blood from these early stages are frequently very small. In such cases one gets the impression that they are the 'special' granules of the leucocytes which have taken up the dye. However, the very coarse and den.se granules which are often seen, especially in the .sections of the vessels from these stages, are identical with the typical dye granules of the macrophages of the tissues, (iranules of tliis type are abundant in the polymorphonuclears of smears from blood or peritoneal fluid which have held the dye in suspension for a longer period of time. Such an experiment with peritoneal fluid will be described later.

Ill the earlier experiments, described in the first paper of this series, attempts to get dye granules in the polmorphonuclears of the subcutaneous tissue, following subcutaneous injections of the dyes, were unsucces.sful. However, good results were obtained with intermuscular injections, which pro\'ed that pohinorphonuelears th;i,t had migrated into the tissues could take up the dyes under certain conditions.

From the earlier experiments it was evident that a polymorphonuclear reaction always follows the subcutaneous injections. But, altliougli there had been no difficulty in getting the polymorphonuclears to migrate into the tissues, it had been impossible to get them to take up the dye, even though the tissue was saturated with it. The .second set of experiments was undertaken maiidy for the purpo.se of clearing up this difficulty. That some progress was made will be shown in the following.

At first the later stages were studied, usually after repeated subcutaneous injections of 1 per cent Pyrrholblau. 5 per cent Trypanblau, or of lithium canuine, filtered and u.-^ualiy (fihited one-half with distilled water. The other two dyes were also alwaj's fihered before being injwted intravenously or subcutancou.sly. This is important in view of the fact that it has been claimed that any considerable difTerence in the size of the particles of the suspension will vitiate the results.

114 HA I. DOWNEY

PolyiiKirphniiurloar leucooytos wrrp seen in many of these lir<pariHit)ii>, i)ut always without dye granules. The niacrophages or histiocytes (Evans. Kiyono) and fibroblasts seemed to have taken up all of the dye wliieh was not bound to the elastic and white fibers.

The early stages, taken within a few hours after a single injection, gave no better results. The tissue frequently contained jiolymorphonuclears, but they were without dye granules. One significant fact however, was noted in these earliest stages, and that was that the elastic fibers were stained ver>' brilliantly (Evans) and that the white fibers had also taken up a great deal of the dye. Various cells of the tissue had also stained difTusely, but no macrophages had deposited the dye in granular form. It was these observations which determined the continuation of the experiments.

The next st<>p was to get a series of continuous stages, beginning with one hour after injection and taken at definite intervals up to the time that the pohiiiorphonuclears disappear from the tis.sues and the dye becomes deposited in granular form in the macrophages and fibroblasts. It was thought deirable to get as

• many of these stages as possible from a single animal and following a single subcutaneous injection, or several injections given

at the same time.

The technique for this is very simple. A few cubic centimeters of the dye are injected into the subcutaneous tissue by means of a 'Record' .syringe. When it is desired to examine the tissue, a slit is made in the skin in the region of injection. With fine forceps a small bit of the softer part of the tissue is hfted up and cut out. This is then .spread out as thinly as possible on a coverglass bj' means of smooth, hne needles. Mopping up the excess fluid with filter-paper will greatly aid in this operation, as it will cause the tissue to adhere more firmly to the cover. Before the preparation has completely dried the cover is floated on Helly's Zenker-fornud mixture and Hxed for irom one-half to one hour. The cover and adhering tissue is then washed in running water for two liours. Tlie fis,sue is gradually dehydrated and iodine added when 7U or 8U per cent alcohol has been reached. Ziehl's

EXPERIMENTS WITH COLLOIDAL DYES 1 1 "»

carlxil-fuchsiii is a pood stuin for thcsf preparations, as it docs not i-han^c tho color of the dyo Kranules.

The following stages have been studied: 2, 3, 4, 5, 6. 8, 10, 12, 14, 16, 18. 24, 30, 36, 48, 116, etc., hours up to several weeks. Not all of the.se stages were ol)tained in any one rat. The twoto six-hour series was run in eaeh of tliree ruts, and tlie eight- to twenty-four-hour series in each of two rats. Different rats were u.sed for the later stages, and some of the earlier and more important stages were olitained at different times from different rats.

Study of the material from these series soon gave an explanation for the absence of dye granules in the poljnnorphonuclears of the previous experiinents. It was found that the subcutaneous injection of the dje was always followed by the migration of numerous polymorphonuclear leucocytes from the vessels, but that the time at which this took place was subject to coasidcrable variation in the different animals. In some eases it occurred as early as five or six hours after the injection, but in others these cells were not found until the twenty- f)r twenty-four-hour stage hatl been reached.

If the reaction occurred during the (>arlier j)eriods the polymorphonuclears contained no dye granules, and this is also true of those cells which were found in the tissue later than the twentyfour hour period. When the polymorphonuclears were numerous at the 12-, 16-, 20-. and 24-hour periods they generally containetl dye-granules, and it seemed to make little difference whether P\'rrholblau, TrA'panblau, or lithium carmine had been u.sed. In one of the most complete series inunerous polymorphs containing dye granules were found at the 12-, 16-, L8-, and 24-h()ur periods. In this .series it was quite evident that the number of cells containing granules was being rapidly reduced in the later stages. In the twentj'-four hour stage there were still many polymorphs in the tissue, but very few of them contained granules. After this time these cells were seen to degenerate rapidly, and in a few more hours they had completely di.siippeared from the tissue. By this time mo.st of the dye had been concentrated in the histiocytes (tig. 3).

1 IG HAL DOWNEY

Tlu- phfiioiiK'Hon se<'uis to be explained by facts which have aln-ady Iwi'ii partly doscribed by Evans and others. It is evident fn)ni tlie alcove that the time at which the poljniiorplionuclears appear is the most unportant factor. Tliis is related to the well-known fact that in the early stages the dye diffuses through the tissue, staining some of its elements from which it is later released. The .storage in the histiocytes and fibroblasts d«)es not take place until later, although the begimiing of this process was seen in several instances as early as five and six hours after the injection.

Figure 2 represents a portion of a field from a thrw-hour stage for wliich no eounterstain was used and figure 1 represents a fourteen-hour stage, stained with carbol-fuchsin. It is quite evident that all of the elements of the tissue have stained intensely with the Trjpanblau wliich was u.sed for the injection in both cases. The cells as well as the fibers have taken up the dye. Figure 2 shows the intense staining of tht elastic fibers, and figure 1 gives an idea of the immense (juantities of the dye which may be ab.sorbed by the hniiphoid wandering cells of the tissue.

If these two figures are compared with figure 3. which is from a celloidin section of the body wall of a fifte<'n-day stage, it is evident that the appe^xrance of the cells and fibers in the earlier stages is verj- chfferent from what it is in the late stage of fifteen days. In the latter the dye has been stored in granular form. and there has been a tremendous multipUcation of cells. The nuclei are fre«' from dye (they are counterstained with carbol fuchsih in this preparation) and the fibers are barely visible.

Considerableshiftingof the d\-enmst have taken place between the stages represented in figures 1 and 2 and the stage .shown in figure 3. Close study of the .series of preparations shows this to be th«' case. The beginning of the storage of the dj^e in granular form is fretjuently seen as early as five or six hours, but at this stage the number of cells containing dye granules is always very small. Their number increases progressively in the later stages, while the cells and fibers which were stained with it in the earlier stag»'s gradually grow paler. In other words, the dye is being liberated bv those elements which had absorbed it soon after

EXPERIMKMTS WITH COLLOIDAL DYES 1 17

the inject ion, and ;it the siinic finic it is bfinn taken np rapidly and stored in the form of granules by fibroblasts and rajjidly multiplying 'macrophages.' While this process is taking place there must be a certain amount of free dye present in the tissue fluid, for if the polymorphonuclears appear at tliis time (twelve to twenty-four hours) they will also store it in the fonn of granules. If th(\v come later the dye has already been disposed of by the fibroblasts and macrophages, and there is none available for the polymorphoiuiclears. If they come too early they get none of it, because it is all bound to the fibers and cellular elements of the connective tissue (figs. 1 and 2).

During the time of storage of the dye in the 'macrophages' the number of Ij'iiiphoeytes in the tissue increases very rapidly. One can easily trace all intennediate stages between those which contain only a few dye granules and those without dye. A.schoff, Kiyono, Evans, and others have claimed that the reactions to colloidal dyes give final proof of the existence of two distinct and independent lines of Ij-niphoid cells. Those of lyiiiph-adenoid (hematogenous) origin presumably never store the dye, while those of tissue origin are marked by their great dye-storing ability.

This reasoning is no more logical when applied to the lymphoid wandering cells of th(> connective tissue than it is for the fibroblasts or polyinorphonuclejirs. For the fibroblast Iviyono admits this very fre<»ly. He cites Ribbert as finding that the number of dye granules in the fibroblasts depended on the amount of injected dye .solution. After from six to eight injections most of the fibrolasts contained granules, but there still remained a few non-granular fibroblasts. With direct subcutaneous injections they all contained granules, but the number of granules in the different cells was still extremely variable.

In conunenting on this, Kiyono concludes that the occurrence of the red gi-anules (lithium carmine) depends on the concentration of the dye in the tissue fluid. Kiyono himself found that in the omentum and loose subcutaneous tissue the number of granules hi the fibroblasts is extremelj' variable. In areas of inflammation he found that th(> fibroblasts which h:u\ l^ecome rounded

lis HAL DOWNEY

off coutuin inon- nuiiuTous ;ind coarser granules, which ho believes is related to the stronger nutritive stn-ani through these cells. His conclusion is, that the intensity of the carmine firanulation differs according to the functional condition of tiio fil)roblasts.

To the writer these two conclusions of Kiyono seem quite logical and well supported by facts. However, when Kiyono will not Jiilniit the application of the same reasoning to the h^nphocytes he is sunMy on \ery unsjife ground.

In the first paper of this series the writer showed that lymjihocytes of a doubly ligatured vessel may contain dye granules, and in this later stud}- of the subcutaneous tissue the siune fact is very evident. Tschaschin and Maximow have also come to the same conclusion, the former from a study of the subcutaneous tissue, and Maxiinow from a study of tissue cultures from lymph nodes and spleen. Why the lymphocytes of the blood stream and lymphoid organs should not take up the dyes under ordinary conditions was explained by the writer in his first paper.

Not all of the polymorphonuclears which arrive in the ti.ssue during the favorable time contain dye gianules. If we apply the reasoning of Aschoff- Kiyono to them we must conclude that there are two kinds of p<)l}^no^pllomK•lear special leucocytes, a conclusion wliich even these gentlemen would liardly venture.

The blood-ve.ssels of the subcutaneous tissue are very active in taking up the injected dyes, as is seen from figure 4, which is taken from the subcutaneous tissue of a rat injected with lithium carmine. This will be discus.sed in more detail later, but for the present it is of interest to note that the polymorphonuclears within the.se ves.sels are frequently gorged with the dye. This is shown v(*r>' clearly in figure 4, where the polymorphonuclears are filled with large masses of the carmine. Exactly the same results wen' obtained with Trypanblau.

.\t the Minneapolismeetingf 1!)I7) of the .Vmerican .Association of Anatomists, .\ddison aiul Trorington gave a paperon theeffects of intraperitoneal injection of Trypanblau. They found numerous polymorphonuclears in the peritoneal fluid, but none of them contained dye, while many of the mononuclears did contain it.

EXPERIMENTS WITH COLLOIDAL DYES 119

III tlir (lisciissioii (if tliis piipcj- the writer siifificstcd tlinf flic l)()lyiii()ri)li(imi('lc>tirs prohubly would Ikivc tiikcii up the dye if it ii.ul l)Oon present in suflieient (|UJintities at the time of their arrival. 'I'he dye disapiiears very ([uickly from the peritoneal cavity. Mueh of it is absorbed direetly by the blood-vessels of the omentum (Shipley aii<l ( 'uiuiinKJiam) and the lymphatics of the central IcikIou of tlic diaphragm, and some is stored by the cells of the taehes laiteuses. clasmatoeytes. etc., of the omentum and by the mononuclear cells which are nonnall}' present in the ix'ritoneal fluid. Unless the poljnnorphonuclcars arrive very soon after the injection they will find very little free dye.

That J)<)l^^norphomIclears which migrate into the peritoneal cavity may take up Trypanblau if it is mail(> available for them is shown l)y the results of the writer's experiment with rats 44 ami 45. These rats wer(> injected intraperitoneally with 5 cc. of a ") per cent Trypanljhiu solution. Sixteen hours later .some of the fluid was removed by means of a fine pipette and examined. It contained numei'ous j)olymoi'phoniiclcai's. l)Ut they were without dye fii'anules. Another injection of 4 cc. of the same solution was then pveu, and fresh prejiarations examined and smears made six hours later. .Vs was to be expected, many of the polymorphonuclears now contained numerous dye granules which were identical with those of the monouclear cells. Many of the granules were suri)risingly large antl dense.

In order to make sure that the marked phagocytosis of the dye on th(> part of the polymorphonuclears was not due to the addition of salt to the .solution, rat 4.3 was injected with Trypanblau made up in distilled water in place of the phy.siological salt solution us((l in mo.st of the experiments. No differences could be (let(clcd lietween the material obtained from this rat and that obtiiined from rat 44 which was injected with Trypanblau maile up in sah snhitinn.

The results of this expi-riment set'in to indicate that, in this ca.se at least, phagocytosis does not depend .so much on the size of I lie particles which irritate the cell nuMnbrane (.\ddison and Thoriiigton) as it does on the availa'nlity of the material to bo phagocytoscd. It is tniethat thesizeof th;Mibject tob,' jihagocytoscd may

THE ANATOUIrAL KECOHP. VOJ.. 15, KO. 3

120 HAL DOWNEY

havo soincthinK li> do witli llii' rcacliDii in so tar as particular types of ri'Us an- iiivnlvcd, as is iiulicatrd l)y Mctclinikoff's classilicatinn of the Icunicytcs into ■iiiacroiiliafics' and 'niicmiiliaKos.' However, lliere are plenty of exceptions to tiie rule. TliusSciiott doscrihes I lie pliatocytosis by jiolyinori)honuclears of ontiro erythrocytes whidi were inject<'d into the peritoneal cavity, and Rowley describes a case of anemia in which all of tiie leucocj'tes of the l)lood were veiy active in the jjliagocytosis of red cells. Evidently pha}j;ocytosis depends on soniethinj; nion' tli;in the irritation of the cell membrane l)y particles of varyinji size.

in view of what has already been reporteil in the literature, it is strange that some investigators still believe tliat the colloidal dyes cannot l)e phagocylosed by leucocytes. Hibbert, who was one of the first to use lithium carmine, rejMjrted that leucocytes which enter atretic ovarian follicles contain carmine granules. Pari, a student of Ribbert's, found that by ligaturing the ductus choledochus he could get ])olyniori)honuclears in the heart blood which contained hthium carmine gnmules. In the same animals large Ijinjihocjies, large mononuclears, and transitional cells also contained cannine granules. Loele, working with Isaminblau, inj(>cted one mouse wliich had a dej)osit of pus on the surfaces of the i)leura, liver, and sjjleen due to pleuritis and peritonitis. Many of the polymorphonuclears contained blue granules. I.oclc's interpretation of this is interesting. Tie believes that the i)olyniorphoimelears do not (wdinarily stain with the colloidal dyes, because their granules are able to destroy (reduce) the dyes as rapidly as they come in contact withthem. Intheperiotonitis niou.se the leucocyte graiudes are .stained because they are .so injured b\- Iiarniful agents that they are no longer able to destroy the dye.

Cloldniann also ol)served dye granules in polymoriiliomiclears and large mononuclears of the blood after repeated injections of Trypanblau. However, he interprets these granules as pathologic foniiations, rather than as examjiles of true vital staining.

In experimental pneumonia Kline and Wint;'rnitz found dye granules in the involved jiorlioiis of the lungs which wen- cut off from the general circulation by plugs of fibrin in ihr capillaries.

EXPERIMENTS WriH COLI.OIDAI, UVES 121

For the lyiiii^hocyfos, Muxiniow scciiis to Ikivc (Icnioiistriifcil hcyoiid (|U(stion that they Jirc ublc to tiikc up the colloidal dyes !ift<T tlicy liavc undcrKoiic further differentiation. (loldnuiun and 'l'-(li:i<clnri >liould :i\«i lie li-lcd anions those wlio l)elieve thai l_\ni|)lioc\tes may phaficx-ylose theeulloidal d\'es, alt hough they I)oth believed that the phenomenon was one of ti'ue vital staining of preformed structures or of structures fonncd under the influence of the dyes.

While hunting through the tissues of different stages for polyinor])honu(lears containing dye granules, some interesting observations were made on the early reactions of the tissue to the dyes. Some of these observations do not agree with what has been previously reported in the literature. This is becau.se most of the previous work has been confineil to a study of the later stages, especially after the animals had been given repeated injections of the dyes.

It has l)een generally assumed that ditTuse staining of the cells, ('s])( cialix- of theii' nuclei, is alwa>'s an inilication of a pathologic condition of the cells or of their death. I'ari and others have shown that injured cells .stain diffusely with lithium carmine, l)Ut according to Pari dead cells do not stain at all with the carmine. These observations, together with those of (Joldman. Kiyoiio, Kvans. etc., all ajiply to the later stages.

If this rule holds good for the earlier stages, then we are forced to conclude that Trypanlilau is tpiite toxic for all of the cells of the connective tissue, for in the earlier .stages they all stain more or less intensely with the dye. Especially the nuclei show a great affinity for the dye soon aft(>r it is injected. Fibroblast nuclei are freciuently .stained when their e\toplam contains none of the dye i ligs. 1 and 2, FbL). The free wandering cells <if the tissue and the clasmatocytes usually ab.sorb much more of the dye, as is .shown especially well in the fourteen-hour stage from which figure 1 was drawn.

Th(> upper, large cell of figure 2 shows the beginning of a i)rocess which is nior*- marked in the later stages, esi)ecially in the fourtecn-hour stage of figure I. The miclei of the free cells are verj' dark from the large (luantities of dye which they have ab

122 HAL DOWNKY

sorlx'ii. 'riif pcriplicnil ixirtimi of the cytoplasm is also very (lark, l)Ut that part of it which iiiiincdiatcly surrounds the nucleus is <'oniparativcly free from dye. The picture resembles (ho so-call( (1 Hiincfcid-IIcnscii pictures obtained wh.-^n 12 per cent cane sugar is added to Anipliibian blood. Meves explains the resulting changes of the erythrocytes as follows. The sugar solution produces a firm precii)itation membrane at the surface of the erythocytes. while the hemoglobin in the interior of the cells remains fluiil. \\ith the death of the cell the nucleus swells, and in so iloing it absorbs th(> more (luid hi'moglobin, while the surface membrane and the marginal band prevent the collapse of the corpuscle.

The free cells of the connective tis.sue are e\'idently similarly afTected by the Trypanl>lau, for in general appearance they are ((uite similar to the Amphibian erythrocytes which have been treated with a sugar solution. In lUbS, the writer described somewhat similar ])icluri's wliicli i-csulted fi'oiii the Iri'atment of mast cells with a(|Ueous solutions. The soluble metachromatic sub.stance of the granules dil'fuscd into the nucleus just as does the hemoglobin in the case of the erythrocytes treated with sugar .solution.

The cells of figure 1 seem to have passed through a similar jirocess. The dye has jirobably coagulated th(> pei-ii)lural cytopla.sm. but enough of it has diffused into lli>" luicleus to cause the lattn- to sw( 11 ami to absorb the sui'rounding mor,' fluid portion of the cytoi)lasm, with the dye which it contained. This condition docs not la.st long, for in the twenty-four-hour and later stages such cells are quite exceptional.

By no means all of the free cells are in this condition. Some of them contain v. ry little (ly>', and in some it has already begun to be deposited in the form of (li>linel dye granules as early as five and six hours after the injection, fn the late stages the numlx r of tin .se latter cells increases rapidly, while the number of the vacuolated cells with the dark nuclei decreases. It is (lifTicuIt to .say just what becomes of all of thes;>. If they all degenerate one would expect to find far more degencj'ating ells in the I)reparati<in than are actually present. .V few degenerating

EXPERIMENTS WITH COLLOIDAL UYES 123

cells are always prosont, even in the early stap^. but tlieir appearance is very different from that of the cells which contain so much of the dye. Their nuclei become pyknotic and stain intensely with the carbol-fuchsin which was used for a counterstain on most of the prcpanitions, while their cytoplasm contains little or none of the colloidal dye. Such a degenerating cell is shown in figure 1, deg. Its nucleus is the only structure in the field which is stained with the carbol-fuchsin.

Most of the fibroblast nuclei of these early stages also contain more or less of the Trypanblau, but it is quite evident that few of these cells degenerate. Intermediate forms between the dye cells of the early stages and the dye-granule cells of the later stages could not be found, so it would seem, that most of the dye diffuses out of these cells to be deposited later in granular form by other cells. It is quite possible that some of the dye may remain in the cytoplasm of these same cells to be worked over into granules later. However, all of it eventually leaves the nucleus, and much of it is probably set free at the same time that the dye is diffusing out of the fibers, for if the pohTUorphonuclears reach the tissue at the time when the nucleus and cytoplasm of the cells in C[uestion have begun to grow pale they are able to get enough free dye to be able to store it in granular form.

In these early stages the endothelial cells of many of the capillaries and smaller vessels are in the condition of the large, dark, vacuolated cells of figure 1. Later they lose every trace of the dye. The statement which has often been made, that endothelial cells of the vessels do not store colloidal dyes, applies, therefore, to the later stages only.

Although it can be shown that cells hke the ones illustrated in figure 1. with the nuclei filled with dye and dark peripheral cytoplasm, later give up the dye and recover, still one could hardly claim that they are normal anil healthy while in this condition. It is quite likely that they have been injured by the dye, and that the staining of their nuclei is due entirely to its toxic effects. The presence of luuiierous cells with df-nse pyknotic nuclei staineil deeply in carbol-fuchsin, cells which are clearly degenerating, is further evidence of the toxicity of the dye when it first

124 HAL DOWNEY

coiiH's in contact with the tissues. It is clear that some cells are killed, and that others are able to rid themselves of the dye, especially that which has been absorbed by their nuclei, and repain their foniier nonnal appearance.

Anions the cells which show the most injurious effects of the dyo are the smaller wandering cells of the connective tissue and the eosinophil leucocytes, which are always abundant in the normal subcutaneous tissue of the rat. The behavior of the latter is of special interest, for in them we have a case of true staining of preformed struct iu"es which is almost invariably followed by the death of the cell.

All through the earlier stages, and in some cases even in the very late stages, granular cells were found w'hose nuclei were in various stages of degeneration. The most common fonii of the.se cells is illustrated in figure G. Its nucleus is very pyknotic and stained intensely with earbol-fuchsin. The cytoplasmic granules have stained with the Trypanblau used for the injection. In many other cells of this same tj'pe the nucleus is still smaller or it has broken up into several pieces. The staining reaction of the granules varies somewhat, although in the cells with pyknotic nuclei they are alwaj's quite dark, and they never take any of the earbol-fuchsin.

Numerous cells of this type had been observed before it became possible to classify them, although it was evident from the first ones observed that they did not belong to the dye-granule cells (histiocytes, macrophages) shown in figure 3. The regularity of size and shape of the granules, and the comparative freedom of the intergranular protoplasm from dye, together with the pyknotic imcleus .stained in earbol-fuchsin and the fre(}uent perinuclear .space (fig. 6), immediately placed these cells in a group by themselves. It was not until cells like the one of figure 5, and others grading from this to the type drawn in figure 0, were found that it be<'aiiie jiossible to classify tliis group.

The only granular leucocytes of the rat which have a ringshaped nucleus similar to the one of figure 5 are the eosinophils. Comparison of the eosinophils of untreated nonnal rat ti.ssue with the cells in question in the vitally stained animals .shows that the latter are eosinophil leucocytes.

EXPERIMENTS WITH COLLOIDAL DYES 125

ThcmicU'Usof tht'eosinophils never stains with Trypaiiblau. but their jaanules are often brilliantly stained, even before the nucleus shows any degenerative changes. As the granules take more of the dye and enlarge somewhat Cfig. 6) the nucleus degenerates rapidly, and finally becomes reduced to a small, dense, pyknotic sphere. This is the condition of most of the eosinophils encountered, although cells in more advanced stages of degeneration are by no means rare.

In the eosinophils we have an example of true vital staining of preformed granules with a colloidal dye. This must be clearly distinguished from phagocytosis aJid storage of the dye as it is illustrated in the cells of figure 3.

Since the cell of figure 6 is taken from an eight-hour stage, it is evident that the toxic action of the dye may cause verj- rapid degeneraton of the eosinophils. However, the cell of figure .5 was found in a preparation of a fourteen-hour stage, from which we must conclude that either the eosinophils of the injected area are not all killed at the same time or that others migrate into the region of greatest dye concentration from outlying regions of lower concentration. The large ninnber of degenerating eosinophils in some of the later stages would seem to favor the latter view.

That true vital staining of preformed structures may occur while other cells are phagocytosing the dye and storing it in granular fonn is by no means a new idea. Evans and Schulemann ('15) admit this very freely when they claim that the granules of the epitht^liimi of the plexus chorioideus, hypophysis, epithelial body, and adrenal body are vitally stained 'secretory droplets.' They compare this staining with that of the elastic fibers and cartilage matrix, for which it is not claimed that it is due to phagocyte wis.

The eflfect of the dye on the eosinoi)hils, the numerous degenerating cells of other types, the staining of the nuclei of the fibroblasts and hmiphoid cells, together with the frequent vacuolization of the cytoplasm about the nucleus of the latter in the early stages, seem to indicate clearly that the dye has a decidedly toxic action when it is fir.st introduced into the tissue. Th(> eo.sinophils, however, seem to be the onlv cells which are unable to recover

12t) II A I. DOWNEY

fmm tlu' «>ff("<'ts of it. Lutrr, when all of the dyt> has been stored in Rranuliir fonn in the fibroblasts and histiocytes it seems to bo conijiarativcly harmless, for degenerating cells are not so numerous, although they are never entirely absent from the preparations.

The contUtion of the blood-vessels of the subcutaneous tissue is of special interest in view of what has been n-ported for the blood-vessels of the omentum by Shipley and Cunningham. These authors proved exp.'rimentally that not only the capillariee, but also the larger veins and arteries of the omentum are verj- active in the absorption of solutions of foreign matter from the ptritoneal cavity.

The writer's experiments with sul)cutaneous injections of colloidal dyes show that the vessels of this tissue possess similar absorptive powers. M(»st of the material which was gathered within from ten to twenty hours after the injection contains snialhr and larger vessels which are filled with the dye. This conditfiu is of sueh fr-cpi^'nt oeeurrenee tliat it ean hardly bo due to accidental puncture of the vessels during the injection. The dye seems to pass through the endothehal cells, for in the earlier stages they are stained intensely with it. I^ater, as in the eighteen-hour stage illustrated in figure 4, the endothelial cells are free from dye, but there is a groat deal of free dye in the lumen of the vessel, and the polymorphonuclear leucocytes of the vessel eontjun dyv' granules. The mat(^rial was taken from a rat injected subeutan^ <»usly with litliium carmine. A mass of free cannine (Car.) is shown in the lumen of the vessel, and the polyninrphonulears (Pm.) contain large cannine granules. The endothi'lial c.-lls of the vessel are stained with the hemalum used as ;• eounterstain, but not with the carmine.

The vessels of nunu rous rats injected with Trypanblau show this sjuiK" condition, so it is evidently not of accidental occurrence. A nirchanism for the rapid distribution of the dye to all parts of the b)dy is. therefore, supplied by the blood-vessels. The exi.stence of >uch a mechanism seems necessary in order to account for the extreme rapidity with which some of these dyes are able lo reach the most remote parts of the body following single

EXPERIMENTS WITH COLLOIDAL DYES 127

intraporitonoal or subcutanoous injoctions. The part phiyctl by the blood-vosscls would !)»' surprising if the work of Shi pl«\v and Cunningham liad not aln'ady demonstrated their al)ility to absorb foreign fluids.

SUMMARY

Poljinorphonuclears are ver>' active as phagocytes for Trj'panblau and lithium carmine, but in order to demonstrate this in the general eirculation the blood from the larger vessels must be examined within from one-half to two hours after the intravenous injection. Pohnnorph<inuelears in the lung, spleen, and liver removed within this time limit may also contain dye granules before the reticular cells of these organs have gathered up any of the dye.

Polymoj'phonuclears in the vessels and tissues of the organs will also phagocytose living bacteria injected intravenously, but those of the general circulation will phagocytose the bacteria only when the latter arc injected into a doubly ligatured vessel or after they have reached a vessel or sinus in which the velocity of the blood current is greatly reduced.

Subcutaneous injections of the dyes always cause the migration of numerf)us pol\inorplionuclears from the vessels, but the time at which this reaction takes place varies in different animals. If the leucocytes are numer<jus during the twelve- to twent3'-four-hour period they usually contain dye-granules.

Pol^^llorphonuclears of the peritoneal cavity will als(» take up Trypanblau and lithium carmine if the ilyes are injected after the leucoc^ies have reached the peritoneal fluid.

Phagocytosis of colloidal ilyes by jiolymorphonuchnir leucocytes depends entirely on the availabihty of the dye rather than upon any inherent physiological lUfference with respect to phagocytosis between these cells and the mononuclear macrophages of the tissues.

The three colloiilal dyes used in these experunents have a decideilly toxic jtction on the tissues when they are first introduced. .Vll types of cells in the connective tissue show diffuse .staining of cytoplasm and nucleus in the early stages. Many

128 11 AL DOWNEY

(t'lls iurv killed, but members of all groups of cells are able to eliiuinnte the dye and rt^cover, except mg the eosmophils. All of the »'<)sim)phils located in the region of the hijection are killed by Tryi)anl)iau, hut degenerative ehanges in their nuclei do not occur until after their granules have become brilliantly stained with the dj'e.

Bl<K)d- vessels of the subcutaneous tissue absorb large quantities of the dyes and probably carry them to other regions of the body. Endothelial cells are stained in the earlier stages, but not in the later stages when the vessels are full of dye. The polymori^honuclears within the vessels contain dye granules.

The experiments have brought additional evidence in favor of the view expres.sed in an earUer paper, that the intra vitam reaction to colloidal dyes is not specific, and that it does not serve to distinguish between blood-cells and tissue macrophages (histiocj^es of Aschoff-Kiyono, macrophages of Evans).

EXPERIMENTS WITH COLLOIDAL DYES 129

LITEKATURE CITED

For titles not listed here see bibliography of the first paper of this series, Anat. Rec, vol. 12, no. 4.

Downey, H. 1913 The development of histogenous mast cells of adult guineapig and cat, and the structure of the histogenous mast cells of man.

Folia Haematol., Archiv, lid. 16.

1917 Reactions of blood- and tissue-cells to acid colloidal dyes under

experimental conditions. .\nat. Uec, vol. 12, no. 4. LoELE, W. 1912 Uber vitale Granulafarbung mit sauren Farbstoffen. Folia

Haematol., Archiv, Bd. 14. Meves, F. 1911 Gesammclte Studien an den roten Blutkdrperchen der .\mphi bien. Arch. f. Mikr. Anat., 1, Abteil., Bd. 77. P.\Ri, G. A. 1910 Uber die .Vnwendbarkeit vitaler Karmineinspritzungen fur

die pathologische .\natomie. Frankfurter Zeitschr. f. Pathologic,

Bd. 4. RiBBERT, H. 1914 Die .\bscheidung intravenos injizierten gelosten Karmins in

den Geweben. Zeitschr. f. Allgem. Physiol., Bd. 4. Rowley, M. W. 1908 A fatal anaemia with enormous numbers of circulating

phagocytes. Jour, of Exper. Med., vol. 10. Shipley and CcN.saNGHAM 1917 The histology of blood and lymphatic vessels

during the passage of foreign fluids through their walls. .\nat. Rec,

vol. 11.

PLATE 1

KXPLA.NATION OK FIGURES

1 Siihcutanoous tissue of rat. fourteen hours after subcutaneous injection of Trypanblau. The pyknotic nucleus of the deKeneratlnu cell, Deg., is the only structure stained with the carbol-fuchsin used as a counterstain. All of the other structures are stained with the Trypanblau. The large dark cells with the clear perinuclear space are of special interest. They are larp- lymphoid cells which have ab.S(>rbed great quantities of the colloidal dye. Fbl., fibroblast nuclei.

2 Subcutaneous tissue of rat. three hours after subcutaneous injection of Trypanblau. No counterstain. Shows staining of all of the fibers and cells of the tissue. A large lymphoid wandering cell which ha.s absorbed a groat deal of the dye is shown in the upper, left-hand portion of the figure. The nucleus cif this cell and of the fibroblast {Fhl.) stained very dark.

i:»

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PLATE 2

EXI'I.WATIDN OP FIOUIIES

3 .\l>ili>minal wall of rat. ("clloidiii section. Six subcutanoous injections of Trypanlilaii nivcn at intervals of from two to three days. Animal killed fifteen days after first injection. Coiintcrstained witli carbol-fuchsin. Macrophages and Unn|ihocytes of various sizes loaded with dye granules. The fibroblasts (two in the center of the field) also contain numerous fine dye granules which are very different from tho.se of the macrophages.

i IJIood-vessel from subcutaneous li.ssue of rat, eighteen hours after subcutaneous injection of lithium carmine. Counterstained with hcnialum. Shows absorption of the dye from the tissue. Car., a mass of free carmine in the lumen of the vessel; I'm., polymorphonuclears containing large carmine granules; Ery., erythrocytes.

5 Eosinophil leucocyte from subcutaneous tissue of rat. eighteen hours after subcutaneous injection of Trypanblau. Counterstained with carbol-fuchsin. (iranules sl.'iined blue with the Trypanblau, nucleus stained red with the fuchsin.

ti Degenerating cosiMophil leucocyte with pyknotic nucleus from subcutaneous tissue of rat. eight hours after the subcutaneous injection of Trypanblau. Counterstained with carbol-fuchsin. CSranules blue, nucleus red.

i:}2

KXPKHIMKNTS WITH CX)l,l^)II)Ar. DVICS

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133

ADTUOK I* Ali-^THACT OF THI^ l'\PCII IIWlCrD HY TBB BIBLIOONAPUIC IBIIVICI. HBPTOIDER 1ft

THE K.MBRYONK' STRUCTURE OF A\ lAX HEART MUSCLE WrrH SOME CONSIDERATIONS REGARDING ITS EARLIEST CONTRACTION

E. D. COXGDO^f Anatomical Department of Leland Stanford Junior University, California

TEN FIGURES

The long-standing differences of opinion regarding the structure of adult heart nuisole have not as yet been brought any nearer to a reconciliation by the study of its simpler make-up in the embrj^o. In the various accounts which have appeared of the histogenesis of muscle in higher vertebrates, the mj'ofibril has been derived in turn from granules in rows or in groups of four, from rods and filaments, from nets combined with granules, and from a honeycomb structure either combined with or free from granules. No one view has ^ecei^•ed the adherence of even the majority of the more recent writers. The obser\-ations which follow relate to the earlier stages in the development of the heart muscle and were made upon chick embrj-os of the first four days of incubation. They suggest a reconciliation of the divergent views regarding the early contractile structure.

Photographs were emploj'ed to mea.sure certain elements of the sarcoplasm and also as a control to the conclusions based upon microscopical study. Several po.ssible errors need to be considered before the trustworthiness of this method of measurement can be admitted. Spherical aberration was avoided by using <inly a small central ar(>a in the microscopic field. Foreshortening was readily eliminated by measuring only those planes and lines all of whose granules were in sharp focus. It is not po.ssible, unfortunatel}', to do away completely with changes in the (liincnsioiis of the sarcoplasmic structures during preparation

TUE ANATUHICAL HECURD, VOL. 15, NO. 3

\'M\ E. D. CONGDON

f<ir thf slid«'. The usual Imlanoc was maintained, howovcr, botwtH'U tho fixiuR agents which contract and wliich swell the tissue. Shrinkage due to the passage of the tissue through the paraffins was reduced to a miiiiinuni by shortening the process to four minutes. The preparati<ins give but slight evidence of this defect.

The fixation was by Zenker's fluid or a combination of osmic acid, pota.ssium bichromate, acetic acid, and normal salt solution. Tiie .second fonnula was found the more serviceable of the two. The best results were obtained with the osmic acid only when its temperature was controlled during fixation. The staining was by iron haematoxylin. Its entire manipulation was carried on in 70 per cent alcohol, as water has an unfavorable action upon the mitochondria. It was impossible to obtain entirely .satisfactory fixation of the older heart because its bulk is so great as to prevent an exact control of the action of the osmic acid. The sarcoplasmic structure if well preserved is so dense that microscopic sections of 5 micra were found too opaque to give a clear view of the details. A thickness of 2| micra was the most satisfaotorj'.

During the developmental period ending with the fourth day a primitive .sarcoplasmic structure is present which gradually undergoes changes adapting it to its function of rhythmical jjulsation. The heart mu.scle in ten-day chick is made up entirely of this primitive type. It can be traced back to the earliest distinguishable heart rudiment. In optical section the .structure is made up of a series of granules staining with haematoxylin and connected by fine lines (figs. 1 and 2). It extends throughout the cj'toplasm. The areas included bj^ the lines are usually parallelograms approaching the form of a square. They are approximately 0.8 micron on a side. Triangles and various tj'pes of quadrilaterals also are seen.

The granules are shown well by an osmic bichromate fixation when followed by haematoxylin. The higher alcohols, oil of biTgamot, and xjlol partially dissolve them. Witliout cnlering into a di.scus.sion of the variations in definition of the term mitochondria, it is clear that it will be in agreement with the views of

KMltinoNK STUl rTflUC OK AVI.W IIKAHT MISCLE 137

I'lci. I riansvrrsc scitinii ul' priiiiilivc stn:ik ni i-liick i IckusciI al surface of spot ion). X (MX).

Fl(J. 2 Transvi'isc section of liearl of a len-soinile chick. X IKX).

Kip. 3 Growth in iilasiiia I'ulttirc of heart tissue from a chick of four days' inciiliation. X -70.

13S K. 1). <'ON(iDON

most of those occupied willi llie siil»jecl if (he term is jippHed to the pramiies.

The network wiiicli lias been deserilx'd could be pinduced as the optical section of a mesh in three tlimensions or of a system of jilanes completely enclosing protoplasmic bodies. The decision between these alternatives in the case of various tissues has long occupied the attention of those attempting to detennine the ultimate microseo|)ic structure of protoplasm. It is well known that the value of ob.servat ions upon the (incr details of fixed protoplasm as a means for determining the make-up of its living structure has been called seriously into (piestion by the di.scovery that various coagulation patterns can be produced in solutions physically similar to protoplasm by the use of difTerent conditions of fixation. It is of interest, then, that the hexahedra are fountl no matter whether osmic, formol, or coi-rosive sublimate fixing fluids are used. Also three observers lunc given good reasons for bs'lieving that it gi'adually changes during development into a structure which few would denj- as an actual element of the living adult muscle, namely, the myofibril. Evidently, then, we are not d(>aling with an artefact and the relative likelihoods of the net and the honeycomb being the reality in the sarcoplasm has a vital interest not only in r.'lation to the make-up of muscle, but also because of its bearing upon tlie ultimate structures of protoplasm.

Of the three observers who describe tiie network in optical section, MacCallum refers to the sarcoplasmic structure of the embrj-onic pig heart as a me.sh. .Vt the same time he apparently believes that it contains flat membranes, since Ik^ speaks of .sarcoplasmic discs surrounded by the net. Briick also uses the word net in his discussion, ^'et he (Icfinitely states that then is a honeycomb structure. Wieman believes that in the chi<'k heart there is a net in three dimensions and applies to it the name cytoreticulum. It is n iufen-etl that they are too dclicati- to I),- visible except when jilaced nearly edgewise to til,' observi r. A ^ireat majority of t he spaces must be six-sided, since their cri)ss-section is so uniformly a (|uadrilateral figure. The structure, then, can well be described as hexaheilral.

CELLII.VK IM)!:i'i:XDi:XCE

A controversy n'gariling the distinctness in the .separation of the cell areas has been waged sine? the time when the cell theory was formulate<i. The d-cision in the adult heart depends on the interpivtation of the intercalated discs. These do not enter into the {[uesiinii for the eiiibryoiiic oi'-ian because they make tlieir a]ii)(arance late in development. It is generally agreed tiial wlun it is first distinguishable the h(>art rudiment, like the mesenchyme, is made up of cells s;'parate except for a few fine proc:ss3s. A iiuiiil)er of observers of heart development in birds and nianmials believe that flu'V soon fuse. In this group are Chiarugi I'STj. Hoycr ("01), Heideidiain (,'9*J), Kurkiewicz ("09), and Duesberg ('10). .V loss of identity is not admitted bj- MacC'allum ('97). Wieman ('07), and Schockaert ('09).

A comparison of sections of youngi-r ancl older heart ventricles indicate the possibility that there are .slight distinctions between the (1. gr. e of ell indcp. ndonce at three succe.s.sive ages. .Vt no time, hoWv'Vi r, was a cell membrane made out. In the ventricle of a tcn-.somite chick (here is a suggestion of cellular independence consisting of an arrangement of hrxahedral structure concentric to each nucl( us. \\hen a region is found, however, where the condition of tile planes can be well made out all the way acro.ss from one imcleus to another, no break in tlidr contiiuiity is discovered or ol h( r modification to indicate a cell boundary. There is a lobatioii of the surfa<'e of the myocardium corresponding to each subjacent nucleus. The heart of .somewhat ohler embryos, including

140 E. D. CONGDON

the tliirtfH^n-somito staRo, contains small clefts in tlic interior nf tlic niyocanlinni partially iliviiiinK coll aroas(fi)i. 'Ji. Kurkicwicz ("OU) has (li'scril)t'(l thcni at Iciifith. l^vidcntly, then, the apparently coniplcto continuity of the sarcoplasni in tiic jjrcvious stage is tleceptive. The four-day heart no longer contains clefts. There are portions where the planes of the liexahedral struclure take a perfectly rectihnear course past several miclei (figs. 4 to 0). If our conehisions were to he l)ased on these localities alone there could be iiltlc doul)! of tlic complete fusion of tlie cells. Other regions have a more irregular ajipearance.

In a previous paper ('15), which describes the migration and growth of four-day chick ventricle in plasma cultures, it was found that the sarcoplasni extended out into tlie clot in anastomosing multinuclear columns whose only indication of cell Ixiundaries (^lig. 'A) are light constrictions of the columns. The complete cohesion of the tissue under the.se conditions certainly speaks for a close union between its cellular elements.

.S'hockaert ('09) gives the only specific statement the writer has found that there are cell membranes in the embryonic heart. She publishes a photograpii of a section from a .seventeen-day ralil)it lieart which contains markings roughly simulating cell boundaries. The finer structure of the sareophisin is too completely lacking to make pos.sible an understanding of its original character. In Wieman's study of the chick lunut he does not dcscrii)e cell memiiranes, l)ut he has obtained mononuclear fusiform .siircoiilasmic bodi(>s by maceration. Thes(> caimot l)e exphiined away as coming from regions of the atria which are tardy in tlieir difTerentiation because some of tiiem are figured with a well-develoi)ed contractile structure. Kvidently, in spite of the indications of complete fusion .seen in the hex.diedral sy.stem .'ind of almost complete coalescence in plasma cultures, .^^ome kind of structural demarcation between cell areas is maintained in embryonic heart. It is in liarmony with this conclusion that although no cell boundaries are visible in the heart of the teii.somite chick, yet later a partial separation takes place by means of clefts.

KMHKYONIC STRUCTURE OF AVIAN HEAHT MUSCLE 141

? '» ^.

4

\

Figs. 4, 5, and 6 I'liotoftruphs at successive optical levels of ;i triilx-ciiln from heart of chick of fo\ir days' incubation.

142 E. D. CONtiDOX

Tin: sTurnriu-: of thi: i^hvthmically-contracting heart

All ('xaminatii)n was iiiudc of elovrn chick cmbrj'os containing from fifteen to scvontcon somites to learn the time of the beginning of i)ulsation. The heart was beating in four wliich were in the seventeen-somite stage, but in none of the younger chicks.

Begiiming witli tlie heart of the sixteen-somite embryo, series of parallel bars were found in the sarcoplasm which were believed to mark areas fixed in a condition of normal function. Their fonnation will be best understood after the sarcoplasmic structure has been described.

The comparison of the sarcoplasm of th(> non-beating heart with a later stage shows surprisingly little developmental change in structure. The volume of the hexahedral spaces is still the same. The granules have increased in size. In some regions the hexahedral structure has an irregular arrangement as in the younger heart. \l\ gradations occur between this condition and a nearly perfect alignment of the planes with intersections approaching a right angle. For the developmental jieriod with which we are concerned alignment is best seen in longitudinal .sections of the trabecular which make up the spong>- portion of the four-day ventricle. Where it is fountl the parallel bars usually also occur. They are associated with a uniform elongation of the hexahedral spaces in a common direction and result from a drawing together of the granules in the planes transverse to the direction of extension (fig. S). The optical section of the spaces in extension frequently have a length of 1.2 micron anil .•I breadth of 0.(5 micron.

Contraction pluises were made out less frequently than the opposite functional condition. It is possible to come upon regions, however, where the hexahedra are elongated transversely to the long axis of the trabeeulae. ' In describing the extension and contraction onl}' the two dimensions of the hexahedra parallel to the plane of t he cover-slip have been referred to. The spaces are so .•^rnall that the corresponding changes in the direction parallel to the optical axis were not satisfactorily determined. It is jjrobable that the greater irregularity of arrangement of hexa

EMBKVONIC STRUCTURE OK AVIAN HEART MUSCLE

143

♦^ >•

^,

■dTcS

10

Fig. 7 DiuRniin of mitochondrial granules and outlines of hexahedrnl spaces of sarcoplasmic structure before the beginning of rhytlimic pulsation.

Fig. 8 Optical section of hexahedral structure in condition of fimctional elongation.

Fig. '.t Tr;insvcr.sp section of heart wall of a Ihirteen-somite chick, showing clefts between cell areas.

Fig. 10 Diagram of a hexahedral space with it^ membranes and granules.

144 E. D. CONGDON

hodra in \hv coinpaot wall than in the traboculac is due to a less uniform diroolion of contraction and extension, since the wall is not only a curved plane, l)Ut has trabeculae of the spongy niyocardium attaching to it in an irregular manner. The trabeculae, on the other hand, are cylinders attached only at the ends and so must contract in the direction of their long axis.

There is a certain amount of disorganization in the siircoplasmic structure common to the wall and the trabeculae. It is probably the result of agonal contractions that have disturbed the normal pattern at the time of death. \'arious methods were tried in order to fix the heart without the breaking up of the regular beat into irregular local contractions, but without success. A second abnormal feature that is to be seen in some regions of the heiirt is taken to be the effect of unsatisfactory killing and fixing. It consists of a massing together of parallel planes into long bands together with a partial disappearance of the transverse planes and the mitochondrial granules. It may be that the mitochondrial substance has become spread upon or through the planes as under these conditions they take a deep stain.

The consideration of the majority of earlier views regarding the origin of the myofibril can be brief, since they are plainly based upon preparations showing but incompletely the structure first seen by MacCallum and described in the preceding paragraphs. It is not necessary to consider separately the work on heart and skeletal muscle or to distinguish between the bird and the mammal, since variations in the character of the myofibrils in all of these instances is but slight. Wagener ('80), Mtodowska ('08), and Krukiewicz ('09j clahn that the myofibrils when first identified appear to a microscopic examination as structureless filaments. Ej'cleshymer f'04) came to a similar conclusion for the skeletal muscle of Necturus. Marceau (02) is not certain whether they are .segmented or not. Bardeen ('11) refers to them as having no definite cro.ss .striation. Since the development of the mitoch<indrial concept many have become convinced that in one form or another it is the precursor of the nnofibril. Meves ('09), Ducsberg ('10), Asai ('14, '15), believe that the myofibrils can be traced back to the mitochondrial rods or

KMHHYONIC STRUCTURE OF AVIAX HEART MUSCLE 145

'chondriokonten,' and Torraca ('14) finds a similar oriKin duririK their rcKciifratidii. IJriick ('()()). flodlowski r02). and Rubasohkin CIO) derive the niyofihrii fnnii granular niitoehoiiih'ia. Luna ('13) finds that Ki'iHi'dc^ ajjpear first in skeletal muscle while in the heart the primitive eondition is an unsefjinented fibril. Altnian in 1894, before the name mitochondria had bet-n introduced, exfiressed a belief in the {^lanular origin of the myofibril.

It is not (lillicull to understand how many observers came to believe that granules, rods, or filaments jm'ceded the adult myofibril. Each of these structures can be found in the siircoplasm if the technique does not bring out the hexahedra and granules in their totality. .\s already indicat(>il. heavy short rods and structureless filaments api)ear when the hexahedral structure is not perfectly pr(>served. Tlie fine granules described by (lodlcwski ('00) were in many in.stances .strung along slender filaments. He saw in part both elements of the sarcoplasmic structure. Schlater ('()(•) distinguished even the optical .sections of entire liexahedra with their granules. He did not, however, obser\'e them to be united in a continuous structure throughout the protoplasm.

It has ahvatly been said that MacCallum ("97), Wieman ('07), and Briick ( '13) related either a structure of filaments or of planes to tile development of the myofibril. In Wieman's ('07) valualile account for the chick he finds mitochondrial graiudes con.stantly at the intersections. MacC'allum founil them less frequently. The figures of the two do not show the predominance of foursided optical .sections or as much regularity in the size and arrangement of tlie sjiaces as was found in the preparations used in the present stuilj-.

A question of greater significance upon which tlie present account is at variance witii their ol)servations is the numner of appearance of the myofibril. Both authors believe it takes origin within rows of the spaces as a residt of their subtUvision into still smaller compartments. Wieman fimls this process to b(> under way in the four-day chick. The measuremeiU of the spaces in the heart at this stage by the writer with the aid of photographs did not furnish any evidence that they fell into two groups in

14() K. 1). COXGDON

rcf«'roric»> Id tlicir siz<>. Hi' luis alsn cxprcsscil tlio Ix-lit-t tliut tlic font met ill' struct uiv at this tiiiu' docs not consist of fibrils, but that it is a slight inodificatinii of the primitive hcxahcdral spaces with their mitocliomhil firaiiules.

liriick i'i;ii (l('scril)e(i in the eiiilirymiie nuisch- cells (if the lainellibraneh Aiiodonta a primitive protoplasnii*- hoiieycoiiib which gives rise to Hl)rils by ihiekeiiing at th(> intersection of the phmes. Mitochondria is not only accunuilated at the intersection of jilanes as in the liigher forms, but is also present scattered along till' (le\-eloping Hbril. Hriiek's observations are all the more valuable as a coiidi-ination of the chief features ofthe accounts of .MacC'allum and Wieman, since one may conclude from his failure to mention their articles that he arrived at this view entirelj- independent of any suggestion from their work.

THE BEGINNING OF CONTRACTION

It has been siiid that rhythmic contraction is first to be seen in the seventeen- or possibly sixteen-soniite chick. It is in accord with the usual history of developmental processes that there siiould b(» a pi't'liminary contraction of a more simjile kind heading up tn it. If the claim had b;^('ii substantiateil that my()til)rils take their origin at a tleHnite period in development rather than by a gradual modihcation of a structure that has been in the heart from the first, ther>' would have been .some reason for anticipating that the contraction also would begin abruptly at the time of the appearance of the contractile structure. One writer jjlacil the liegimiing of pulsation at a time after the aj)pearance of the myofibril, another makes the two contemporaneous, and a third believes that the contracticm comes first. None of them discu.ss a jjossible gradual assmnption of the contractile function. Wieman has pointed out that there is no longer a question regarding the time of appearance of a niyofibril, since it is a gradual ililTcrentiation of a struclure pn'seiit in the youngest heart cell. This coiisidci-ation suggested to MacCallum that the contractile function also may be very gradu.il in its beginning.

.\ search was made for evidence of contraction in preparations fixed before the begiimiiig of rhythmic pulsation. It was found

p:mbryonic structure of avian- heart muscle 147

thiit ill flic licurt of the ti'ii-sdinitc chick siiuill ;ir.';is of licxaiicdnil structur.' often show cloiiKutioii. They uiv of too liriiilcd extent to bo oxphiinod as the result of stretching durinK th ■ handling of the tissue. It is not impossihle, then, that they are the expression of local niuscle contractions. If this !);> true it is still a (piestion whetlier the contraction occurred spontaneously or due to the stimulus of the fixing fluid.

Roux has de.-<cril)ed under the name 'faml)rosia' a widespread resj)onse of various typ;'s of embryonic cells to unfavonxble conditions which consist in their ilrawing away from each other. This can be seen in the early chick heart and is an undoubted expression of a protoplasmic contractility still iiio7\' primitive than ai)i)areiitly occurs in tlii' ten-somite heart.

SUMMAin'

The he.iil of I lie chicks younger than the sixteen- or seventeen-.somite stages, when rhythmical contraction begins, has a sarcoplasmic structure whose optical sx'tion is a net consisting of two systems of parallel lines intersecting to cut ofT .spaces approaching a square form. They iiuuisure in the fixed material about O.S micron on a side. The appi'rent net is probably produced by three .systems of membranes each pai'allel among themselves which inter.sect to form hexahedral compartments. At all intersections of three planes are .small uniform mitochondrial granules.

There is some slight indication in the arrangement of the planes of a division of the early myocarilium into mononuclear cell areas which corresiioiid to lobations on its exterior. For a period including the thirteeii-somite stage clefts appear j)artly separating cell areas. Yet at no time can cell walls be made out or any interruption of the planes cro.s.sing the region intermediate between two nuclei excej)t tlu^se occasional clefts. The myocardium of the four-day chick shows no clefts. It was oijserved in tissue cultures to migral<' out into the clot in I he form of anastomosing multimiclear columns which gave no eviilenc of breaking up into cells other than slight constrictions of th(> columns. In

14S E. D. CONC.DON

spitr of this a|)pc;iniii('c of struct iim.l coiitiiiuity throuf^lioiit the siircoplasiu ill the four-diiy cliick, since Winnan has boon ablo to divide it into iiiononucloar liodics l)y iiiarcratioii and since temporary clefts ai)pear in tlic myocardial tissue it is to be concluded there is proi)al)ly some kind of demarcation into cell areas in the early myocjirdium.

The hexahedral structure of the i)uisatinfi heart throujrli the fourth day of incubation tiitVers from its earlier condition in the more rectilinear arrangement of its planes and in functional changes consisting in the elongation of the hexahedra over consiilerable areas in a eonmion ilirection. This brings the granules of the j)lanes transverse to the dinction of elongation closer together so that they give the appearance of bars. The opposite phase to contraction is apparently in part due to an elongation of the hexahedra in a direction transv(>rse to the original extension. The complete understaiuling of the functional changes in the form of the hexahedra was prevented by the inability to determine the extensions and elongations parallel to the optical axis of the microscope.

Several con.siderations suggest that the ili>thmical beat is precedetl by a less highly organized type of contraction. In the first jilace. the changes in the contractile structure through a period including the beginning pulsition and up to the fifth day are not very nuirked. Then also <ni;tl! arras in wliicli I lie liexahedral structure is elongated in a common din'ction can sometimes be found in the heart of a ten-somite chick. It nuiy be that these are the primitive local contractions brought about either spontane(msly or through the stimulus of the fixing fluid. The creeping apart of embryonic cells described by Koux and observed in the early heart is evidence for the existence of a VQTy primitive protopla.smic contractility.

EMBHYOmC STRUCTURE OK AVIAN HEART MUSCLE 149

LITERATURE CITED

Ai.TMAXN. 1{. 1S94 Dio Elemcntarorganismen und ihre BMichiingcn zu den

zi'llcii*. 2(i ed., Lcipzii;. Asm. T. I!)1I HeitriiKe ziir Histologic und Histogencsc dcr qucrgcstreifteii

Miiskrliitur dcr SiiUKcticrc. Arch, mik .\nut,. Bd. STi. Bardek.v, C K. 1900 The development of the iiiusculuturc of the body wall of

the pig, including its histogenesis and its relation to the myotomes

and to the skeletal and nervous apparatu.s. Johns Hop. Hospital, Rep.,

vol. 11. Bkxi>.\, C. Wcitcre Mittcilungcn iiher die Mitochondria. \'erh. physiol. Cles.

Berlin. JalirK. 1,S'.).S-1,S'.W. BrIVk. a. 191.'{ Cher die Muskelstruktur und ihre Entstehung, .sowie uber die

Verbindung der Muskein niit der .'^(■h:ile bci den Mu.scheln. Zool.

Anz., Bd. 42. Chi.micoi, Ci. 1887 Delia condizione anatoniische del cuarc al principio della

sua funzione c contribute all istogenesi delle cellule muscolari cardi ache. .\fti R. Accad. Fisiocritici. Siena. CoxoDOX, E. D. 1915 The identification of tissues in artificial cultures. .\nat.

Rec, vol. 9. DuESBERC!, J. 1910 Les chondriosomes des dellules embryonnaires du poulet,

et leur role dans la genesc des myofibrilles avec quelques observations

sur le devcloppement des fibrillcs musculaire striees. Arch. ZcUforsch.,

Bd. 4. Eycle.shy.mkr, .\. C. 1904 The cytoplasmic and nuclear changes in the striated

muscle cell of Xecturns. Am. Jour. Anat., vol. 3. CioDLKWsKi. E., Jun. 1902 Die Entwickelung des .Skelett- imd Herzmuskelge wcbes der Siiugetiere. Arch. mik. Anat.. Bd. 60. HfiDENHAi.N. M. 1899 Bcitriige zur Aufklarung des wahren Wesens dea faser forniigen Differenzicrungen. Anat. .\nz.. Bd. 16. HoYKR, H. June. 1901 Tber die Continuitat der contractilen Fibrillen im den

Herzmu.skelzellen. Anz. Akad. \Vi.ss.. Krakau. Kt'HKiEWirz 1<X)9 Zur Kenntnis der Histogenese des Herzmuskels der Wir beltiere. Anz. Akad. Wiss. Krakau. LiNA. v.. 1913 .Sulla importanza dei condriosomi nella genesi dei miofibrille.

Arch. ZcUforsch, Bd. 9. MacC'alia'm. J. B. 1897 On the histology and histogenesis of the heart muscle

cell. Anat. Anz. Bd. 13. Marikat, v. 1903 Recherches sur la aatructure et le devcloppement compares

des fibres de Purkinje et drs fibres cardiaque.s. Bibl. .\nat., T. 10. Meves. F. 190<.) t^er Neubildung quergestreifter .Muskelfa.iern nach Beo barhtungen am Hidinorembryo. Anat. .\nz., Bd. M. Mtiiuowska, J. 1908 Zur Histogenese der Skeletmuskcln. Bull. Acad. Sc.

Cracovie. RriiAscHKiN, \V. 1910 ("hondriosomcn und DifferenzierunKsprozcise boi

.S'iugetierembryonen. .\nat. Helfte.. Bd. 41. Sfiii.ATKR, (i. 1906 Histologische rntcrsuchungcn uber da.i .Mu.xkolgewebe,

II. Die .Myufibrille de.« enibryonalen Hiihnerherzcns. .\rch. mik.

Anal.. Bd. 69.

150 E. D. CONGDON

ScHocKAEKT, A. 1900 Nouvelles recherchos conipariitivea siir le texture et le

devcloppement du myocnrdc chez les Vertebres. Arcli. Biol., T. 24. ToRBACA, L. 1014 II comportaniento del condriozomi nella rcgencrazionc dei

iiiiiKcoli striati. Arch. Zcllforsch., Bd. 12. Waoener, t!. H. 1S80 Uber die Kntstehung der Qiierstreifen auf den Muskein

mid die davon ahhiinRigen KrclieinunReii. .\rcli. .\nat. Physiol.,

Aniit. WiEMAN, H. L. 1907 The relation between the cytoreticulum and the fibril

bundles in the heart muscle cell of the chick. .\m. Jour. Anat., vol. 6.

AmiOK a AlMTItAlT !>»' Tllllt fAPEft I1I0I KD BT THE milLlonitAPIIIC AKRVICE HrpTCUUCR 16

rilACOfYTOSIS OF (WRBOX AND TARMrXE CinMLKS IX THE TRAXSPARKXT TAILS OF TADPOLES

ELIOT K. CLARK AND ELEANOR LINTON CLARK Anatomical Laboratory of the University of Missouri

OXE PLATE

The present study is part of a series dealing with the growth and reactive powers of the tissues and cells in the transparent tails of tadpoles toward extenuil stimuli. These experiments were begun with the especial oliject of studying the behavior and growth-regulating factors of lymphatic endotheUuin, but necessarily included the observation of blood vessels, leucocytes, and mesenchyiuc cells. One of the authors ('()9) had previously shown that the lymphatics react positively toward red blood cells extruded into the tissue, sending out new sprouts which grow toward them and actively engulf them. Also, the authors ('17) found that lymphatic capillaries will react positively' toward fatty subst.ances introduced into the subcutaneous tissue, growing toward the injected oil globules, and. in this ca.se with the aid of leucocytes, taking part in their ab.sorption. It is well known that lymphatics play an important jiart in the disposal of coal pigment, in anthracotic lung tissue, and in the absorption of foreign particles introduced into the peritoneal cavity and, for this reason, we thought it migiit prove profitable to test the response of lymphatics, in the transparent tails of tadpoles, toward injected gramiles of carbon and carmine.

The experiment of injecting small quantities of India ink into the tissue spaces of the tail lins of tadpoles was firstcarri(>d out in 191 3 in the course of other studies on growing lymphatics. 1 1 was found that th(> carbon granules were taken up i\v leucocyt<>s and by mesenchyme cells, but that lym|)hatics and Ijlood vessels

l.il

TUB ANATOUU-M. IIEtOIID, VOL. 15. NO. 3

1.V2 KLIOT H. CLARK AND Kl.KANOU LINTOX CLARK

wvTv apparently iiulifforont to the iiroseiipe of foreign partieles of this nature in their vieinity.

Altlioiiuli the presence of cjirhon uninules in th(> tissue spaces did not prove to be a stimuius for the growth of lyinpliatics, it soenied advisable to record briefly the reaction of the various cells and tissues toward this substance and especially that of the niesenclmne cells, in view of the fact that the phagocytic powere of these cells has been disregarded or ilenied by most investigators of the subject. The region of studj* — the transparent tail of the tadpole — where each individual cell can be watched, recordetl, antl followed for days, or weeks if necessary-, and the method of chloretone anesthesia and observation in the upright chamber which permit of study in vivo under approxijnately nonnal conditions, both possess marked advantages over many of the regions and methcxls selected by others for the study of phagocj'tosis.

The tadpoles used for these experimejits were the larvae of Bombinator, Rana pipiens, and Hyla pickeringii. The method of introducing small amounts of foreign substances was the same as that described previou.sly for injections of paraffin oil (E. R. Clark, 'l(i) and fat (E. R. and E. L. Clark, '17). The injection materials — India ink, diluted one-half with tap water, and powdered cannine, suspended in distilled water — were placed in small vials, which were set in a di.sh of boiling water for half an hour. The tadpoles were anesthetized in chloretone, 1 to MM), and small amounts of the ink or caniiine were injected into both tail fins, through fine glass cannulae, under the binocular microscope. These su.spensions of granules were injected at ilifTerent points in the fins in some ca.ses near the margin and, on a few occasions, they were injected directly into the lumen of a lymphatic capillary. The tadpoles were then transferred to the obser\-ation duunber, devised and described by one of the authors ('09, '12) and studied under the compoimd microscope in a 1 to 50()() solution of chloretone. Records of the site of injection, including all the cells and vessels in the neigliborhood, were MKule soon after the injection with the ai<l of the Ix'itz drawing eye-piece. The tadpoles were then returned to fresh water.

PHAGOCYTOSIS IN THE TADPOLE's TAIL 153

The injrc'ted repons were obsorvcil on succooding days, rofords wore made of the same cells and vessels and of the changes which occurred.

The results obtained from injecting carbon, in tlie fonn of India ink, and carmine granules proved to be identical, and a single description will suffice for them both.

Leucf)cytes were the first cells to respond to the presence of the carbon and camiine granules. About an hour after the injection some of these cells, cfmtauiing retl or black granules, could be seen near' the injection site. For several days after the injection, leucocytes continued to migrate toward the point of injection and to take up the injected granules. These cells became loaded with the carbon or cannine to such an extent, in some instances, that they resembled merely balls of pigment with no cellular matter visible, ^fany of the leucocytes, after taking up the ink or cannine, moved over to a near-by blood vessel and flattened out on its wall. In some cases such a pigmented leucocyte was obser\-ed to crawl through the vessel wall. But the removal of foreign particles by this method was evidently very slow, since many cells, loaded with the granules, remained close to the exterior wall of blood capillaries for long periods before entering the ves.sels. Other pigmented leucocytes did not migrate to blood vessels or Ijinphatics, but instead they wandered away through the tissue spaces of the tail fin. In the cjvse of cells which had taken up carbon granules, this process was difhcult to follow, owing to the fact that wandering cells containing black pigment are usually present in the tadpole's tail, under nonnal conditions, but it was easy to trace the wanderings of the leucocytes containing cannine, and these cells were found scattered througli the tail fin at long distances fr<im the site of injection.

Records made ten days and two weeks after injection, showed collections of leucocytes still present near the site of injection, all of them filled with black or red pigment and most of them adlu'rent to the walls of blood vessels. By this time the munber of leucocytes present in this region had diminished somewhat. iSiiice the phagocytosis of foreign j)articles by wandering cells

151 ELIOT R. CL.\RK AND ELEANOR LINTON CLARK

and leucocytes is such a. well-kiKJWii phenomenon, it seemed superfluous to inv«>stigate the cliaracter of these cells, in the tadpole's tail, and tlicir response toward the injected substances in anything but an incidental manner.

The response of the mesenchyme cells — and by this term we mean the stellate connective-tissue cells— was much slower than that of the leucoc>'tes and wandering cells, and for several hours after an injection no reaction was noted. On the day following the injection, an occasional granule of carbon or cannine c !uld be detect etl along the processes of some of these connective-tissue cells. From this time on the number of granules taken up by the cell processes of the mesenchyme cells increased and, after three or foiu* days, clumps of granules were visible in the cell bodies near the base of the processes as well as on the processes.

After ten days or two weeks the nmnljer of mesenchyme cells ct)ntaining red or black granules and the amount of foreign pigment present in these cells had both increased. This increase occurred at a tune when the number of mobile phagocytes had begun to diminish. The carbon or carmine persisted within the mesenchjnne cells, at the site of injection, for as long as the tadpoles remained under observation.

One of the authors ( '12) has made a careful study of the movement of these so-called 'fixed' connective-tissue cells. He found tliat their shape is continually changing: processes are withdrawn on one side and sent out on the other, and that by this means these cells are capable of a slow amoeboid locomotion. The sajne kind of movement was noted in the records made of these cells before and after taking up the foreign particles, but in compaiison with the behavior of tlu^ leucocytes this migration of the mesenchyme cells is quite negligible. The granules inside of leucocytes were conveyed to blood vessels and eventually transported by the blood stream to other portions of the bodj' or they were carried througli the tissue spaces of the tail toa relatively great distance from the site of injection. On the other hand, those cannine and carbon granules whose fate it was to be picked up by me.senchyme cells remained re'atively stationary, neai' the apot at which they had been originally introduced into the tail.

PHAGOCYTOSIS IN THE TADPOLE's TAIL 155

Figure 1 is ;i drawing made two weeks after an injection of a suspension of canuine gi-anules. The number of leucocytes in this region has diminished by this time and several wandering cells containing red pigment can be found scattered througli the fin at some distance from the point of injection. Those that remain for the most part have congregated near the wall of a near-by bloo(.l cai)illar3- and two of them are shown in the act of entering the vessel. Almost all of the mesenchyme cells of the region contain carmine granules. Figure 2 is a high-power drawingof such a mesenchvine cell, selected from a similar specuuen, which shows the arrangement of phagocytized carmine granules. As may be seen, these red granules have collected f)n the processes and in the cell body near the base of the processes. Figure 3 is a drawing of a number of mesenchjm^e cells made Hvq days after an injection of India ink into the subcutaneous tissue of the tail fin and .shows an identical response on the part of this type of cell toward the granules of carbon which have collected, in a suiiilar manner, on the branched processes and within the cell bodies near the base of the processes.

It is evident, therefore, that in a transparent region such as the tadpole's tail where each individual cell may be observed in tietail, in the living, and under practically nomaal conditions and where the different cell types may be distinguished with ease, that the mesenchyme cells are actiAC phagocj'tes of foreign particles, such as carbon and cannine, present in the tissue spaces. Their reaction toward these foreign substances is less rapid and intense than that of the leucocytes, but it is nevertheless definite alid probably important. The present observations gave the impression that gianules housed within the mesenchyme cells were more permanently located than those which had been engulfed by leucocytes, since that portion of the foreign pigment remaining at the point of injection two or tlu-ee weeks after it had been introduced is, for the most part, retained in the interior of connective-tissue cells.

We have made the statement that the lymphatics of the tadpole's tail showed no visible reaction towanl these foreign particles present in the tissue spaces. The sending out of new

Ifid KI.IOT R. CLARK AND EI.KANOR LINTON CLARK

sprouts wliicli p-«'\v tuwjinl tlic iiijcctod finuiiilcs ^thc churactoristic response of the Ij-mplmtics towjinl extruded blood cells and injccteil fat k1<>I'ii1<* — wjis never observed iix the case of the injections of carbon and cannine granules. H(wever, in repeating these exp(>rinu'nts during th(> past year, we found that the IjTnphiitic endothelium, although not stuuulatcd to grow toward these foreign substances when they were at a distance, would react toward them when they were in \erv close proximity. On a few occlusions, a small amount of India ink was injected directly into the lumen of a Ij'mphatic capillar}'. When such a tadpole was exiiniinetl on the following day, carbon gr-amdes were found to be enclosed within the endothelial cells of that h'mphatic which had been injected with ink. They were present as small black spots and as large clumps of granules within the areas surroimding the nuclei of the endothelial cells — the same region which stains with vital dyes (E. K. Clark, '09; Wislocki, 'IG).

A similar behavior on the part of blood-vessel endothelium was not noted.

That foreign particles, such as carbon, carmine, cinnabar, etc., introdueetl into the blood strean; or subcutaneous tissue are quickly phagocytized within the body has been knowTi for many years. Thus, Ponfiek ('Gi)) and Sicbel ('89) showed that such substiuices as cinnabar and indigo injected into the blood stream quickly disappeared from the circulation and collected in the liver, spleen, and bone marrow, where they were taken up bj' cells with large round nuclei, resembling leucocytes.

MetchnikoiT ("83), in his studies on iiiflaiiunation, described the phagocytosis of carmine granules and of red blood cells injected into Triton larvae and into the tails of tadpoles, by leucocyt«'s which have migrated from the blood stream antl by wamlering cells of the tissues. Later ('92), in describing further observations, he groups these two types of cells together as macropluiges or cells whose chief function is the phagocytosis of foreign {)arli(les and i)f cell debris.

Muscatello ('95) and MacCalhun ('03) found that carmine and carbon gramih^, injected into the peritoncjil cavity, were conveyetl to the lymphatics of the diaphragni by means of leuco

PHAGOCYTOSIS IN THE TADPOLES TAIL 157

cytos which colloctotl in the poritonoal cavnty in largo niimbrrs and actively cnRulfod the foroign jjarticlos. These results diffennl from th(^ conclusiijii of Von Recklinghausen that such i)articles reached the lymph vessels in the free- state tlirougli large openings, or stoiuata, in the endothelial wall.

Recent studies on anthracosis of the lungs made bj- llaythorn ri3) and Klotz ('14) abandon the older view that the coal pigment is taken up by the epithelial cells of the alveoli and that it frofjuently reaches the h7iiph glands draining the lung in the fr(M' state, cutting through the walls of the h^nph vessels by means of the sharp comers on the granules. These authors find that all the coal pigment in anthracotic lung tissue is present inside of phagocytes, wliich, according to Haj'thom, are probably 'endothelial' leucocytes, which pick up the coal pigment in the alveoU, convey it to the IjTnphatics and l>Tnph nodes and also to the connective-tissue septa. They state that the pign\ent remains indefinitely intracellular.

The origin of these large mononuclear wandering cells and leucocytes is still undecided, but their importance as phagocj-tes of foreign particles and cell debris is unquestionetl.

The phagocytic power of the specialized endotheliiun in certain regions of the body has been emphasized by many authors. The endothelial cells of the liver and bone marrow and the reticul()-tMidt>thelial cells of the spleen and Ijinph glands have long been recognized as phagocytes of foreign material, and Evans ('15) has grouped this type of phagocyte with the large mononucloiir wandering cells and the clasmatocytes as macrophages. Mallory (14) even takes the extreme view that the whole group of mononuclear wandering cells and leucocytes, which are phagocytes of foreign particles and cell debris are derived from the endothehiun.

MacCallum ('03) found that the endothelial cells lining the l\^nph vessels of the diaphragm were packed with carbon granules aft(>r injections of India ink into the peritoneal cavity. The obs«'rvation of one of the authors (K. R. ("lark, '00), on the t;iking up of red blood cells from the tissue spaces, showed that the gi-owing lymphatics of amphibian larvae are phagocytic. In

158 KLIOT R. CLARK AND ELEANOR LINTON CLARK

tliis inst;iiice the foreipi suhstuiu-c, ui the form (jf reel blood eells. was taken into the liuuen of the lynii)h vessel. The observation of Wislocki ('16), tliat the colloidal dye trypan blue is stored in tlu" fonii of pianules in the iierinuclear areas of the hinphatic endothelium of tadpoles, rendei-s it hiphly probal)le that all the endotheliikl cells of the lymphatics in amphibian larvae possess the power of phagocytosis. This conclusion of Wislocki's is based on the work of Evans and 8chulemann ('14. '15) which apiH'ars to jirove that the storage of acid azo dyes, in the form of aggregations of dye granules, is evidence of phagocytosis on the part of cells which display this b(>ha\i()r. The present observations on the fate of carbon antl cannine granules introduced into the lumen of hinphatic capillaries demonstrates this phagocytic power of the Ijinphatc endotheliuju, for these granules were taken up by the endotheUal cells lining the l>inphatics and stored in tlie perinuclear areas.

The phagocj'tic power of the mesenchyme cells has not been so generally recognized in the literature. The ability of these cells to ingest foreigii material was not studied in detail by the earlier writers, while recent in\estigators frequently have doubted or denied its existence. That deposits of foreign pigment, such as coal jiigment in anthracosis, metallic silver in cases of argyria, etc., are j)r('sent in the connective tissues has long been known, but recent authorities claim that such material is stored solely in the resting wandering cells or in mononuclear leucocytes or in endothelial cells, such as the KuptTer cells of the liver, and not in the connective-tissue cells proper, or fibroblasts. MacCallujn ('03) is an exception to this statement, for in his description of the results <if injecting India ink into the peritoneal cavity, he mentions the i)resence of carbon gnuiules in true coiuiectivetissue cells.

Haythom (M.'i), in his studies on the histology of anthracosis, describes wanilering cells or endothelial leucocytes as the only cells which phagocytize the coal pigment. Carbon granules present in the connective-tissue septa he beUeves to have been conveyed tluTe by the mobile phagocytes and to remain pennanently within these cells. .Metclmikon' I's:^). in studying tlu

PHAGOCYTOSIS IX THE TADPOLE's TAIL 159

pffpots of injecting carmine giiinules into the tnins|)urent tails of Triton hir\ae and tadpoles, notes and figures these foreign granules inside of niesenchynie cells as well as in the wandering cells and leucocytes. However, in later studies ('92) he recedes from the opinion that the ronnective-t issue cells are phagocytic and states that mesenchyme cells which contain foreign pigment are cells which have ingested it while they were leucocytes and that leucocytes containing ingested pigment nuiy wanfler off antl become transfonned into cells which resemble mesenchyme cells. In a still later publication ('05) he denies the power of true mesenchjinc cells or fibroblasts to phagocytize foreign matter. Similarly, Buxton :md Torrey COO), in describing the disposal of carbon granules injected into the peritoneal cavity, note the rapid jiliagocytosis of these particles by the macrophages and consider that the carbon granules, wich are found several hours later in the connective tissue, have been conveyed there by the.se macrophages, wliich then proceed to transfonn themselves into 'trailers.' However, the authors are not absolutely convinced that these 'trailers,' wliich are practically stationary and which store the granules indefinitely, are in\-ariably transformed macrophages and they state that these cells are frequently indistinguishable from ordinary connective-tissue cells.

Recently, Jones and Rous ('17) have denied the phagocytic power of niesenchpne cells. Their rather elaborate method of study consisted in making suspensions of individual cells by digesting with tryp.sin the clot of proliferating tissue cultures of embrj'onic chick material. The indivitlual cells were then freed from the tissue mass by filtering tlirough gauze, and new cultures were then made to which finelj^ ground carmine was added. .\ slight amount of phagocytosis occurretl, but mainly on the part of large cells which the authors believed to be endothelial hi nature. The authors concludetl that the connective■ tissue cells proper possess no power of phagocytosis."

' Since writing this article, the recent work of Leo Loeb and Fleisher ('\7) has come to our attention. These authors, in studying the behavior of connectivetissue cells in tissue cultures, question the decision of Jones and Hous that the large cells which phagocytize foreign particles, in their tissue cultures are endo

Kid KLIOT K. Cl.AHK AND ELEANOR LINTON CLARK

In tln' i»n'.s(»nt oxpi'iimciits, witli u roRion of study such as the t ranspHrcnt tail fin of Amphibian larvae and with the aid of chlorotone anaesthesia and the special observation chmnber, it was possilile to si'e and to keep records of the individual cells of the area at the time of the injections of India ink and of cannine pranules. and to follow these cells and to observe their subsequent beha\-ior with jierfect clearness and in tlieir normal environment. When the living cells are observed in this manner, it is perfectly clear that mesenchjnne cells possess the power of phagocj'tizing foreign particles of carbon and carmine and of retaining them indelinitely. It is very easy, by making records of individual mesenchjnne cells, to see that these same cells which were jiresent as connective-tissue cells at the time of injection later contain gninules of carbon or cannine and that they retain their characteristic identity as 'star-shajied' cells after taking up the pigment. -\lso it is clear that the wandering cells and leucocytes which ingest the foreign particles do not become transformed into connect ive-tis.sue cells, even in the cases where they wander off througli the tissue spaces, but that they persist as round or amoeboid cells, easily distinguishable from the branching connective-tissue cells.

CONCLUSION

The present observations show that three types of cell present in the transparent tails of tadpoles display the power of phagocytizing granules of carbon and cannine injected into the subcutaneous tissue. These arc: leucocj^tes (including wandering cells), stellate connective tissue-cells, and the endotlielial cells of the l>anphatics. The leucocytes actively migrate toward the sit«> of injection, while the me-senchjane cells and Ij-mpliatic endothelial cells apparently ingest only those particles which are in close proximity to them.

tlielial in nature. Locb and Flcishcr consider such cells to be fibroblasts which have increased in size during the process of regeneration and which are capable of ingesting foreign particles and cell d6bris.

PHAGOCYTOSIS IN THE TADPOLE's TAIL 161

LITERATURE CITED

Buxton and Toniiicy I'JOC Absorption from the peritoneal cavity. Jour. Med. Res., vol. 15, p. 3.

Clark, E. R. 1909 Observations of living growing lymphatics in the fail of the frog larva. Anat. Rec, vol. 3, no. 4, p. 183.

1912 Further observations on living growing l\^npllatics: their relation to the mesenchyme cells. Am. Jour. Anat., vol. 13, p. 351. 191G .\ study of the reaction of mesenchjone cells in the tadpole's tail toward injected oil globules. Anat. Rec. vol. 11, no. 1, p. 1.

Clark. E. R. and E. L. 1917 .\ .study of the reaction of lymphatic endothelium and leucocytes in the tadpole's tail toward injected fat. Am. Jour. Anat., vol. 21, no. 3, p. 421.

EvAN.s, H. M. 1915 The macrophages of mammals. Am. Jour. Physiol., vol. 37, p. 343.

Evans, H. M., and .S<hdlemann 1914 The action of vital stains belonging to the benzidine group. Science, X. S., vol. xxxix, no. 1004, p. 443. 1915 t*bcr Xatur und Gene.se der durch saure FarbstofTe enstehenden Vitulfiirbungsgranula. Folia Haem. Arch., vol. 19, p. 207.

Haythorn, S. R. 1913 Some histological evidence of the disease importance of pulmonary anthracosis. Jour. Med. Res., vol. 29, p. 259.

Jones and Rous 1917 The phagocytic power of connective-tissue cells. Jour. Exp. Med., vol. 25, no. 1.

Klotz, O. 1914 Pulmonary anthracosis — a community disease. Am. Jour. Pub. Health, vol. 4, p. 887.

LoEB, Leo and Fleisher 1917 On the factors which determine the movement of tissues in culture media. Jour. Med. Res., vol. 37, no. 1, p. 75.

MacCallum, \V. G. 1903 The relation between the lymphatics and the connective tissue. Johns Hop. Hosp. Bull., vol. 14, p. 1. 1903 On the mechanism of absorption of granular materials from the peritoneum. Johns Hop. Hosp. Bull. vol. 14, p. 105.

Mallgry, F. B. 1914 Principles of pathological histology.

Metchnikoff, E. 1883. L'ntersuchungcn iibcr die mesodermalen Phagocyten einigcr Wirbelthicrc. Biol. Cent., vol. 3, p. 560. 1892 Lemons sur la pathologic comparfe de I'inHammation. Paris. 1905 Immunity in infective diseases. Cambr. Univ. Press. Trans, by Binnie.

MuscATEi.Lo 1895 Uber den Bau und das .\ufsaugungsvermdgen des Peritoniium. Virchow's Arch., vol. 142, p. 327.

PoNFicK, E. 1869 Studien iiber die Schicksale korniger FarbstofTe im Organisnuis. Virchow's Arch., vol. 48. p. 1.

SlEBEi.. \V. 1886 l*ber das Schicksal von Frcmdkorpern in der Blutbahn. Virchow's Arch., vol. 104, p. 514.

Slavjanskv 1869 Experimentclle Beifnige zur Pnetiraonokoninsifj, Virchow's Archiv., vol. 48, p. .326.

WlSLOCKi. (i. B. 1916 The staining of .\mphibian larvae with boniidine dye with especial reference to the behavior of the lymphatic endothelium. .\m. Jour. Physiol , vol. 42, no. 1, p. 124.

PLATE 1

EXPLANATION OF FIGURES

I'ig. 1 Cameru-lucida drawing of a region in the dorsal Kn of a tadpole, into wliirli a su8|)cnsioii of cacniino Rrannlos had been injected two weeks previously. The sketch shows that the red granules have been taken up by leucocytes and connective-tissue cells, a. Leucocyte, containing carmine, on the point of entering a blood Vessel. Enlargement = X 275.

Kig. 2 High-power drawing of a single mesenchyme cell, showing phagocytized carmine granules, distributed on the cell processes and inthe bod}' of the cell, near the base of the processes.

Fig. 3 Camera-lucida drawHng of a number of mesenchyme cells from a region of the tail fin into which India ink had been injected five days previously. Carbon granules have been taken up by the connective-tissue cells and are lodged on the processes of the cells and in the cell bodies near the base of the processes. Enlargement = X 300.

102

SLIOT a. CLABK AMD £LIA.NOR LINTON CI.ARK

PlJlTE 1

\K^

-^; ^ "^

. , '^w >4'>^ i^ / Mr "

f

ju.

Id.-)

ON THE TIME OF THE POST-NATAL OBLITERATION

OF THE FETAL BLOOD-PASSAGES (FORAMEN

OVALE, DUCTUS ARTERIOSUS, DUCTUS

VENOSUS)

HICHAKD E. SCAMMON AND EDGAR H. NOURIS Institute of Anatomy, University of Minnesota

THREE PiaURES

It is gcnorally rocognized that two separate processes are involved in the occlusion of the fetal blood passages after birth. The first is the simple functional closure which takes place, in the great majority of cases, at or immediately following birth; the second is the permanent anatomic obliterati(jn which occurs at a later period. The mechanics and histology of the latter process are generally discussed in our larger treatises on obstetrics and pediatrics, and in our major an.atoTnical texts; but the thiie of postnatal obliteration is often unmentioned, although in a number the statement is made that the obliteration takes place in the first few days or, at most, weeks after birth.

The origin of the current concepts as expressed in these larger texts can be traced, we think, to the first statistical study on this subject, which was pubUshed ninety years ago by the French clinician Billard. This investigator (H)Ilected data on the obliteration of the ductus veno.sus, ductus arteriosus, and foramen ovale in a series of one hundred and twenty-eight children who died in the first eight days of life. He found instances of the obliteration of ail these pas.sjiges on the first tlay after birth. The foramen ovale and ductus arteriosus were closed in over fifty per cent of his cases on the eighth day while the ductus venosus was clo.sed in a still larger* immber. He therefore concluded that the obliteration of the fetal blood-passages proce<'ded very rapid!}- in th<' first few days of life- an opinion in accord witii that

16,i

TBI ANATOUICAI, RBCOIIO, VOL. 15. NO. 4 NUVKUBKK, 1018

166 RICHARD E. SCAMMON AXD EDGAR H. NORRIS

held by a niimbor of wTiters in the eighteenth century. The n^sults of Billard's study were published in his 'Traits des maladies des enfants nouveau-n6s' in 1S2S. This work was extremely popular in its time; it passed through a number of French editions, wa.s translated into Italian and German and twice app<>jired in .Vmerican editions. In several publications of the middl.' of the last century Billard's figures are cited and his name is quoted in connection with them. In later works, howevei", the same opinion has been repeatedly expressed but its sourc(^ apparently has been forgotten.

A large amount of data concerning the chronology of the postnatal obliteration of the fetal blood-passages has accumulated since the time of Billard. This material is scattered through the periodical literature of legal medicine, obstetrics, and pediatrics and is also uieluded in a number of rather inaccessible brochures. In the following pages we have collected as much as possible of these scattered data and have arranged them in tabular and graphic fonii. In so doing we have confined oiu-selves to those records in which series of consecutive cases have been assembled. These records include ob.serA^ations on children who were born at term, and also a few cases of children who were prematiu-ely born. In most instances, however, investigators have failed to separate these two groups. A comparison of the records of the few known cases of prematurity with those of children known to be born at term shows no appreciable difference in the time of obliteration of the fetal blood-passages. We hav(> therefore combined them in our tables. In all cases the patency of the fetal passages was te.sted either by injection or by probing, excepting those of Faber ('09) which were examined micro-scopically.

The results made apparent by this combined series of observations are at variance with the current concepts on the subject as expressed in most of our larger texts, and also with the results of Billard. Billard's observations were confirmed by Bernutz ('65) who found the ductus arteriosus clo.sed in fourteen cases in a series of twenty-one children who died between the tenth and twentieth daj's, and in thirty-six out of thirty-eight children dying between the twentieth and sixtieth days. Since this time

OBLITERATION OF FETAL BLOOD-PASSAGES 167

no observer has substunfiatod these findings. .-ilthouRh a number of series niueli larger than those of Billard and Bernutz' have been collected. Thus Elsasser ('52), in a series of nearly three hundred observations upon children of the first month, found obliteration of the ductus arteriosus in about two per cent and of the foramen ovale in about three per cent of his cases; and Alvan^nga ('69) found practical!}' no instances of obliteration of the foramen ovale or ductus art^-riosus before sixty days. The findings of later obseivers (Alexeieff ('00), Theremin ('87-'95), KuchefT ('01) and others) agree essentially with those of Elsasser and Aharenga although they have noted some instances of earlier obliteration of the pas.-^ages.^

THE FOR.\MEN OVALE

The compiled data on the obliteration of the foramen ovale are shown in extenso in table 1. Table 2 is a summary of these data giving bj^ periods the total number of observations and the number and per cent of cases obliterated. Graphic representations of these data are shown in figure 1 curve A. and in figure 2. In the summary and curve the data of Billard are omitted because his findings are so directly opposed to those of all other investigators that we conclude that either his method of investigation was defective or that his definition of obliteration was entirely different.

As will be seen in table 2. less than one per cent of the foramina are completely closed in the first week of life, and less than two and one-half per cent in the second week. In the latter part of the first month the figures indicate that the obUterative process takes place more rapidly as the opening is impervious in about one-eighth of all cases of this period. The rapiditj' of this process mcreases during the second month, for the interatrial

' The cases reported by Bernutz were not examined by him personally but were oollected iit his instance by the interne of a colleague in the Hospice des EnfantsTrouv<5s.

' Haberda ('96) studied the obliteration of the ductus venosus and ductus arteriosus in a considerable series of infants and children. As his data are not given in numerical form we are unable to include them in our summary. The general statements of this writer indicate that his findings were somewhat similar, as regards these two vessels, to our own.

168

RICHARD E. SCAMMON AND EDGAR U. NORRIS

TABLE 1

Data on the obliteration of the Foramen ovale. Numerals enclosed in parentheses indicate number of obliterated cases

ornanTU AXD

DAT*

r

i

< a

5

o

i

9

1!

•4 O

5

a

1

a

O

at

o a

5

3

S

3:

o

s

f*

i<

i!

H

H

i

O

8

S

g

i!

^s

«o

7

6

11

21

25

46

68

15

3

2

1

1

Alexeieff. '00.

3

(1)

(3)

(6)

(3)

(2)

(1)

(1)

Alvarenga,

'67

44

19

17

33

28

12

23

(2)

18

(1)

1

4

12

(4)

2

(1)

Billard, '28...

118 (17)

20

(11)

British Anat.

Soc, '98...

23

(11)

9

(7)

30

(23)

286 (217)

Bitot, '37...

34

63

(23)

(49)

EUasser, '52.

150

(1)

63 (4)

62 (9)

23

(2)

3

(1)

Fawcett

6

(3)

209

(207)

Letourneau,

'58

8

(1)

1

Ogle, '57

62 (49)

Theremin '87

70

76

63

49

55

45

81

65

11

7

18

5

(10) 4

(13) 6

(17)

(18) 4

(47) 8

(51) 6

(9)

1

(7)

4

Theremin '95

6

(1)

(2)

(3)

(6)

(6)

(4)

Wallraann,

'59

2

o

4

291 (170)

Zahn

293

171

152

121

107

86

158

157

27

20

43

12

42

711

(552)

Totals

1712

(2)

(4)

(19)

(16)

(18)

(21)

(55)

(64)

(12)

(11)

(21)

(8)

(28)

(1228)

• 1 to 15 years t 7 to 10 months.

OBLITERATION OF FETAL BLOOD-PASSAGES

169

TABLE 2 Obliteration oj the Foramen ovale (SB58 cases)

Birth to 8 days...

8 to I.'} (lays.. . .

15 to 32 days

32 to 46 days

46 to 61 days

61 to 91 days

3 to 6 months.. 6 to 9 months. .

9 to 12 months. . 1 to 5 years . . . . 1 fo 15 years. . . . 5 to 10 years ....

10 to 20 years .... 15 years and over 20 years and over

TOTAL

HOHBIB or

FEB CZNT or

HmiBBB or

CABCa

CAIIU

ctam

OBUTBRATED

OBLTTBnATBD

293

2

0.7

171

4

2.3

273

35

12.8

107

18

16.8

86

21

24.8

158

55

34.8

157

64

40.7

27

12

44.4

20

11

55.0

43

21

50.0

93

52

55.9

12

8

66.6

42

28

66.6

1840

1320

71.7

1712

1228

71.7

comnumication is oblitoratcd in approxiinatelj^ one-.sixth of all cases in the first half of this poriod and in about one-fourth in the latter half. During the third month somewhat less than ten per cent of the cases are obliterated so that by the end of the first trimester the foramen or {do is finally closed in about onethird of all cases. After the end of the third month the process again goes on more slowly and the average of obliteration in the second trimester is about forty per cent, that in the third trimester about forty-five per cent and that in the fourth trimester about fifty-five per cent. Our figures for the period between one and five years show an average obliteration of fiftj-five per cent, which is five per cent less than that of the last trimester of the first year. This difference is due. no doubt, to the small number of cases tabulated in these two periods, and does not represent a real increase in the number of patent foramina. The figures of the .second five year interval indicate that twothirds of the foramina are clo.sed: but the small amount of data for the period between the first and tenth years makes it impossible to say with certainty just when this increase in obliteration

170 RICHARD E. 8CAMMON AND EDGAR H. NORRIS

is bntuphf about. In the srcond drrado the percontago of open foramina is the same as in the period l)et\veen five and ten years and tlie foramen ovale is found to be obliterated in about seventytwo per cent of individuals of twenty years and over.

Curve A of hgure 1 expresses graph ieallj' the frequency of obliteration of the foramen ovale during different periods in the first year of life. This curve is readily di\'isible into three parts. Tlie first portion, which extends from birth to the middle of the

" ' >• Ol »3 W go 120 IBO JTO 360.1^

Fig. 1 Three curves representing the average percentages of obliterated fetal blood-passages at different periods in the first year of life. A, dotted line, foramen ovale, B, solid line, ductus arteriosus; C, broken line, ductus venosus. These curves are based upon the material summarized in tables 2, 4 and 5.

second week, is a short segment expressing the obliteration of a Uttle over two per cent of the ca.ses. It is followed by a longer segment rising abruptly and terminating in about the middle of the third month. Nearly half of the ca.ses which are finally closed are obliterated in the period represented by this segment (sixtj- days), l^he third segment, extending from about the middle of the third month to the end of the J'ear, shows a very slow but continuous rise and expresses the obhteration of about ten per cent of the total number of cases.

OBLITERATION OF FETAL BLOOD-PASSAGES 171

Figure 2 is a curve illustrating the froquoncy of tho oblitfration of the foraiiu'n ovale throughout life. The details of the obliteration during the first year, which have just been described, are masked in this figure by the diminution of the time unit. Here again three periods can be recognized which correspond roughly to infancy, childhood, and adolescence and maturity.

TO

%

60

^^^■-^ lS-80y«»rsTl.T

SO

-r^'^

40

SO

n

10

I.I 1 1 1 1

1 i to LSyur*

Fig. 2 .\ curve showing the average percentages of cases of oblit«rated foramen ovale at different periods throughout life. Based upon the material summarizcd in table 2.

During the first period the curve rises with extreme abruptness to a point at which fifty per cent of the cases are obliterated. The ri.-^e is continued but is much less rapid during the second period which extends from infancy well into childhood. In the third period, which extends to extreme old age, the curve rises very slowly to about seventj'-two per cent. In all probability this final percentage of obliteration is reached in early maturity although the character of our data does not permit a graphic representation of this point.

172

RICHARD K. SCAMMON AND EDGAR H. NORRIS

THE DUCTUS ARTERIOSUS

Tabic 3 shows tho compiled ihxta. upon the post-uutal obliteration of the ductus arteriosus, and table 4 summarizes these data by periods. The graphic presentation of this material is shown

TABLE 3

Data on the frequency of post-nalal oblileralion of the Ductus arteriosus. NumeraU enclosed in parentheses indicate number of obliterated cases

OMBBTBB AKC DAT!

Alverenga, '69 .

Bernutz, '65 — Billard, '28... Elaaaser, '52....

Faber, '12

Gerard, '00

Kucheff, '01...

Letourneau, '58 Theremin, '87..

Theremin '9.t Totals

19

118

(16)

150

(1)

52

12 67

II

429 (17)

11

«

5

P

<

<

a

fi

O

!•

H

t*

(O

o

«

N

CO

15

28

(1)

17

1

66

168

(14)

2

(2)

61

(6) 4 (2)

143 (18)

8 (1)

23

(1)

56

(8) 6 (3)

117 (13)

38 (36)

2

(2)

2

(2)

40

(18)

50 (26)

75

(28)

43

(23) 4 (1)

57 (27)

14

(5)

9

(3)

15

(14) 2 (2)

68

(55) 8 (8)

92 (70)

53

(48)

1

(1)

63

(52)

6

(6)

16

(12)

54 (54)

12 (12)

89 (84)

7

(7)

23

(18)

13

(13)

4

(4)

47 (33)

• 10 to 20 days.

•• 21 to 60 days.

t 2nd month (data for other periods not available).

■' to 30 days.

OBLITERATION OF FETAL BLOOD-PASSAGES

173

TABLE i Obliteration of the Ductus arteriosus (1095 cases)

Birth to 8 days 8 to 15 days. .. 15 to 22 days... 22 to 32 days... 32 to 46 days... 46 to 61 days... 61 to 91 days... 91 to 120 days.. 120 to 365 days.

10TAI.

KUHBIB or

CUM

311

148 143 117 75 57 92 63

HUHBIB or

CAIES OBLtTSBATBO

1 3

16 13 28 27 70 52 84

ram csirr or

CABIS OBLITUATSO

0.3 2.0 11.2 11.1 37.3 47.4 76.0 82.5 94.5

in figuro 1, curve B. As in the ca.se of (he foramen ovale, and for the same reasons, we have omitted Billard's data and also those of Bernutz from the summarized table and from the curve.

During the first week of life the percentage of obliteration of the ductus arterio.sus is even less than that of the foramen ovale (three-tenths of one per cent). In the second week the percentage rises to an average of two and in the third and fourth weeks to an average of a little over eleven. From this tune on to the end of the third month the process of obliteration is e.\tremely rapid; in the first part of the second month it averages over thirty-seven per cent, in the latter part over forty-seven, and in the third month seventy-six per cent. During the fourth month the average obliteration is eighty-two per cent. Thereafter the percentage increases quite slowly until the end of the year. The a\erage percentage of obliteration in the last three-quarters of the first year is nearly ninetj'-five.

The data a\ailable for the period after one year are small in amount and, with the exception of certain instances in Faber's series, all cases of this period were obliterated. It is quite possible that Faber's material included several specimens which, while containing remnants of the original lumen, were obliterated at other points. His method of examination might easily classify such cases iis patent. It is probable, therefore, that table 3 shows a much lower per cent of obliteration for this period than is actually the case.

174

RICHARD E. SCAMMON AND EDGAR H. XORRIS

Curve B of figure 1 is the graphic presentation of the data summarizes above. Like the curve for the foramen ovale already doserihod and repr(>s('ntrd in the .same figure, thi.s graph is reaciily divisible into three portion,s. The first segment extends from birth to the middle of the second week and shows a temunal obliteration of two per cent of the cases. The second .segment ri.ses abruptly, cros,ses that of the foramen ovale, and teniiiimted at about .seventy-five per cent in the middle of the third month. The third segment, which extends from the middle of the third month to the end of the first year, shows a continuously decreasing rate of obliteration and terminates at nearly one hundred per cent. Probably all normal cases are closed shortly after the first year, although there are numerous records of individual cases of the anomalous persistence of the lumen of the ductus arteriosus in later life.'

THE DUCTUS VENOSUS

The material compiled upon the obliteration of the ductus venosus is shown in table 5 and is represented graphically in curve A of figure 1.

TABLE S

Obliteration of the Ducltis venosus (,76i

cases)

Aoa

TOTAL IfDHBBB OF

oiaas

KDUBBB or

cASn

OBLRBRATBD

FIB CENT OF

CASM OBUTBKATBD

Birth to 8 dara

211

150

169

103

37

20

41

19

12

6

27 64 78 36 20 41 19 12

2.3

8 to 15 dava

18.0

IS to 22 dayK

37.5

22 to 32 days

75.7

Second month

97.3

Third month

100.0

Fourth, tifth, and sixth months

100.0 100.0

100.0

• For the literature on the subject of persistent patency of the ductus arteriosus the reader is referred to the papers of Poynter ('16), Wells ('08) and Gerard ('00').

OBLITERATION OF FETAL BLOOD-PASSAGES 175

The procrss of oblilcnitidn is much more rapid in the ductus venosus than in the other fetal passiiges. During the first week the average is two and three-tenths per cent, in the second week it is eighteen per cent, in the third thirty-seven and one-half per cent, and in the last ten days of the first month about seventysix per cent: During the second month the percentage rises to nearly one hundred and thereafter all cases are obliterated.

The curve shown in figure I, while nuich more abrupt than that of the foramen ovale and th(> ductus arteriosus, is of the same general character and shows three segments. The first segment corresponds to the first few days after birth, and terminates between two and three per cent. The second segment rises with extreme abruptness to ninety-seven per cent in the middle of the second month. The third .segment, which is very short, rises gradually to a full hundred per cent by the end of this month.

THE HATE OF THE OBLITERATIVE PROCESS IN THE SEVER.\L

PASSAGES

In t)rder to study the actinty of the obliterative process in the various fetal blood-passages we have calculated from the graphs shown in figure 1 the average daily rate of obUteration for a series of periods in earlj' life. This was done by determining from the graphs the initial and terminal percentages of obliteration for each given period. The initial percentage was then subtracted from the terminal one and the figure thus obtained divided by the number of dajs in the period. For example, in the case of the ductus arteriosus the percentage of obliteration at the beginning of the second month was fift>--seven and at the close wjvs seventy-nine and one-half. Thus twenty-two and one-half per cent of all cases were obUterated in this period of thirtA' days and the average daily rate of obliteration was seventy-five hundredths per cent. Table (5 shows the results of these calculations for the three passages and the curves in figure 3 express them graphically.

It will be seen by the examination of these curves that they have certain characters in common. Each starts with a low rate

176

RICHARD E. 8CAMMON AND EDGAR H. NORRIS

TABLE t

Approximate rate of daily and f

obliteration of the Ductus venosut, Ductus arteriosus, 'oramen ovale in early childhood

FSRIOD

ODCTDB VEHOSnS

DUCTCS

ARTiBioem

roBAuaN

OTALI

0.33

3.00

0.60

0.04 0.92 1.00 0.75 0.07 0.04

0.10

1 week to 1 month

0.60

0.45

0.40

3 to 6 months

0.07

6 to 12 months

0.04

1 to 5 vears

0.005

of obliteration, rises rapidly to a peak or niaxinium, and then declines to the base-line which represents the cessation of obliterative activity. In all cases the portion of the curve representing the decline in activity is less abrupt than is the initial rise.

Considering the curves individually, it will be noted that in the case of the ductus venosus both the initial and maximal rate of obUteration is much greater than that of the other two passages and that consequently the entire obliterative process is completed much sooner. The curve expressing the rate of obliteration of the ductus arteriosus is very similjir to that of the ductus venosus although the initial and maximal rates are lower and the apex of the curve falls at a later period. While the curve of the rate of obUteration of the foramen ovale shows the three conmion characters indicated in the preceding paragraph it is markedl}' different from the curves of the \cs.sol.s. It ri.scs less abruptly to a lower maximum rate of obliteration which is maintained with but little loss for a much longer period, so that the curve presents a plateau which is entirely absent from the curves of the vessels. Following this plateau, the curve falls at first rapidly and then gradually over a long interval to the base line.

The results of this study may be sunmiarized as follows:

OBLITERATION OF FETAL BLOOD-PASSAGES

177

IstwV.

lukrl mo.

2nd mo.

3rd mo.

15-6 mos. 6-12mo».

l-5yrs

Fig. 3 Three curves showing the approximate average daily rate of obliteration of the fetal blood-passnges in early life. A, dotted line, foramen ovale, B, solid line, ductus arteriosus; C, broken line, ductus venosus. Based upon the data summarized in table 6.

17S RICHARD E. 8CAMMON AND EDGAR H. NORRIS

SUMMARY

The tinio of oblitoration of the three fetal blood-passages (the ductus venosiis, the ductus arteriosus, and the foramen ovale) is distinctly hittr than is cojnnionly assumed. The prnocss of oblitvniliim in each of these passages shows three fairly- distinct periods: an initial period with a low rate of obliteration, a middle period in which the rate of obliteration rises and the majority of cases iire closed, ami a tenninal period in which the rate of obliteration is again slower.

ObUteration is most rapid in the ductus venosus. Although slow in the first week, the process reaches its maxiniuni before the end of the first month and in the third month and thereafter all cases are closed.*

The ductus arteriosus closes more slowly. The obliterative proce-s-s, which is very slow during the first two weeks of life, does not n>ach its maxiiimm until the second month. Threefourths of all cases arc closed at the end of the first trimester and over ninety-five per cent by the end of the first j^ear.

The period of the obliterative process of the foramen ovale is a matter of years rather than months. Beginning ver)' slowly the process reaches its maximum activity near the close of the first nxmth and continues with a slightly diminishing rate during the remainder of the first trunester. At the end of this time approximately one-third of all cases are closed. During the second trimester the rate of obliteration declines rapidl}' and thereafter decreases verj- slowly for an indefinite period — probably until early maturity, although few cases are closed after childliood. At the end of the first year about one-half of all cases are closed, ill th«' second deccnnium about two-thirds, and in maturity about seventy-two per cent.

^ * It has been shown by the studies of Wertheimer ('86), Nitdtin COl), Fontan ClI) and others that the vein which sometimes occupies the center of the ligament of the ductus venoaus in older children and adults is a new vessel developed after the obliteration of the ductus venosus and is not derived from the remains of this trunk.

OULITERATION OF FETAL HLOOD-PASSAGES 179

POSTSCRIPT

Aft or this pii,pc>r was in press we secured :i summiiry of onrhundn (1 ;ind eighty-*,'Veii obs Tvations by Parrot on the obliteration of the ductus jirteriosus. Parrot's findings are in general agroeint nt with thos.^ of other observers which we have sununarized above. Fnfortunatt'ly his cases under one year are grouped in such a way that wc? eaiuujt include them in our table 4, but if this were possible they would evidently affect our averages very little. Parrot found the ductus arteriosus patent in four ca.ses in thirty-three of one yejvr and in one in fifty-four of two years. In seventeen crises of tliree y<>ars and over the the <luctus wa.s always obliterated.

BIULIOOHAPHY

Alexeieff 1900 The foramen ovale in tlie child. Diss. St. Petersburg. Alvarenga, p. F. DaC. 1869 Considi^rations et observations sur IV'poque de

I'occlusion du trou ovale et du canal arti^riel. Lisbon. Ber.ndtz 1865 [Quoted from Gc/rard, (1. ('00«).l BiLLARD, C. M. 1828 Traitd des maladies des enfants nouveau-n^s et k la

mamelle, fondi^ sur de nouvelles observations cliniques et d'anatomie

pathologique, faites & I'hopital des enfants trouvos de Paris. Paris. BizoT, J. 1837 Hecherches sur le coeur et le systtoe art(?riel chez I'homme.

Mem. .Soc. Med. d'Observation, T. I. EL6A8SER 1852 t'ber den Zustand der Fotuskreislaufwcge bei neugeborenen

Kindern. Zeitschr. f. Staatsarzneik., Bd. LXIV. Faber, \. 1912 Die anatomischen und physikalischen Verhiiltnisse des Ductus

Botalli. .Vrch. f. .\nat. u. Ent. Fawcett E. AND Blachford, J. V. 1900 The frequency of an opening between

the right and left auricles at the seat of the foetal foramen ovale.

Journ. .\nat. and Physiol., vol. XXXV. FoNTAN, C. 1911 Lc canal d'Arantius (i^tude anatomique). Th^sc, Lille. G£rard, G. 1900 Lc canal artrriel. Etude anatomique. Journ. de I'Anat.,

T. XXXVL

1900' De I'oblitt^ration du canal art^'riel (les th^-ories et les faits).

Journ. de I'Anat., T. XXXVL Uauerda. A. 1896 Die fotalen Krcislaufswege des Neugeborenen und ihre

Vcriinderungen nach der Geburt. Wien. Klobb 1859 (Amtl. Ber. XXXIII Versamml. deutsch. Xaturf. u. .\rzle zu

Bonn, 1857.)

• Depaul, Diet. Encyclo|x5d. d. So. M6d., 2. a6i., T. XIII.

ISO RICHARD E. SCAMMON AND EDGAR H. NORRIS

KucHEFF, N. E. 1901 (The ductus Botftlli in cliildren.) Diss. St. Petersburg

(Quoti'd from summnrips in Jnhrcsl)cr. f. Annt. u. Entwickl., Bd. VII

(N.F.), and from Ciundohin: Die Besonderheiten des Ivindcsalters,

1912.) LETorRNTiAU 1858 Quelques observations sur le nouveau-n<5. ThJse, Paris. Parsons, F. G. and Keith, A. 1898 Seventh report of the committee of collective investi(?iilion of the .\nntomicaI Societj- of Great Britain and

Ireland, for the year 1896-97. Journ. of Anat. and Physiol., vol.

XXXII. NiDTiN, A. A. 1901 The duct of Arantius in childhood. Diss. St. Petere burif. Ogle, J. 1857 On certain cases in which the foramen ovale was still patent in

the adult. British Med. Journ. PoYSTEB, C. \\. M. 1916 .\rterial anomalies pertaining to the aortic arches

and the branches arising from them. University Studies, (U. of

Nebraska) vol. .XVI. Quincke, H. 1885 Ueber die Entstehung der Gelbsucht Neugeborener. .A.rch.

f. exp. Pathol, u Pharmakol., Bd. XIX. THfREMiN, E. 1887 Xote sur I'involution des voies foetales. Rev. d. Mai. d.

I'Enfance, T. V.

1895 £tudes sur les afTectioos congenitales du coeur. Paris. Wallma.vn, H. 1859 Ueber das Offenbleiben des Foramen ovale cordis bei

Erwachsenen. Vierteljalirschr. f. d. prakt. Heilk., Bd. LXII. Wells, H. G. 1908 Persistent patency of the ductus arteriosus. Amer. Journ.

Med. Sc. N.S., vol. CXXXVI. Wertheimer, E. 1886 Recherches sur la veine ombilcale. Journ. de I'Anat.,

T. XXII. Zahn, (Cited by .\bbot, M. E. in: Osier and McCrae, Modern Medicine, vol.

IV, 1908.)

ltTIU>H»' AH.-»TH\CT fV Tilt* P*PI:K I^hUCD KV TIIK blULIuaHAPIIIC AKflVICC, OCTOUKK It

iiii; i;Ai;i.\ AiTKAirwch; of iiii-; aslma.s oi' iiii-: I'AUs nuKHAi.is IN iiii: iivpophvsis of i'iif-:

ClIKK

UAYNK J. AfWKLF- AND IDA SITLKU

Dr.pnrliiiriil •>) Aiiiilninii, Meilinil Sihiiiil nf the I'niverxili/ nf Miih!ijiin

FIVK PICIHKS

[t is now well n'('()>riuz((l lli:it tlic cpithcliiil portion of tlip liyiJophysis consists of three ilistinct parts. The pars anterior propria is the principal cpitheHal lobe and constitutes the main l)iilk of the ulaml, the i);'.rs int(>nii( dia is a thin lavt'r. epithehal in iiatun". which becomes iiitiniately associated with the neural lobe. 'rh(» nicst recenth" rec(jgnizeil epithelial lobe is the pars tul)eralis — so named by Tilney ('13) on account of its close relation to the tuber cinereum. It extends forwaril from the junction of the pars intermedia anil the pars anterior propria, surrounding the infundil)ular stalk and spreading out for .some di.staiice under the brain floor.

The jnirs tubcralis has been .sometimes confu.sed with the pars intermedia- Lothringer ('86) and Herring ('08) — but recent studies have .shown conclusively that these two parts are different both in adult structure and in developmental historj-. Tilney, in summarizing the develo])ment of tlie pars tuberalis in the chick and the cat, states:

In atUlition to (In- histological ditTorcnccs between these two parts, llic ontogenesis of tlie organ as observed in the cat and the fowl still furtluT einpiiasizes the fact .that the pars tubcralis and the pars infmnlibularis (pars inlernuMlia) are morphologically distinct elements. Till- |)ars infiiiidibularis makes its appearance inniiediately after the aiiianc of the buccal portion of the liypopliysis is fornietl. The pars tubcralis ai'ises as a relatively late stniciiirc. It has its origin in two sccond.aiy ilivcrliciila or sprouts from the ImkIv of the pituitary sac. These sprouts, the tubcral processes, ultimately fuse with each other across the median line, displace the body of the pituitary sac vcntrad and thus secondarily a.ssume their juxta-iiein-al position.

181

TUB ANATOMICAL RI.CiiKD, Vol. 15. SO 4

IS2 WAYNK J. ATWKM- WD IDA SITLKR

Out" (if lis Al\V(>ll CIS) — ill ;i recent study of tlie tlevelopiiii'iit of tlie liypopliysis in the nil)l)if has obtainod somewhat (lifTereiit results. Wliile afireeiiifi with Tilney as to the distinctness of the pars tul)erahs and the pars intermedia, and also conHrminK the statement tliat the pars tulieralis is late in acquiring its adult relationship with the tuber cinereum, it has been found thai in the rabbit the anlagen of the pars tuberalis may 1)1' discerned \erv early. They were found to precede* the definite pars intermedia by a considerable period of time.

It was with the lio|)e of throwiiifi some light upon this point that the j)resent study was imdertaken. Accordingly we have been led to construct a luimber of wax-plate models of the epillielial hypophysis from chick emiiryos, l)eginning with .stages in whicii the tuberal processes might be recognized easily and then j)roceeding to succes.sively younger (>mbryos in an effort to determine the earliest appearance of th<' anlagcu.

Tlie literature relating to the lateral lobes and the pai's tuberalis in the hypophysis of the chick is not extensive.

Rossi ('{)()) speaks of a median and two lateral parts in the early hypophj'sis of the chick embryo, .\ccording to Ro.ssi the lateral lobes are secondary structures.

iM'onomo ('!)!)) observed a pair of "Seitenspro.ssen' in the hypophysis of the dove and of the domestic fowl. In dove embryos the sprouts appear b(>tween the fourth and seventh days of incubation. \o definite statement is made concerning the first ai)pearance of the sprouts in the chick.

Tilney ('13) first ob.served the 'tuberal processes' in a chick embryo of o days and 20 hours of incubation. From this stage the jiroces.ses were traced to the formation of the pars tuberalis of the adult fowl, .\lthough a reconstruction was prepared from an embryo of fourda3's of incubation the tul)eral proces-ses were not seen in this stage. Embryos younger than four days were not studied.

Woerdeman ('14) notes that the lateral lobes are forming in a chick embryo of about 72 hours of de\-elopment. The thickened epithelium which lies in front of Hathke's pocket is constricted off from the mouth cavity by two lateral folds. Woerdeman con

PARS TUUER.\US IN HYPOPHYSIS OF CHICK 1S3

siders that the lateral lobes so formed arise independently of Rathko's pocket.

Bruni ('15) observes the presence of two 'lobi laterali' in the chick at 82 hours of incubation. He also figures and describes the latrral lobes in older embryos but does not trace them into the formation of the pars luberalis.

OBSERVATIONS

We have prcpan-d wax-plate reconstructions of the epithelial portion of the hypophj'sis from cliick embryos of 48, 59, 67, 72, 96, 120 and 144 hours of incubation. A relatively high magnification was chosen for the construction of the models in order that all details of structure might be shown as accurately as possible. For all younger stages, including the 72 hour embryo, the magnification was 300 diameters. For the older embryos the magnification was reduced to 200 diameters.

Chick embryo, 4^ hours of incubation {21-2 pairs of primitive segments). Fig. 1. The hj^jophyseal pouch is well formed but opens widely into the mouth invagination. There is no indication of the lateral lobes. The anterior end of the fore-gut, which will later form Seessel's pouch, extends farther cranially than does the hj-pophyseal pouch. At this time the oral membrane is intact.

Chick embryo, 59 hours of incubation (30 pairs of segments). The hypophyseal pouch (Rathke's pocket) has deepened and now exhibits two lateral enlargements near its attachment to the oral epithelium. As later models show, these are theanlagen of the lat«'ral lobes from which the tuberal processes develop. .As may be seen from figure 2, Rathke's pocket is shghtly constricted just above the lateral lobes. The lateral lobes have the fomi of blunt ridges which protrude laterally and also somewhat nas^iUy. Their long axes lie parallel with the long axis of the entire h.vpophyseal pouch. This embryo shows one small perforation in the oral membrane.

Chick embryo, 67 hours of incubation. The lateral lobes are more prominent at this stage due to the fact that the hypophyseal pouch is beginning to be constricted somewhat from the

184

WAYNE J. ATWELL ANU IDA SITLER

_l.p.

PARS TUBERALIS IN HVPOPHYSIS OF CHICK 185

oral ojivity. The constriction of Rathkc's pocket dorsal to the lateral lobes is also more distinct than previously. Each lateral lobe contains a lumen communicating with the cavity of the main h}7iophyseal sac. Seessel's pouch is in contact with the dorsal wall of Hathke's pocket for a considerable area. This is the eeto-entodennal fusion which has been recorded by numerous observers.

Chick embryo, 72 hours of incubnlion, figure 3. The hypophysis anlage is closely appUed to the brain wall, causing the nasal surface of the pouch to be sharply concave. The lateral lobes are more prominent than in the preceding .stage. The lumen of the pouch extends well into each lateral lobe. One striking feature is the extensive degree of communication between the cavity of Seessel's pouch and the hypophyseal sac. The two open into each other almost to the summit of the ecto-entodemial fusion. This cau-ses the opening of the hypophyseal sac into the oro-phuryn.\ to be relatively larger than in pre\-ious stages. From an examination of embryos of this age alone the impression might be gained that the lateral lobes are being added to Rathke's pocket. A critical comparison of this and younger stages, however, indiates strongly that the lateral lobes of the chick do not arise independently of Rathke's pouch, but that they are formed from it. In this we support the observations of Rossi.

Chick cinhryo, 96 hours of incubaUon. The principal feature of interest in this stage is the beginning recession of Seessel's pouch and its separation from the hj'pophyseal .sac. The lateral lobes are more sharply marked off from the superior part of the hypophysis, but otherwise this stage does not exhibit any striking differences from the preceding. .

.\11 figures represent wux-pliite recon-structions of the epithelial hypophysis as viewed from in front and from the left side. S, Seessel's pouch, R, Rathke's pouch, /./., lateral lobes, t.p., tuberal processes, st., hypophyseal stalk.

Fig. 1. Hypophysis region from chick embryo of 21-2 pairs of primitive segments (end of second day of incubation). X 100.

Fig. 2. Hypophysis from chick embryo of 30 pairs of primitive segments (59 hours of incubation). X 100.

Fig. 3. Hypophysis from chick embryo of 72 hours of incubation. X 100.

Fig. 4. Hypophysis from chick embryo, o days (120 hours) of incubation. X 75.

Fig. 5. Hypophysis from chick embryo, 6 days (144 hours) of incubation. X 75.

186 WAYNE J. ATWELL AND IDA SITLKK

Chick embryo. 5 days (ISD hoiirn) of incubation. By tliis lime a definite h yp< )pliysoal stalk has been fonned. It is hollow and affords a coniniunioation between the lumen of the hypophysis and the oral cavity. The lateral lobes have increased in size so that the transverse diameter of the gland, measured between the lateral extremities of the two lobes, is almost twice the transverse diameter of the superior part of Rathke's pocket. The lateral lobes ar<^ united by a prominent ridge around the inferior and nasal enil of the hj-jiophysis. This solid median protuberance doubtless corresponds to a vestigial '\'orraum' or 'corpus I«)buli bifiu-cati' of other vertebrates as described by Woerdeman. The lateral lobes are beginning to be solid, also. At this stage they sometimes contain lumina, which, however, no longer conmiunicate clearlj- with the main h3^pophyseal cavity.

Seessel's pouch is represented by a solid bud of epithelial cells just dorsal to the hypophyseal stalk (S, fig. 4). Curiously enough Economo labels this bud the remains of Rathke's pocket.

Chid: embryo. 6 days (144 hours) of incubation. The hypophyseal stalk is much elongated and has become solid. Near its connection with the oral epithelium may be seen the bud-like remains of Seessel's pouch. The superior, or distal, half of the hjT)ophysisis is bent dorsalh" and forms an angle of about ninety degrees with the inferior or proximal half of the gland. The constriction near the middle of the gland is pronounced. Distinct 'tuberal processes' have formed from the lateral lobes. Instead of projecting so much laterally-, thej' are now directed toward the brain wall. The tub(>ral proc(\sses are not located at the extreme nasal end of the gland but are seen to protrude from about the middle of the inferior half (fig. 5).

SUMMARY

The lateral lobes, from which the tuberal processes arise, may be distinguished in a chick embryo having 30 pairs of primitive segjiients. From a careful study of stages preceding and following the rupture of the oral membrane it is evident that the lateral lobes are not fonned independently of Rathke's pocket

PABS TUBERALIS IN HYPOPHYSIS OF CHICK 187

and later added to it, but are rather fonried secondarily from the nasjil wall of the early hypophyseal anlaK*'.

The lateral lobes, in all foniis studied, appear earlj- in devel«»pment. This would indicate that they and their derivative in higher vertebrates, the pars tuberahs, are of fundamental phylf>genetie importance. Thus gwat interest is attached to the broad homologies drawn by Woerdeman ('14).

LITERATURE

Atwell, Wayne J. 1918 The development of the hypophysis cerebri of the rabbit (Lepus cuniculus L.). Amer. Jour. Anat., vol. 24, p. 271.

Brum, A. C. 1915 Sullo sviluppo del lobo ghiandolare dell' ipofisi negli Amnioti. Internat. Monutschr. f. Anat. u. Physiol., Bd. 31, S. 129.

EcoNOMO, C. J. 1899 Zur Entwicklung der Vogelhj-pophyse. Sitzber. d. kais. Akad. d. Wiss., math.-naturw. Classe, Bd. 108, Abth. 3, S. 381.

Herring, P. T. 1908 The histological appearances of the mammalian pituitary body. Quar. Jour. Ex. Physiol., vol. 1, p. 121.

LoTBRiNGER, S. 1886 Untersuchungcn an der hypophyse einiger Saugetbiere und des Menschen. Arch. f. mikr. Anat., Bd. 28, S. 257.

Rossi, U. 1896 Sui lobi latcrale della Ipsofisi. Monit. Zool. itul., 7, p. 240.

TiiJJET, Frederick 1913 An analysis of the ju.xtra-neural epithelial portion of the hypophysis cerebri, with an embryological and histological account of an hitherto undescribed part of the organ. Internat. Monatschr. f. Anat. u. Physiol.. Bd 30. S. 258.

Woerdeman, Martin W. 1914 Vergleichende Ontogenie der Hypophysis. Arch. f. mikr. Anat., Bd. 86, ,S. 108.

THE IDENTIFICATION OF ENDOTHELIAL LEUCOCYTES IN HUMAN TISSUE

THIRD REPORT OF STUDIES ON THE MONONTJCLEAR CELLS OF THE

BLOOD

F. A. McJUNKIN Deparlmenl of Pathology, Marquette Univerrily School of Medicine

TWO FI0UTIE8

In an earlier report by the writer ('18) it was shown that the phagocytic mononuclear cells present in the peripheral blood arise by mitosis from the endothelium of the blood vessels. The method that was devised for this purpose consists of the intravenous injection of lampblack suspensions and is not, therefore, applicable to human tissues. The tis.sues of animals injected with carbon suspensions in which the endothelial leucocj-tes and cells are characterized bj' carbon particles ingested by phagocj-tosis are, however, well adapted for testing the action of various stains on these cells. It has been found that the staining method u.sed by Craham ('16) colors these leucocytes in a characteristic way in both animal and human tissue. Since paraffin or celloidin sections caiuiot be used, Graham emploj-ed frozen sections for his stain but owing to their thickness, they are not suitable for accurate cell identification. The purpose of this paper is to record a new method of tissue imbedding for obtaining thin sections to which the stain is applicable.

The .staining method of (iraliam depenils on the action of solutions of alphanaphthol on parts of the cytoplasm of cells. It was shown by O. Witt ('82) that a blue dye (indophenol blue) is formed by the oxidation in dilute alkaUne or acid solution of alphanaphthol and dimethyl-para-phenylenediamine. Winkler ('07) and others found that myeloblastic cells react in a characteristic way in tissues treated with these two substances and the

189

190 F. A. MrJUNKIN

grunulcs of the reacting rolls wore said to contain an oxidizing frrniont (oxydase or peroxidase) that oxidized (he two conii)ounds and caused them to unite with the production of a blue color in the neutrophilic and eosinophil granules. Later Loele ('14) found that the treatment of mjeloblastic cells with alphanaphthol solutions alone produced the same blue color in the granules. The part played bj- the cell granules has usually been regarded as an oxidizing one ('oxydase or peroxydase reaction). As applied by Loele the phenomenon consists of a pur])lish or bluish color in the cytoplasmic granules of the cell produced by treatment with old alphanaphthol solutions. If a dj'ostuff is produced by alphanaphthol alone, and this seems likely, an aromatic compound or compounds must be supplied by the leucocytic granules. If such is the case "indophenol reaction" is a better term than "oxydase reaction."

Leucocytic granule stain (Ciraham). Since hydrogen peroxide is added to the alphanaphthol solution to make it immediately activ(> and the swollen granules are heavily and permanently stained by treating the preparations with an aniline dye, the method of Graham is better than the other indophenol staining methods devised. To applj' this method, remove thin sections of formalin-fixed tissue attached to slides from the distilled water and stain in dilute Q-5) hematoxylin (Dclafield) for five minutes; wash them in distilled water, place in a saturated solution of lithium carbonate for five minutes, wash in distilled water for two minutes, and stain for ten minutes in 10 cc. alphanaphthol .solution to which 10 drops of 1 per cent pyronin (Griibler) have been added immediately before placing the preparations in it. Prevent evaporation by covering the dish. Wash the sections in distilled water, place in saturated aqueous solution of lithium carbonate for from five to ten minutes, wash in water and differentiate and dehydrate in 95 per cent alcohol for one-half minute. Complete the dehydration by inniiersing the slides in xylol and raising them above the surface of the liquid two or three times and blotting with smooth, soft filter paper. A flat oblong .staining di.sh, the .size and width of a slide, is used for the staining and differentiation of the slides. Mount in

ENDOTHEUAL LEUCOCYTES IN HUMAN TISSUE 191

coloplioniuni-xylol or acid-fr(M> balsam. Tho tinu' that tlio prpparations remain in tin* saturatod lithium carbonate aftor troatmont with alphanaphthol is important because this removes the excess of pyronin. With some tissue better results are obtained by staininp only five minutes in the alphanaphthol-pjTonin solution and differentiating for a shorter time in the lithium carbonate after this stain. If the one per cent pyronin solution is to be kept for some time, sufficient formalin should be added to make it 10 per cent formalin.

The alphanaphthol solution is made by dissolving 1 gm. alphanaphthol (Merck Reagent) in 100 cc. 40 per cent ethyl alcohol (made from absolute alcohol) at a temperature of 50°C. and adding 0.2 cc. hydrogen peroxide. Commercial hydrogen peroxide containing approximately 3 per cent hydrogen peroxide, as detennined by titration with decinomial potassium permanganate, should be used.

Soap method of imbedding. 200 grams transparent glycerine toilet soap are placed in a 5(X)-cc. Erlenmeyer or Florence flask, that contains 200 cc. distilled water. The soap must be so hard that it is brittle and cracks apart when cut with a knife, otherwise the soap solution will not be of the proper consistency. The flask is placed in the paraffin oven at 52°C. overnight in order to dis,solve the soap. Remove it from the oven and place for three hours in an incubator at 37.o°C. The contents should be a syrupy liquid and should solidify when a small amount is poured into a paper boat and allowed to stand at room temperature for one-half hour. If solidification does not take place, 20 grams of soap should be added to the flask, and the contents again melted in the paraffin oven. After the correct consistency has been obtained the soap solution is placed in 100-cc. wide-mouth bottles with cork or glass stoppers, with about 50 cc. to a bottle.

To imbed the tissue small pieces are taken from 10 per cent fonnalin and dropped into the melted soap contained in one of the bottles which is placed at 37.5°r. two to three hours and occasionally shaken. The liquid soap in the bottles usually become solid jifter remaining at 37.5°C. for a day or more. To melt the solidified soap, the bottles are placed in the paraffin oven for

192 F. A. McJUNKIN

an hour, the s(jap coolctl to A't'^C, the tissue added, and the Inittles rephic«'d in the incubator at 37.5°C. The soap solution with the tissue in it is eniplitd into a box of suitable size made from paper as in paraffin imIjediLing and the tissue arranged on the bott«.>m of the box with forceps. The box should be made from paraffined paper or the paper may be coated by pouring melted paraffin into it. At the end of about one hour the paper is removed, the soUd soap trinuned with a knife to thi; desired size about the tissue and the blocks attached to a heated metal disk just as paraffin blocks are attached. The blocks after about one hour are dropped into a siiturated solution of sodium chioriile in a pint Mason jar, and the jar placed hi the incubator at 37.5°C. overnight.

With forceps remove the block from the saturated salt solution, attach to rotarj- microtome and cut away the block until the tissue is reached. Carefullj' trim the block and allow to dry for from three to six hours until a ribbon 6 to 8 microns thick cuts perfectly. The di.sks maybe detached from the microtome and after the proper drj-ing again attached, so that the ribbon comes from the very surface of the block. The ribbon is placed in distilled water in a fiat dish more than 6 inches in diameter, and sections floated on shdes on which there is a thin coating of fixative made by adding 4 cc. of a very thick .syrupy celloidin to 16 cc. oil of cloves. The preparations after being pressed out and carefully blotted with filter-paper are placed in the paraffin oven for fifteen minutes, when they are removed, washed in 95 per cent alcohol for thirty seconds, and placed in distilled water where they remain less than five minutes. It)nization in the large volume of water in which the soap sections Jire first placed develops only a shght alkalinity, and in the thick soap solution ionization is practically absent. The sjiturated salt solution hardens the blocks since it prevents hydrolysis by mass action. If the tissue is quite fragile the ribbon may be placed in sjiturated salt .solution instead of distilled water.

Reaction of sectio7i.s obtained by the soap method of imbeddimj to the stain. The nuclei are blue and the granules of myeloblastic cells, eudothehal cells and endotlielial leucocytes arc red. (iraham

ENDOTHELIAL LEUCOCYTES IN HUMAN TISSUE 193

notrd fh<' red granuli-s in both ciidothclijil cells and ondothcliiil leucocytes and ixpluincd their presence there as the result of ingestion by phagocytosis of niyeloblastic ceils or the cytoplasmic granules of these cells. He does not make it clear whether the grannies were found in all endothelial cells and leucocytes.

In the thin .soap sections it is evident that many cells of endothelial origin contain the granules and that they are not present here as the result of an accidental phagocytic phenomenon is shown by the small size of the granules and their even distribution in the cell cjioplasm. The granules difTer from the myelobla.stic granules .seen in neutrophiles and eosinophiles in being f(>wer in number, snudler in size and more di.scretely distributed. In ti-ssues in which only mature ipol\Tnorphonuclear) niyeloblastic cells are present a casual glance is sufficient to distinguish the endothelial leucocytes containing the red granules since they are mononuclear. In ti.ssue containing myeloc.vtes, the heavier staining and greater number of granules of the neutrophilic and eosinophihc myelocytes separate them from the endothelial leucoc\-tes. The differentiation between myelocytes and endothelial leucocytes is well shown in sections of bone-maiTow

(fig. 2).

The characteristic action of the stain is best .seen in sections of the liver or other organ of an animal that has received intravenous injwtions of a lampblack suspen.sion according to a method devised by the wTiter ('18). Endothelial cells containing carbon and definiieiy lining the sinusoids have nnl granules scattered in their cytoplasm (fig. 1). Likewise carbon-containing endotheUal leucocytes in the vessels show the discrete red granules. The neutrophilic and eosinophilic leucocytes are more conspicuous than the cells of endoihdial origin owing to the greater number and larger size of their granules. There are a certain number of neutrophiles, eosinphiles, and endothelial leucocytes that do not take the stain; anil it is only in the endotheUal cells with a distinct amount of visible cytoplasm that the granules may be distinguished. The failure of some C(>lls to stain appears to be due to an error in technic but all attempts to correct this have failed.

194 F. A. McJUNKIN

BIBLIOGRAPHY

Witt, O. 1882 J. Soc. Chem. Ind., p. 255.

Winkler, F. 1907 Folin Hematologia, vol. 4, p. 323.

LoELE, W. 1914 Folia Hematologia, vol. 18 p. 581.

Uraiiau, G. S. 1916 Jour. -Med. Research, vol. 35, no. 2, p 231.

McJuNKi.v, F A. 1918 Am. Jour. Aiiat., in press.

PLATE 1

EXPLANATION OP FIOtTRES

Both figures drawn with camera lucida and oil-immersion lens from indophenol stains of 7-micron soap sections.

1 Liver of a dog that has received intravenous injections of lampblack suspensions, a, two endothelial leucocytes containing small particles of carbon; 6, an endothelial leucocyte near a mass of carbon hi a sinusoid.

2 Bone-marrow (human), a, endothelial leucocyte or cell, 6, myelocyte; c, polymorphonuclear neutrophile. All leucocytic granules are a bright red; those in the cells of endothelial origin are smaller and more discrete than those in the myeloblastic cells.

KN'IX)llli;l.l \l. I.i:i COCVTKS IN HI MAN TISSIK I'l.ATK I

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195

aothor'h AiMTiiAcr or iiim patbr ihubd or TiiE Hiui-KKiHAPUio anivtoi. October 8

THE SCAPULA OF TRAGULUS

FRANK BLAIR HANSON Zoological Laboratory of Washington University

NI.VE PiaUREB

During tlic suiiunor of 1!)17 tlio author was examining the collection of nuiminalian scapulae in the U. S. National Museum, Washington, D. C, seeking data in regard to an entirely difTorent problem than the subject of this present paper, ^^^lile handUng this material his attention was attracted to a scapula, whose suprascapular region was quite unlike anything else in the whole mammalian series under review.

This scapula was of an adult mounted specuncn of one of the species of Tragulus; also known as Pigmy Deer, Deerlet, and Chevrotain. There is very Uttle in the literature concerning these small door-like animals, which have also some points in cojumnn with lioth pigs and marsupials. Their sy.stemic po.sition is doubtful, but Beddard ('02), whose brief account is the most extensive one known to me, puts them with the Ruminants since they possess .Vrtiodactyle feet. In many respects, dentition, feet, .stomach, and placenta, they are intermediate between the pigs and the Ruminants.

.Milnc-I]dwards ('04) has a few notes on the structure of these aiiinKtis.

Parker ('68) in his Monograph on the Shoulder-Girdle and Sternum, does not figure the scapula of Tragulus at all. but gives the following brief, and with one exception, correct account of it:

III an adult Tragulus javanicus the whole shape is broader, the neck short, and very narrow; the deep spine foiins a rifiht aiiRJo witii the suprasrapular liordcr of the hone: tiiis border is nearly straight; the prac.scapiiiar region has an arcuate outline, and its fos.s!» is only onefifth the widtli of the widest (upper) part of the infra-spinous space.

m

TlIK ANATOMICAL RECORD. VOL. 15, NO. i

198 FRANK BLAIR HANSON

The supriiscjipula ossifies very late; the spine grows downwards into a straipht, sharp acromion; tlic coracoid process is well shown, but is short, flat, cinarginate, and incurved.

The exception taken to Parker's account is rcf^anling the ossification of the suprascapula. The authorities of the U. S. National Museum placed at my disposal several specimens of foetal and .idult Tranulidae, four of which are seen in figures 1-4.

Figures 1 and 2 are tkawings of foetal scapulae of Traguius, one from Borneo, and the other from S. W. Borneo. They were from foetuses about 4 J inches in length, and there is no essential cUfference between the two spechnens. The suprascapula is relatively large and as yet no center of ossification is present. The entire shape is essentially adult, except that the cartilaginous region around the glenoid fossa extends some little distance up the neck of the scapula ; also the neck of the scapula of figure 1 is much thicker than in the adult.

Figures 3 and 4 are of adult scapulae. The first one is from Borneo, the secimd from Java. Here appears the peculiarity in the suprascapula, of which this paper is a brief record. In the center of the cartilaginous supra-scapula and occupying roughly one-third its area is a patch of bone. The bone is entirely surrounded by cartilage of the suprascapula, and as the animals were obviously fully matured, it is not likelj' that this center of bone formation ever unites with the scapular blade.

The fact has been mentioned that in several respects Traguius is intermediate or transitional between the Suidae and the liigher Ungulates. It wf)uld secjn that in the scapula also we have another character pointing to this intermediate position. In figures 5 9 are shown a series of vertebral borders of scapulae, begimiing with the pig in which there is never any ossification in the suprascapula, not even in an aged boar, as I have recently had the opportunity of ascertaining. In this series of sketches (fig. 5-9) Traguius seenxs to fit in between the .Suidae and the Red Deer, in which the suprascapula is poorly ossified over its entire extent (Flower); this in turn leiids to the Carnivora, with the merest tip of cartilage rejuaining; and so on up to the Primates, where the vertebral bonier has lost the last vestige of cartilage in the mature adult.

THE SCAPULA OF TRAGULUS 199

Tims ill .Ml ascciidiii)!: pliylngcnotic series of unijiials the sraj)Uhie arniiigc fhemsolves witli roKurd to a (liiiiinisfiiiiK siiprascapula in the precise manner in whicli this reduction takes place in the oiitofteny of the scH|)iila in uny one of (he hi^lier inanmials.

1 desire to exjjress niy deeji ajipreeiation of the courtesy accorded me by Mr. (lerritt S. ^liller, Curator of the Division of Mammals, U. S. National Afiiseum, in permitting me to examine and dissect this rare and \'aluaf)le material.

LITKH.VTUHK CITED

Hkdoakd, V. K. 1!K)-' Maiiimalia. Ciiiiil>. N:it. Hist., vol. 10. Fi.owEK, W. H. I88.'> Osteology of the .Munmialiii. London. Mii.\-k-Kdwards 18(5-1 TrnRiiIiis. Ann. Sci. Nat. (5) ii. Pakkkr, W. K. 1868 The Shoulder Girdle and Sternum. London.

•_'(MI

KHANK Itl.AlK II WSON

li({. 1 ScMpula of footus of TriiKuliis from Borneo, iiicilinl side. X S. V. S. .\';it. Mum. six^rimcn No. 17S.

Kig. 2 >Sr!ipulu of foetus of Tragulus from S. \V. IJoriu'o. laUTiil sidi'. X 8. U. S. Nat. Miia. 8|)cciraen No. 153906.

Fife. ■'< .Scapula <if adult Tragulus from Borneo, sliowinK bone formation in rentre of rarliliipnouH suprn.srapula. X 1. V. S. Nal. Mus. specimen No. I<»7ti72.

Fig. ^ .Scapula of adult Tragulus from Java. X 1. 1'. .S. Nat. .Mus. s|M.-eiiiMii No. 1.5(i289.

.tHBRKVIATIONS

«, Bono

X.F., Nerve foramina

.S'.Sr., .Supraseapula Sc, .Scapula

THE SCAPl'LA OF TRAOrM'S

201

SSc

Kin. •"> Wrlebral Murder of ailull pin. SupriuscHimla never iissifies. Two ranches of spinal nerves pass throuiel' foramina in the cartilage. Fig. 6 Kiilarged view of verlcliral liorder of scapula in ligurc 3. Fig. 7 Suprascapiila of Red Deer. Modified after Flower. Fig. 8 Vertebral scapular border of adult male cat. Fig. Vertebral scapular border of man.

Resuniido por ol autor, Waro Nakahara.

Algunas obsen'aciones sobre el crecimiento de los ovocitos

de Perla ininarginata Say, con especial menci6n del

origen y funci6n de las estructuras nucleolares.

El nucleo del huevo ovdrico de Perla inmarginata contiene dos clases de cstnioturas nucleolares, a saber: I'n nurleolo unico, de gran taniano (nuclcolo principal) y otro multiple mas pequeiio (accesorio). Las observaciones efectuadas por el autor indican que el primero existe en el nucleo desde los primeros estados del crecimiento, pudiendo emigrar algunas veces, fuera de 61 demostrando esto su origen endog^nico. Los nucleolos accesorios se derivan, con toda probabilidad, de los nucleos vitelinos que se encuontran primero en el area citophlsinica y mas tarde penetran dentro del nucleo. Estos hechos demuestran que las teorias endog^nica y exog^nica de las estructuras nucleolares, no son necesariamente contradictorias. Si consideramos las estructuras nucleolares como representaci6n de substancias que pasan a trav^s del nucleo durante el metabolismo, los nucleolos endo'g^nicos deben considerarse como pertenecientes al lado catab6lico, los exog^nicos al lado anab6lico del proceso.

Traiulstion by Dr Joai Noaidei, Columbia t'niversity.

AUTHOR fl AOflTRACT or TIIIH PAPKR IMt'EO DT THB BIBUOaRAPIlIC BBKVICE. NOVCUBEIt IH

SOME OBSEU\ A IK >XS OX THE (IROWIXG OOCYTES OF THE STONEFLY, PERLA EMMARCINATA SAY, WEfH SPECTAL RECARD TO THE ORR'.IX AND FrXCTIOX OF THE XUCLEOL.VR STRUCTURES'

WARO NAKAHARA Biological Laboratories, Cornell University, Ithaca, N. Y.

NINE FKJCKES

CONTENTS

Introduction. . . : 203

Technique 204

Observations 204

General consideration 207

Review and discussions .* 210

Summary and conclusions 214

Literature cited 214

INTRODUCTION

There are two conflictinp; views regarding the origin of the nueleolus. According to Korschelt ('89) and Montgomery ("98), the nucleolus represents a sort of nutritive substance derived from the cytoplasm, and therefore it is extranuclear in origin; while, on the contrary, a niunber of recent authors, including Obst ('99), Walker and Tozer ('09), et al., maintain that it is of intranuclear origin. The general contention today that the nucleohir substance is a passive product of the nuclear activity, and hence intranuclear in origin, seems premature, in view of the fact that data cited as su})porting the exogenic theory of the nucleolus have not been adequately studied.

In the course of my study of the ovarian eggs of the connnon stonefly, Perla iimnargiuata Say, I found some facts which are

' In the preparation of this paper, the author is under deep obligation to Dr. Wm. A. Riley, now of the University of Minnesota, for the critical examination of the manuscripts.

203

Till ANATOMICAL RKCORD. VOL. IS, NO. S DICUIBER, ISIS

204 WARO NAKAHARA

(lirrctly related to the ciuostion at issue, and arc of a little value, if my interpretation is correct, in showing the possibility of bringing the twd conflict inp views in hannony.

The following note sunnnarizes the results so far obtainctl in the study, which has been carried on in the biological laboratories of Cornell I'niversity. until it was discontinued through my leaving the institution. Although there are many details remaining to be worked i>ut, pt)ints brought out in the following lines seem clear.

TECHNIQUE

Although several other fixing fluids were employed, the one that pr<iv(Hl to be mo.st satisfactory and hence most used is Flemming's chromo-aceto-osmic. Ovaries, dissected out in the nonnal salt solution, were fixed in this fluid for ' twenty-four hours.

For staining, the best result was obtained by a modification of Flenuiiing's "triple" method, which is as follows:

1. Stain in a mixture of equal parts of saturated aqueous and saturated alcoholic solutions of safranin for fi\-e minutes.

2. Rinse in water.

3. Stain in 1 per cent gentian-violet, about ten seconds.

4. Rinse in water.

5. Stain in 2 per cent solution of orange G for two or three minutes.

0. Rapidly dehydrate with absolute alcohol; differentiate with clove oil under the control of microscope; clear with xylene; mount in balsam.

Iron hematoxylin and borax carmine countcrstained with Lichtgrtin were also used for a few slides.

OBSERVATIONS

Figure 1 represents a group of early oocytes and some follicular cells. In the nucleus of the oocyte, the chromatin granules, which stain deeply with gentian-violet, are seen arranged more or less linearly. The nucleolus is very prominent, always being situated near the center of the nucleus. Before the growth

OOCYTE OF PERLA

205

Fig 1. A portion of 'egg tube,' showing early oocytes with prominent nucleolus. X 670.

Fig. 2. \ portion of 'egg tube,' showing early oocytes and a growing oocyte. T«'o early, oocytes are seen in the process of degeneration. X 670.

Figs. 3 to 6. Early ovarian eggs. In figures 4 and 5 follicular cells are not shown. The yolk-nurloi are represented by black masseii around the nucleus. The single nucleolus Lt seen near the nucleus, except in figure 5, which does not .show this structure. In figure -I a peripheral nucleolus is seen half way through the nuclear nienibrane. X 670.

206 WAKO NAKAIIAKA

of llu' <»<ipytos conuiionocs, sonio of them always seem to be connnittcd to chuciuTatioii. In liKun' 2 two cells are shown to he in this process. The nuclear structure of the normal oocyte at this jieriod is very much the same as l)efore.

In its \t>ry early state (fips. 2 to G) the ovarian egg is more or less irregularly oval. I'ollicular cells surrounding the eggs are few in niunber. 'rh(> nucleus of the egg cell is usually oval or spherical, containing chromatin granules, which stain less intensely than in the oocyte. XucleoU are of two sorts, a large principal nucleolus and a nmnber of smaller nucleoli. The larger nucleolus, which seems doubtlessly derived from the original oocytic nucleolus, is ahnost always single and is situated near the center of the nucleus. Its substance is usually homogeneous, with dearly defined outline, taking acid color (orange G) when treated with Flennning's triple stain, and seldom containing a vacuole.

The smaller nucleoli varj' in nmnber; a section usually showing two or three, but sometines more — up to six or seven. They VSLTV also in size, although none of them were found to be even half as large as the larger nucleolus. In shape, they are in most cases .spherical, or more rarely much elongated. They are very characteristic in being situated ju.st beneath the nuclear membrane, or at least not far from the latter. The substance of the smaller nucleoU distinguishes it.self from that of the larger nucleolus in choosing safranin out of Flemming's triple combination.

The mo.st characteristic structure in the ovum of this stage is the irregular masses of dense material in the cytoplasm, always closely appo.s<'d to the nucleus. AMiether the masses in question, which are apparently identical with a certain kind of yolk-nuclei, lie within or without the nucleus, is in many eases f|uite difficult to detennine. For instance, in two cells repn^ ftented in figure 3, it has been unpossible for me to detect the nuclear membrane between the areas of the nucleus and yolknucleus. It is still more interesting that the substance of the yolk-nuclei agre*- with that of the smaller nucleoli in staining reaction. These facts, taken together with the peripheral posi

OOCYTE OF PEHLA 207

tion of llic miclcoli, are suggestive enough of some very close relation between these two kinds of structures.

Ova of the next stage in the growth are represented in figures 7 and S. H<'re the increuse in size is most marked in the nucleus, although the follicular epithelium is also attaining much development. The larger nucleolus is somewhat larger than in the earlier stage, but in other respects, it does not show any ai> preciable difference according to the stages. It may, however, sometimes migrate out ()f the nucleus in the well-known manner for the phenomenon (fig. 7). The smaller nucleoli are seen to be much increased in number, usually about twice as many as in the last stage, and not infrequently more than fifteen, being present in a single section. There is no 'yolk-nucleus' situated close to the nucleus. Sometuiies, as represented in figure 8, less deeply stained and ratlier indistinct masses inay be seen scattered near the periphery of the cell, but this is not a constant feature of the ovum at this stage. The nature of these masses is unknoAvn, but they may be, in some way, connected with the yolk-nuclei. A few yolk-gi-anules are seen to appear in the cytoplasm (fig. 7).

This stage of growth is followed by another, at which the volume is increased in the cell-body, rather than in the nucleus. At this stage, the follicular epithelimn is attaining its full growth (fig. 9). The nucleus remains in nearly the same size as in the preceding stage and the larger nucleolus shows no special change. Most of the small nucleoli are now moved from the peripherj' toward the center of the nucleus, and they are somewhat less in numi)er than in the last stage. The cell-body grows umnensely. Indeed, the growth of the cell at this stage may be ahiiost entirely attributed to that of the cell-body. The c>-toplasm is rather homogeneous, and no structure resembhng the yolkmicl(>us is foimd. The yolk-gi-anules are present in a fairly larg(> number at this stage.

C.ENEU.XL CONSlDIiR.VTIOX

From the facts described in tlie last section, it is evident that the growth of the ovarian egg oi Perla is eflfected first by a rather marked growtii of the nucleus, and th(>n by an immense increase of cyto]iiasni.

Fig. 7. An ovarian crr in the stage of nucU-iir growth; follicular cells arc not drawn. Note the increase of the .smaller nucleoli. The larger nucleolus is here seen passing out into the cytoplasm. X 070.

Fig. 8. The same stage. Indistinct dark masses are seen scattered in the cytoplasm. X 670.

Fig. 0. Ovarian eggs in the next stage. The cell-body is greatly increased in volume. Note the less numerous smaller nucleoli. Deposition of yolk-granules in the cytoplasm is also illustrated. X G70.

208

OOCYTE OF PERLA 209

The appearance of the 'yolk-nuclei' always precedes the nuclear {^owth. During the growing period of the nucleus, the yolk-nuc'loi more or less diminish and finally disappear, and the growth of the cytoplasm is rather insignificant.

It has been observTd that the yolk-nucleus and the peripheral nucleoli show very similar staining reactions, and that in some cases the fonner is so closely apposed to the nucleus as to make it impossible to draw a line between the nuclear area and the area of the yolk-nucleus. In fact, many cases were found where the only possible interpretation was? that the peripheral nucleolus and the yolk-nucleus are the same substance, and it ma}' pass through the boundary of the nucleus when the latter is not provided with nuclear membrane. As far as these facts are concerned, it might be considered either, 1) that the nucleoli migrate into the cell-body and give rise to the j'olk-nuclei, or, 2) that the latter are taken up by the nucleus and constitute the former there. However, since the yolk-nuclei appear preparatory to the growth of the nucleus, the first theory does not seem to be acceptable; therefore, the substance of the yolk-nuclei is to be considered as, at least in part, a provision for the growth of the nucleus, to be taken up by the latter to constitute the peripheral nucleoli.

In this connection, a few words might be said regarding interpretation of the 'yolk-nuclei.' The data accumulated by Hubbard ('04), CalkinsCoS), Henneguy ('96), Foot ('96). Xem?c ("97), Van Bmnbeke ('9S), Munson ('98) and Crampton ("99) from various animals indicate, in a general way, that the j'olknucleus and a certain nuclear substance are ver>' closely allied to each other, if not exactlv identical, and that later the volknucl(>us breaks up into smaller and smaller granules, which scatter through the cell-body. Wilson ('00), in his excellent r('\i('W, expresses his opinioTi that the yolk-nucleus may be a product of the nuclejir activity, being directh' or indirocth' derived from the nucleus, and it many contribute to fonned elements of the cytoplasm.

In his extensive comparative study on the subject, Munson ('12) speaks of the type of yolk-nuclei under tli.scussion as

210 WAUO NAKAIIAHA

'niotaplasni.' According to him, thr metaplftsm is a product of fennent action of karj'ohnnph, which comes out of the nucleus, ujion unassiiiiilatcd, ingested food in the cytoplasm, and it is gracUialiy absorbed as food i)y the sphere.

To the case of Perla egg, no one of these interpretations is applicable, because here, iix all ])r()])abiUty, the yolk-iuu'l<>i are, at least in part, provisions for nuclear growth. It may be that the yolk-nuclei in this case are derived from the degenerating cells, wliich are rather abundant at the time when the growth of tlie oviun starts, and they may be entirely different in nature from the yolk-nuclei of other fonns studied. However, the whole subject obviously needs a careful re-examination. Regarding the larger nucleolus, nothing ven,-^ definite can be said as to its origin. Indications as a whole, however, seem to favor the view of its uitranuclear origin. It is very probable that the nucleolus was formed in the nucleus after the last oogonial division, and lianded down through the stages of growth of the oN'arian egg.

REVIEW .\ND DLSCUSSIOXS

Korschelt' ('89), working on the ova of Epeira, Dolomedes, Plialangium, Spinther, and Ciona, came to the conclusion that the nucleolar .substance is in close connection with the nutritive process of the cell, and probably it is derived from the cytoplasm.

This theory is strongly maintained by Montgomery ('98), on the basis of his observations:

In all the Ciuses oliscrvod by nic, the nucleus appears to assimilate a substance or substances from the cytoijlasm, and after this substance has entered the nucleus it apparently underp;oes there a chemical change, and becomes deposited on the inner surface of the nuclear membrane in the form of ma.sses of varj-ing dimensions, which may be eilhcr Klobiilar f)r irregular in shape, according as they are fluid or viscid in consistency. In the case of the ova of the nemcrteans. the substance taken up into tlie inicleus, and which there becomes deposited in till' form of nucleoli, is sometimes exactly similar to the substance of the yolk-bails, whifii lie in the cytoplasm; in other cases, it is probably similar to tho.se metabolically changed portions of or inclusions of the cjioplasm. out of which the yoik-balls arc later differentiated. In Linens, indeed, the yolk-balls may often be found halfway through the nuclear membrane, and their appearance is

• OOCYTE OF PERLA 211

exactly siniihir to that of the; nucleoli. In the mescnchyin cells of Ccregrattiliis, the substance of the nucleoli appears to be identical with that of the numerous nutritive granules which arc dispersed in the cytoplasm; the latter globules arise in the cjloplasm before the nucleolus appears in the nucleus, and as soon as they become numerous in the nei(;lil)orii()(td of the nucleus, peripheral nucleoli Ix'nin to appear in the latter. In the subcutical gland cells of PLscicola. the nucleolus, at the time of its most rajiid growth, is ajjposed to the nuclear membrane; but wiieii this period of volume-increase has cf;a.sed. it is never founil in this position. Furthermore, the paranudeoli of Rodalia appear first in contact with the nuclear membrane.

The greater part of the above statements are in accordance with my observation on Porla egg, and in my mind there is little d()ul)t as to the appropriatcne.s.s of Montgomerj-'s interpretation. Regarding the case of subcutical gland cells of Piscicola only, I am rather inclined to disagree with him, because of the fact tliat the nucleoli migrate into cytoplasm in great abundance at the time of the fonnation of secretion granules — a fact which apparently speaks against the theory of extranuclear origin of the imclcoli. ^lontgomery also cites Schwalbe's ('76) ob.servations that in some vertebrate embjTos, the nucleoli first arise as thickenings of the inner surface of the nuclea" membrane. However, any Uterature published at as early a dat<' as 1S7G on 'nucleolus' can hardly be expected to be of much value on account of the poor technique employetl and indefinite use of the term 'nucleolus,' so such early observations as these are perhaps to be intentionally excluded from our consideration.

Obst ('99) considered that the nucleoli are intranuclear in origin and, more especially, they are derived from the chronuitin indirectly by some chemical change. He observed in Unio, K\M ira, and other fonns that in resting nuclei the basophile chromatin granules become acidophile, and then they fuse together to fonn nucleoli.

Page anil Walker ('OS), in the nerve cells of several mammals, ^^ alker and iMubleton ('08), in the cells of Hydra, described the nudtiplieatiim of the nucleoli by regiUar budding. The imcleoli then pass out of the nucKnis and evei\tually become ab.sorbed. Walker and Tozer ('00) deseribeil similar observations in the

212 WARO NAKAHARA

vegetative cells from SponRilla, Planaria, Clepsine, the rabbit, and the bean plant, and stated that "the regular multiplication of nucleoli within the nucleus, and their suhscciuent history, sei>ms to exclude tlic possibility of the extra-nuclear origin suggested by Montgomery," at least in the case of the cells they considered. A similar conclusion may be reached also in the case of silk-gland cells of insects, since the numerous nucleoli in functional cells are derived from a single nucleolus of ordinary type by budding (Vorhies, '08), and they then pass out into the cell-body to form secretion granules (Maziarski, '11, and Nakahara, '17).

Medes ('04) and Dederer ('07), in the male germ cells of Scutigera and Philosamia, respectivelJ^ observed that the true nucleolus first appears in a mass of chromatin. McCJill ('06), studying the ova of the dragonfly, expresses her opinion that the double nucleoli in that form are formed through the condensation of the basichromatin rouiul the oxyphile nucleolus. Wilson ('05), Boving ("07), Randolph ('08), Stevens ("OS), and Payne ('09), in the male germ cells of various insects, observed that the sexchromosomes are usually closely associated with the nucleolus, when they first appear. The idea of a close genetic connection between the nucleolus and at least of a certain chromosome thus developed has been finally put in a somewhat definite fonn by Cioldsmith ('16), who concludes, from his study on the spermatocytes of P.selliodes, that the true nuclco-sus is formed by the accmimlation of linin about the sexchromosomes."

It is vcr>' unfortunate that many of the recent authors are rather too skeptical toward Montgomery's theorj-. Goldsmith ('16) went even so far as to say that the peripheral position of the nucleoli in the nucleus was the only evidence of the theory set forth by Montgomery, and that since many nucleoli do not come in contact with the nuclear membrane throughout their entire history, the evidence does not hold. The more recent author se(>ms to have forgotten that there are some other facts, CdiTclated with the pcriplicral position of the nucleoli, pointed

OOCYTE OF PERLA 213

out by thf othor author, in makinp; his sufcgostion. If anyone goes over Montgonier} 's work very carcfullj' he would find it impossible to escape from the conclusion that the 'extranuclear thoorA-' is undf)ubt('dly acceptable, at least in certain cases.

I have stated that in the ovarian epg of Perla the nucleoli are of two types, namely, a large single nucleolus and a number of smaller ones. The former increases in size with the general growth of the egg, and sometimes passes out into the cell-body, thus indicating its po.s.sible intranuclear origin. The nucleoli of the second type are absent in the early oocyte, but they are newly introduced in the nucleus at the time when the 'yolknucl«M' make their appearance around the latter, and are in all probability directly derived from such extranuclear substance.

These facts suggest that the nuclear structures designated as nucleoli can be divided into two classes, according to whether it is derived directly from outside of the nucleus, hence probably associated with anabohsm, or is secondarily produced within the nucleus from substances which have already been there, hence in probable connection with katabolism. A hjTJothesis might be put forth that the nucleoli represent substances that are going through the nucleus in metabolism. Nutritive substances taken up into the nucleus may or may not be in the form of distinct bodies, and when they are visible as such in sections, they constitute the nucleoli of extranuclear origin. For the nucleoli of intranuclear origin, the current interpretation that they represent a passi\e product of the nuclear, or more especially chromatin acti\ity, may be a proper explanation. Viewed from this standpoint, the two apparently conflicting views are nothing but partial expressions of a whole truth, and recent authors' critical remarks upon Montgomery's conclusion are just as unrational as the latter author's premature generalization.

Cases where the extranuclear origin of the nucleoli is demonstrated are not ai)undant. Asiile of those early ovarian eggs of certain annuals, Conklin's ('97) figure of a cell from the dorsal wall of isopod intestine, shiowing nutritive substances projecting

214 WAUO NAKAHARA

by iiiaiiy processes into the nucleus, some of which apparently are being set fn^ into the nucleus to fonn nucleoli, is the only puhlished observation I know of this phenomenon.

SUMMARY AND CONCLUSIONS

1. In the ovarian ep;g of Perla immarginatii, two typ(>s of nucle<ili are present, namely, a large single nuoeolus and a number of smaller peripheral nucleoh.

2. The larger nucleolus increases in its dimension with the general growth of the o\'um, and it may sometimes pass out into the cell-body hi^nee, possibh'^ of intranuole.ir origin.

.3. The smaller nucleoli are in all prol)al)ilily fonnetl directly of the portions of the yolk-nucleus migrated into the nucleus — hence, extranucle.ar in origin.

4. Different views concerning the nucleoli, and especially the question of their origin, can be brought into haniiony under the hyphothesis that the nucleoli represent substances going through the mieleus in metabolism.

5. .Vccording to this hypothesis, nucleoli may i)e fonned directly of a material taken up by th(> nucleus or may be produced from some sub.stances within the nucknis in the course of metabolic processes.

LITKKATUKE CITKD

Van Bambeke, C. 1893 Elimination d'616ments nucl6aircs dans I'ooiif ov.irion

du Scorpaena scrofa. .\rch. de Biol.,_T. 13. BoRl.NG, .\. M. 1907 A study of the sperraatogeMcsi.s of twenty-two species of

the Membracidae, Jassidae, Cercopidae and Fulgoridae, with special

reference to the behavior of the odd chromosome. Jour. Exp. Zool.,

vol. 4. Calkins, G. N. 1895 Observations on the j'olk-nucleu.s in ttio egg.s of I.um bricus. Trans. N. Y. .^cad. Sci. CoNKLi.v, E. G. ISO" The relation of nuclei and cytoplasm in the intestinal

ccll.s of land isopods. .\mer. Nat. Cramptox, H. E. 1809 The ovarian history of the egg of Molgula. Jour.

Morph., vol. l.i, suppl. Dederer, p. H. 1907 Spermatogenesis in Philosamia cynthia. Biol. Bull.,

vol. 13. Foot, K. 1896 Yoik-nucleu.-) and polar rings. Jour. Murph.. vol. 12. GoLnsMtTH. W. M. lOlfi Relation of the true nucleolus to the linin network in

the growth period of P.selliodes cinctus. Biol. Bull., vol. 31.

OOCYTE OF PERLA 215

Hen.veocv, L. 1'. 1S96 Lemons sur la cellule. Paris.

HuBDARD, .J. W. 1894 The yolk-nucleus in Cymatogaster. Proc. .\mcr. Phil. Soc, vol. 33.

KoRSCHELT, E. 1889 BeitrJigc zur Morphologic und Physiologic des Zellkcrnes. Zool. Jahrl)., .\bt. .\nat., Bd. 4.

Maziarski, S. 1911 Rechcrchcs cytologiques sur les phdnomjncs 86cr<5toire8 dans les glandes fili^res des larvcs des Ldpidoptdres. Arch. f. Zellforsh., Bd. 6.

McGiLL, C. 1906 The behavior of the nucleoli during oogenesis of the dragonfly, with especial reference to synapsis. Zool. Jahrb., Abth. Anat., xxiii.

Medis, G. 1904 The spermatogenesis of Scutigera forceps. Biol. Bull., vol. 9.

Mo.NTtioMERY, T. H. 1898 Comparative cytological studies, with especial regard to the morphology of the nucleolus. Jour. Morph., vol. 15.

MuNSON, J. P. 1898 The ovarian egg of Limulus, etc. Jour. Morph., vol. 15. 1912. A comparative j-tudy of the structure and origin of the yolk nucleus. Arch. f. Zcllforsh., Bd. 8.

Nakahaha, W. 1917 On the physiology of the nucleoli as seen in the silk-gland cells of certain insects. Jour. Morph., vol. 29.

Nemec, B. 1897 Cber die Structur der Diplopodeneier. Anat. Anz., Bd. 13.

Obst, p. 1899 Untersuchungen iiber das Verhalten der N'ucleolen. Zeit. f. wiss. Zool., Bd. 46.

Page, M. \V., a.nd \VALKi:R, C. E. 1908 Note on the multiplication and migration of the nucleoli in the nerve cells of mammals. Quart. Jour. Exp. Phys., vol. 1.

Pay.se, F. 1909 Some new types of chromosome distribution and their relation to sex. Biol. Bull., vol. 16.

Randolph, H. 1908 The spermatogenesis of the ear-wig, Anbolabia maritima. Biol. Bull., vol. 15.

Stevens, N. M. 1908 A study of the germ cells of certain Diptera. with reference to the hetero-chromosomcs and the phenomena of synap.sLs. Jour. Exp. Zool., vol. 5.

VoRHEis, C. T. 1908 The development of the nuclei of the spinning gland cells of Platyphylax dcsignalu.-s Walker. Biol. Bull'., vol. 15.

Walker, C. E., and Embleton, .\. L. 1908 Observations on the nucleoli in the cells of Hydra fusca. Quart. Jour. Exp. Phys., vol. 1.

Walker, C. E., and Tozer, F. M. 1909 Observations on the history and possible function of the nucleoli in the vegetative cells of various plants and animals. Quart. Jour. Exp. Phys., vol. 2.

WiiaoN, E. B. 1900 The cell in development and inheritance. Revised ed. New York.

1905 Studies on chromosomes. The behavior of the idiochromosome in Hemiptera. Jour. Exp. Zool., vol. 2.

|{csiiini(l(i |)i)r el iiutiir. II. it. Hunt.

\ arialiiiidad de las arterias ear6tidas comunes del gato

donn'stifo.

VA alitor ha oxaniinadd vontiocho s^itos. jiara dotonninar los limitos y freciinicia do las vuriaciones tlel origen dc las arterias earotidas conumes. Kn seis easos (22 por eiento) la arteria innominada se dividia en dos ramas, la subela\ia dereeha y un vaso (jiie se hifiirea en las dos arterias earotidas conmnes. En nueve ejeiiiplares {'.i'2' '( ) la siibelavia dereeha y las dos earotidas comunes tenian un niismo origen en el extremo distal de la arteria innominada. lui doee gatos (43'7) I'l earotida eoim'in iztiuierda se orininaha en varios piintos posteriores al origen de la ear6tida eonnin dereeha. Vn ejemplar maeho |)re.sentaba una eondiei6n an6mala l)ien patente, jiuesto ([ue la earotida e<inii'in iziiuierda partia direetaniente de la aorta, de un modo miiy seniejaiite a la disposieion existente en el homhre. Kl autor propone una explieaeioii de la \arial)ilidad del origen de las ear^tiilas eomunes.

Trnnfilntioii hy Dr. Jo^ Xoiii'lii. Columbia rnivprsity.

Ai-Tiinn a AOflTRACT or this papkr ivued ur

THE IlIDLIOOItAnilC SCIIVICE. NOVCUUEIt \H

\.\lil.\Hll.llV IX IHI'; COMMON CAKO'lll) AKTliK 1 KS

OF iiii': i)OMi;sTrc cat

llAKUl.SUN 1{. IILNT Department of Zoology, West Virginia University

FIVE FIGURES

\'ariati()ns in tho oripin of the common carotid urtcrios of the domestic cat ha\(' long been known. The conditions shown in figures 1, 2, and 3 are the usual variations and have been figured and described in the text-books on the anatomy of the cat. (Mivart, '81: Reighard and Jennings; Davison, '03.)

Twenty-eight cats have been examined to detennine more precisely the limits and frequency of the variations in the origin of the common carotids. These individuals were of both sexes antl being selected at random showed a considerable degree of variation in age and size. In six cases (22 per cent of the total number) the innominate artery split into two branches, the right subclavian and a ves.sel which divided into the two common carotids (fig. 1). Nine individuals (32 per cent) showed the conditions illustrated in figure 2, where the right subchuian and the two carotids have a common origin at the distal end of the innominate artery. In twelve cats (43 per cent) the left common carotid arose from the innominate at various points post<»rior to the origin of the right conunon carotid (fig. 3); in several animals the left carotid came off as far forward as m.

In one male animal a distinctly anomalous condition was foiuid which does not appear to hav(> been pnniovisly reported. The left common carotid of this animal was attached directly to the aorta (fig. 4), thus closely resembling the condition found in man. The above facts seem to show that figiu^s 1 aiid 4 represent th(> extreme limits of variability in the origin of the common carotid arteries.

217

218

IIARKISON R. HUNT

'I'lic cauM* of those vurisitions is pr()l);il)ly some v;iriiit)lr drvelopnu'iitul factor. Possibly the history of the loft coiniiioii (•jirotid in tho cjit oloscly rrs('iiil)los tii:it in the pig, whorr Iho left ciirotid is iit first coniu'ctrd with thr left syst('nu<' urch, but later shifts to the right jvrch (Lohniann, 'O(j). Such ;i migration of the left carotid of the c;it might, conceivably, be accomplished in the embryo by a sj)litting, of variable extent, of the left sys

.r -1

-s

-i

-s

-m

-S

sa

Figures I, 2, 3, and 4 are sketches made from actual dissections. Ii)(urc 5 is a diagram to illustrate the hypothetical cause of the variation in the carotids. I, innominate artery; /, left common carotid artery; r. right common carotid artery; », subclavian artery; sa, systemic arch.

temic arch, beginning at point e, figure 5. The condition shown in figure '.\ would obtain if the si)lit extend<'d to point a in figure o. Figure 2 would be produced bj' a furtlun* .split to point b, while a split either to c or d would result in the conditions •shown in figure 1. This hypothesis retjuires testing, of course, by studies on the embrj'ology of the cat.

Whatever the cause of these variations may b.\ the above observations indicate the frequency and lunits of variability.

CAROTID ARTKRIES OF DOMESTIC CAT 219

LITKKATURE

Davison, A. VM'.i Miiiiiiiiiilian unaloiiiy with speciul reference to the eat.

]'. HliikiHton'8 Son & Co. Lehviann, 11. litOO On the embryonio history of the aortic arches in nianitnals.

Zool. Jahrl).. Ablh. Anat. uiid Ontogenie, Bd. 22, p. 387-434. MivAUT, St. C. 18.S1 The eat. liKKiiiAKL) A.ND jEN.MNtis. Anatomy of the cat. Menry Holt & Co.

Resumido por el autor, H. R. Hunt.

Ausencia de un rin6n en un gato domdstico .

El gato macho estudiado por el autor carecia hasta del mas ligero vestigio de rin6n en el lado derecho del cuerpo, si bien existian en dicho lado la glandula adrenal, el testiculo correspondiente y un ureter muy corto. El autor no ha podido hallar ni arteria ni vena renales. El rin6n izquierdo estaba muy hipertrofiado.

Trvulatlon by Dr Joab Nonidei, Columbia University.

ADTHORS AUftTHACT or THIB PAFKR BSCBD BT THE IIIULIOaKAPUIC KKRVICC, NOVEUOEK 18

ABSENCE OF ONE KIDNEY IN THE DOMESTIC CAT

II. H. HUNT Department of Zoology, West Virginia University

TWO FIOCRES

The absence of one kidney, its ureter, and other parts of the urogenital system has been found in many human individuals by Schaffer, Ballowitz and others, v. d. Broek ('07) has reported a human male subject in which the whole right half of the urogenital system was absent. Lyon ('17) has described similar conditions in a female himian individual, which, however, possessed a ureter on the side of the body lacking the other urogenital organs.

Recently the writer found and dissected a full grown male cat (cat 1) in which not even the slightest vestige of the right kidney was visible to the naked eye in the position nonnal to this kidney (fig. 1). The testes and the adrenal glands were present. The right ureter, 6, was attached to the urinary bladder in the nonnal fashion; it ran oephalad for about 1.5 cm., and ended ahrui)tlj', showing nothing at its anterior end which suggested an undeveloped or degenerate kidney. It is evident that the agencies which prevented the nonnal development of the right uri'tcr and kidney began to operate after the ureteric evagiuation of the Wolffian duct had been fonned in the embrj-o.

Neither the right renal artery nor the corresponding renal vein could be identified. Win / (fig. 1) dramed the region supplied by the adrenolumbar jirtery, and is therefore th(> adrenoUinibar vein, though it does not cross the adrenal gland as nonnally.

As far as could bv deterniiiied by a gross dissection, the internal structure of the left kidui-y was nonnal.

•-'21

222

11. H. lll'NT

Tin- size of <ho kidufy of cat 1 unci of the kidneys of several nomml male cats (cats 2, 3, 4 and 5) was determined by immersing the ki(in(\v in a graduate containing water, and then reading

Fig. 1 o, adrenal gland, b, right ureter, r, riglit phrenic artery, d, small artery to the adrenal gland, e, adrcnolumbar artery. /, adrenolumbar vein. g. postcava. h, right internal spermatic artery, t, left phrenic artery, j, left adrenolumbar vein, k, left adrenolumbar artery. I, left renal vein. m. left renal artery, n, left kidney, o, left .spermatic vein, p, left ureter, r, inferior mesenteric arterv. .i, dorsal aorta.

the increa.se in the volume of the graduate's contents. Cat 1 weighed 2:i')() grams; the volume of its only kidney was 27 cc. The weight of eat 2 was 2280 grams, and the volume of each kidney was 12 cc. The two cats weighed nearly the .same, yet

ABSENCE OF ONE KIDNEY IN DOMESTIC CAT 223

the kidney of cat 1, having; about twice as much work to d(j as either kidnej* of cat 2, was about twice as large as the latter. Figure 2 shows the relative sizes of the kidneys from these two animals.

The body length of cat 1 was from 3 to 3^ cm. less than the body lengths of cats 3, 4 and 5, yet one kidney from each of the last three measured 15, 21 and 9 cc, respectively. These measurements clearly demonstrate the hjTJertrophied condition of the kidnej' in cat 1.

b

Fig. 2 a. the two kidneys from cat 2. 6. the single kidney from cat I.

LITERATURE CITED

V. D. Broek, .\. .1. p. 1907 Ein Fall vollkommener .\genesie des reehten Urogenitalapparates. .\natoinischer .\nzeiger, Bd. 31, S. 417-423.

Lyon, Marcus \V. 1917 .\n hereditary case of congenital absence of one kidney. .\nat. Rcc, vol. 13, pp. 303-304.

Rosuniido por el autor, Adolf H. Schultz.

Observaciones sobre el canalis basilaris chordae.

En los huesos basioccipitales de dos adultos de raza blanca, ha encontrado el autor un canalis basilaris chordae completo, resto do la j)arte craneal de la cuerda dorsal, y el mismo canal, parcialinente obliterado, en el crilneo de un filipino adulto. Tanibi(n ha encontrado la misma estructura en los cnineos de un nino bianco y cuatro ninos negros. En uno de los casos obscrvados en el adulto, la anomalfa citada coincide con un os Incae, y en uno de los ninos con un canal cranio-faringeo.

TransUtloD by Dr. Jo»t Nonidci, Columbia Unlverfllty.

aothor'b abstract or tuu paper dmcsd nr

THE niOLIOOHArnir SERVICE, NOVCMIiEH IS

OBSERVATIONS ON THE CANALIS BASILARIS CHORDAE

ADOLF H. SCHULTZ Carnegie Inslitulion of Washington

THREE FIGCRBS

Out of twenty-two adult himian skulls' examined by the wTiter, a coinploto oanalis hasihiris chordae seu medianus, which perforates the basioccipital bone in a sagittal direction, was found in two whites, and the same canal, partially closed, in one Filipino. In a material consisting of thirty-eight skulls (twenty-six negro and t wehe white) of fetuses and infants, ranging in age from the eighth month of prenatal to the second month of postnatal life,= the canalis basilaris, represented only by its posterior part, was found in four negroes and one white.

The canal was first described by CJruber in 1S80, since which time a luunber of new cases of more or less complete canaUs basilaris have been reported by Romiti ('81), Fusari ('89), Staderini ("00), Para^•icini ('03), Le Double ('03), and Perna ('OG). The last-mentioned writer states that it occurs in 2.47 per cent of adults and 4.22 per cent of children. The greater frec|uency in children would indicate that the canal may become obliterated during the process of gro^N-th, but its remaining patent is not necessarily influencetl by age, inasmucli as the author found it complete in a woman about 75 years" of age.

The canalis basilaris has been explained as a trace of the cranial part of the chorda dorsiilis, which nonnalh' disjippears after or during the third month of intra-uterine life. The similarity between the course of the chorda in the cranial ba.se

' Tlii.M mtilcrinl l>clong8 to the .Anntomical Department of the Johns Hopkins Medical .ScIhmiI. I wi.sh to thank Dr. W. II. Lewis for his kind permi.ssion to utilize the same.

' Hi-lonniiin 111 the Cariiepie I.jiboratory of Kmbryologj'.

225

of fotusos suul tliiit of the (muhI in jidults (Tig. 1) is so slrikiun as to make this tlu-ory apjM'ar more than probable.' In ft'tvisos the chorda (iorsaUs, enierKiiig from the dens epistrophei, enters the basioccipital plate on its dorsal side, extends forward and downward, running for some distance beneath the base of the cranium between the latter and the dorsal wall of the pharnyx, after which it again enters the skeletal tissue to extend up toward the dorsum sellae turcicae. A detailed description of these conditions is given by Huber ('12) in his excellent paper on the chorda dorsalis and pharyngeal fossa. The relative distances from the points where the chorda enters and where it leaves the

Cana\ti bos

Chcrda don

Fig. 1 Modiiin sagittal section through the basioccipital and basisphenoid bone of an adult Filipino with incomplete canalis basilaris, and through the corresponding region of the head of a 10-\veeks fetus. (Lower <lra\ving is schematic.)

basilar part of the occipitale to the anterior border of the foramen magnum may vary in different fetuses, just as the location of the terminal points of the canalis basilaris may differ slightly in individual cases, although thej' are found always in the niidsagittal plane.

The canalis basilaris chordae was foinid to be widest and most typical in the skidl of a white man approximately 40 years of age (fig. 2). In this case the posterior opening of tiic canal is 3 nun. in diameter, its narrowest width 0.9 nun., and its length 19 mm. Its anterior end opens into a fossa pharyngea. .Just

' Foramina nutritia may occur at about the same phicc; therefore care must be taken not to diagnose this condition as incomplete canalis basilaris.

CANALTS BASILARIS CHORDAE 227

lu'liiiul tills oiHiiiiifj; is n w cll-dcvdoped tubcrculuni phHiyiiKcimi. Ciinjilrs t'ondyloidci aro absont.

Ill tlic skull of a white woman 75 years of age the canal allows flic [Kiss.-ific of only a bristle ().() mm. thiek ffig. 3). The length of the eunal is 13 mm. No tulu'rculuni or fossa pliar^'UKcul were found. On each side there i.s a wide canalis condyloideus.

The canalis basilaris in the skull of a Filipino approximately 30 years of age is partially obliterated (fig. 1, upper drawing). The small inner opening is (i mm., and the ventral opening 17 mm. fiom the anterior border of the foramen occipitale. The eanal is patent except for a distance of 3 mm. in its middle portion. Se\en millimeters anterior to the ventral opening there is another median foramen, pointing for a short distance in the direction of the pituitary fossa. A shallow furrow combining tli(^ two ventral foramina makes it certain that the most anterior foramen is a continuation of the canalis basilaris. A tulxTculum pharyngeum is present in this case, but only slightly developed; there is also a wide canalis condyloideus on each side. It may be mentioned that this skull shows another anomaly, i.e., a persistent sutura occipitalis transversa, which fonns an os Incae verum, which changes the form and size ()t the occiput and the entire cranium. These chang<>s were demon strated by the author ('15) on fourteen skulls showing the same anomaly. In the three skulls described herein the posterior bonier of the foramen occipitale is thick, slightly elevated and forms a discernable arch — a manifestation of an occipital \-ertebra, which Kollman ('07) sees also in the canalis basilaris.

As mentioned above, the posterior end of the canalis basilaris was found in five skulls of infants and fetuses. One of these is especially interesting, because combined with it was the likewise very rare persistence of a canalis cranio-pharj'ugeus. This combination was found also by Perna on the skull of a yt)ung Tuscan. These two abnonnal canals have several features in common. They are both ri>mnants of enil)ryological structures which normally disappear during the first half of intra-uterine life; both occur more freciuently in children than in adults, and both are often found in an incomplete that is, a partially

itblitomtod, statf. As the explanation of atavism was denied by the author ('1(5) in the ease of the eanalis cranio-pharyngeus, he likewise does not eonsider it probahle tliat the eanalis basilaris chordae is a pure atavism. It is niueii more likel.y to occur in con.sequence of the coincidence of abnonnal earlj' or rapid ossification of the basioccipitale and the late disappearance of the chonla dorsalis. However, many more cases of the presence of the eanalis basilaris in man. where it is known to give rise to tumors, and also in other mammals* will have to be reported before definite conclusions can be reached as to wlicthcr it plays a phylogenetic role.

LlTKHATUItE CITKD

P'usAHi, K. 1SS9 Dulleprincipuli viiriptftprc-ciilalc ilclhi n.s.-iii dol irdiiciMildUi

Icsia I'.-istcnIi iicl Miisco Aniitoniico tlolla 1{. I'liiv. tli Messina. .Sicilia

Mcdica. .\niio'l, Kasc. 4.

1S91 Dollc priiicipali variciti od aiiomalic pro.scntato dalle osHii dclla

testa e del troncocsistciiti iiel Musco .■\natomieodella Univ. di Ferrara.

Meinor. dell' Accad. Mcdieo-Chirurg. di Ferrara. (iiniiKK, W. 1S80 ( ber den nnomalcn Canalis basilaris medianus dcs Os

"coipilale beini Monsehen. Mc^moircs dc I'Aeademic Ini]). des Soienccs.

St. Potcrsburg, Ser. 7, T. 27. IIl'BER, C. Ci. 1912 On the relation of the chorda dorsalis to the anlage of the

pharyngeal bursa or median pharyngeal recess. .\nat. Rec, vol. fi.

p. 373. KoLLMAN.v, J. 1907 Variantcn am Osoccipitale, besondersinder Umgegcnd dcs

Foramen occipitale magnum. Anat. .'Vnz., Bd. 30. Le Double, A. F. 1903 Traitd des variations des os du crfinc <k- I'lioinnio

Paris. Pabavici.vi, G. 1903 Fori e canali del basioccipitale nei 296 crani del .M.-mi comio di Milano in Mombello. Rend. 1st it. Lumbardo di >Sc. e. Lett..

T. 36, Ser. 2. Peii.na, Ci. 1006. Sul canalc basilare mediano e sul significato della fossctta

faringea dell 'osso occipitale. Anat. Anz., Bd. 28. UoMlTl, C!. 18.H1 Canale basilare mediano dell' o.sso occipitale. \'erbali della

.Soc. Tosc. di .*<cienze natural!. Pisa. •ScHULTZ, A. H. 191.5 Kinfluss dcr Sutura occipitalis transversa auf Cirosse und

Form des Occipitale und des ganzen Gehirnschadcls. .\rchives suisses

d'Anthrop. g(n(?rale. T. l.No. 3.

1910. Ocr Canalis cranio-pharyngcus pcrsistens beim Mensch und bei

den .\fTcn. Murphol. .lalirbiich, B<l. .V), No. 2. Staderini. R. 1900 II canale basilare mediano e il suo significato morfologico.

Monil. Zool Ital.. .Anno II. No. 4.

' The author found the canal in a few- skulls of guinea-pigs; Fusari COD found it in the skulls of a colt and a calf.

CANALIS BASILARIS CHORDAE

229

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Rrsimii(li) p(ir Ins aiitoros, Eloanor T.. y Kliol H. (lark.

Solni' las ivaocinnos dc cicrtas (•('hilas do la i-ola del rcnacuajo l)ajn la accioii df Ins coloraiitcs vitalcs.

V.n el traiiscurso tie las ohservaciones sobro ol crooimiento y capafitlad dc ipacci6n de las (•('lulas y tpjidos do la porciOn traiispaiviito dc la cola del rcnacuajo, los aiitorcs ban crcidn couvcnicntc estudiar los cfcclos dc la coloraciou vital sobre cstas cc'lulas. Los renacuajos fueron colocados en soluciones de tUvorsos oolorantcs y despu6s de tenitlos fueron observados en iin micro-acuario, anestesiados con doretona. Han enipleado oasi exclusivaniente tres polorantes, d rojo neutro, pardo de Bismarck y el aziil trj'pan, a causa de la semejanza de su acci6n y por tcfiir todos cUos el cndotclio linfatico con cs])ccial claridad. Los nuis ilitusibles de estos colorantcs — rojo neutro y jiardo de Bismarck — tifien las celulas nuis rapidamente que el azul trypan, colorante coloidal, coloreando pec[uenos gninulos rcf!;ulares, prcformados en aparicncia, existentes en las celulas cpitlcrmicas. Ailcnuis, cl rojo neutro tine con brillantez el contenido dc un sistema subepiderniico ricaniente raniificado. ]']1 azul trypan no tine la ei)idcniiis. Los trcs colorantcs tificn un jrninulo no constante, probai)lcnicnte jjivformado, existente en las paretics tie los vasos sangiifneos. Tanto el rojo neutro como el parilo de Bismarck y el azul trypan se depositan en forma de acumulos frrainilarcs tint/'jrcos en las areas perinucleares de los linfaticos, en cicrtas celulas cniif^rantes y Icucocitos y en los apcndices de las celulas mesenquimatossis. Las relaciones fisiol6gicas (juc la reaccion liacia estos colorantcs vitalcs jjone de nianificsto en estos difcrentes tijios de celulas, se debe probablcnicnte a la j)ropietlad fagocitica comun a todas ellas.

Tianabtlon by Dr Jos* Nonidei, Columl.'lB I'niveralty.

Atrrnoiw' AiiHTn\rT nr thw rAPmi imrsD bt

TIIR IIIIIM'ifllKPflir HRIt%'l('lt, NOVKUnril IH

OX THH UI;A( TIOX OF ("KHTAIX CKLLS IX THE TADPOLE'S TAIL TOWAliO VITAL DYi:S'

ELEANOR LINTON CLARK AND LLIOT R. CLARK From the A nalumical Laboratory of the University of Missouri

TWO FIGURES

Li llio course of studies on the growth and reactive powers of Hviiig iyiuphatic anil Ijlood-vessel endothehum, mesenchyme cells, and wandering cells in the tadpole's (ail, it seemed advisable to try the effect of various vital dyes on these cells and tissues. The following experiments were started with the twofokl object of determining which vital dyes can best serve as aids in the microscopic study of these cells and of throwing more light on the nature of these cells through a knowledge of their response to the various vital dyes. Needless to say, a region such as the transparent tail of Amphibian larvae, where all the cells can be observed in detail, hi the living animal, is an advantageous place to study the effects of vital staining.

Ehrlich ('94) used the tadi)ole in making his first test of neutral red as a vital stain. He mentions briefly that tadpoles are stained very intenselj- after remaining for a tlay in a 1 to 10,000 to a 1 to 100,000 solution of the dye.

Arnold ('00) and Fischel ('01) stained Amphibia with neutral red and methylen blue, but neither of them gave a description of the effect of vital staining on the cells of the transparent tails of the larvae.

One of the authors (E. R. Clark, '00) used neutral red in liis studies on the growth of living lymphatic capillaries in the tadpole's tail, and noted that the granular areas surrounding the nuclei took up the stain.

A\'isl()cki ('\{\. '17), in his valual)le studies on (lie aclion n|' the

' .Vccoplod for |)ul>lic:itioii Juimniy, lillS.

231

Till ANA10MIC.M. KCrOKD, VOL. 15, NO. 5

232 ELEANOR UNTON CLARK AND ELIOT II. rr.ARK

acid azo dye, trypan hluo, on Aiiiphil)ian larvao and on toleosts, nuikcs the interesting observation that trypan l)Iue stains the perinueiear areas of the lymphatic eiulothehinn hrilliantly and specifically.

The points of similarity between Wislocki's description of the staininp of lymphatic endothelimn with trypan blue and the observations of tlieir appearance after staining with neutral red, made it seem worth while to compare the results of using these two stains and to study the efTect of various other vital dyes on the cells of the tadpole's tail.

The tatlpoles used for these experiments were chiefly larvae of Rana pipiens. The method of chloretone anaesthesia and observation in the upriglit chaml^er, devised and previously describeil by one of the authors (E. H. Clark, (09, '12) were employed. The vital stains used were neutral red, Bismarck brown, trj-jmn blue, gentian violet, and methylen blue.

NEUTRAL lUCD

Neutral red, a basic dye soluble in lipoids ami readily diffusible, was first used by Ehrlich ('94) and has been used by many investigators, perhaps most extensively by Fischel ('01). He found that salamander larvae, stained for several hours in neutral red and then transferred to fresh water, retained their red color for as long as eleven months. Fischel found that this dye continues to be absorbed, and that animals left for a long time in the dye solution become so densely stained as to be almost black.

In the present experiments, tadpoles were placed in neutral red solutions of dilutions varying from 1 to .5000 to 1 to 200,000. The ciunulativo effect of the dye can best be appreciated when it is stated that the larvae left overnight in a 1 to 200,000 solution were more intensely stained than those left for an hour in a stain of 1 to .5000. The best results for the .study of the cells in the subcutaneous tissue of the tail were obtained when the tadpoles were allowed to remain for one or two hours in a 1 to 10,000 solution.

VITAL STAINING IN THE TADPOLE's TAIL 233

The propross of the stain can best be watched by placing a tadpole in a 1 to 1(),()()() soUition of neutral red for five to twenty minutes and then removing it to'a chloretone solution (in the observation chamber), for the stain continues to penetrate the tail after the animal has been removed from the dye. The stain appears first as small granules in the cells of the epidermis, as descril)ed by .\rnold ('00) and P'ischel ('01), and as large round bodies in an irregular branching system immediately beneath tlie epiilennis. After two hours of observation, with occasional additions of minute quantities of the stain, the epidermis and this subepidennal system become brilliantly red while the subcutaneous structures are still unstained. Next, the perinuclear areas of the lymphatic vessels and certain large wandering cells take up the stain in the form of numerous granules. Two hours later, a few isolated red granules are visible in the endothelial wall of some of the blooil capillaries. A day or two later, red granules make their appearance on the processes of the connective-tissue cells.

After the stained larvae have been removed to fresh water, the red stain in the subepidennal system gradually changes to orange and, at the end of a week or two, fades to a pale yellow. However, the neutral red remains in the other stained cells and tissues for more than a month — as long as the tadpoles were kept under observation — with no diminution in its intensity.

The cells of the epidermis, after staining with neutral red, contain red granules of unifonn size, regular contours, and of fairly even distribution. These stained granules have been figured by Arnold ('00) and by Fischel ('01). No stain was ever found in the nuclei.

The peculiar system in the sutjepidennal region, which shows np so strikingly with neutral red, was observed in li)().S-()*) by one of the authors, ami. curiously enough, appears not to have been described by any of the nuin<>r<)us investigators who have made use of this dye in the study of Amphil)ian material.

This subepidennal system is made up of a series of large, centrally placed, irregularly shaped nuclei, from which radiate hollow-liranched iirotopiasniic |)rocesses f)f a delicacy so extreme

234 KLEANOR LINTON CLARK AND ELIOT R. CLARK

that tlu\v can be followed only with the greatest difficuily. These hollow processes are tlividod into small segments by delicate partitions, and contain Huiil, as was shown by the fact that now and then a ])i>nnent granule was observed in one of the compartments, in active Brownian movement. Such granules were not seen to migrate from one. compartment to another. These structures lie hmuetliately under the epidermis, and are found generally over the body and tail. In the tail, they are rather sparsely distril)uted near the margins of the fin and the tip of the tail, increasing in immber toward and over the central muscular portion. They have been observed in a immber of species of toad and frog larvae, the pattern, differing in different species. The system shows most strikingly in young Hyla i)ickeringii larvae. Its history in later stages and in frogs has not j-et been followed.

\\'hen first treated with neutral red, the fluid contents of the branched processes are deeply and uniformly stained. Soon, however, the unifonnity is lost, for some compartments lose their stain entirely, while in others the stain becomes more dense. .\fter a day or two, the jjrocesses have the appearance of irregular strings of pink beads, since the walls of intervening, unstained compartments collapse and are seen only with great difficulty. A casual observation at this stage shows what might be described as a 'freckleil' appearance, since the compartments form spheres of (lifTerent sizes, apparently isolated. The stain gradually fades out from this system and, after a few days, has disappeared entirely. It was not stained by any of the other dyes used.

The lymphatic endothelium is brilliantly stained with neutral red and the dj-e is lodged in the area around the nucleus. One of the authors, (E. R. Clark, '09, '12) has given a detailed description of the appearance and behavior of these granular or nuclear areas in living lym])liatic capillaries. In the unslained tadpoles he observed that the nuclear and perinuclear areas merge imperceptibly into one another, and he showed the relationship of the two by first drawing the lymphatic in the living and then drawing the same vessel after fixation and staining. The stain with neutral red differentiates the two i)ortions of the gramilar

VITAL STAINING IN THE TADPOLE's TAIL 235

areas — the nnHcar and porinufloar — in tho living tadpole, since the nucleus, which fails to stain, then shows np as a dear lensshaped region surrounded by a reddish area containing many deeply stained granules. The stain is confined to this area around the nucleus, the rest of the cytoplasm remaining clear (fig. 1, .4). The stained granules vary in size and shape, some of them an' large and refractile, others small and dark. Black and brown granules can be seen between the red ones. If a stronger stain is used, the red granules are found to be more numerous as well as larger and darker in color, and they frequently occur in irregular clumps and masses.

Not all of the wandering cells are stained with neutral red. Here and there in the tail, large round cells, containing brilliantly stained red granules, can be seen (fig. 1, C). These cells often contain black pigment as well. Small wandering cells, as a rule, contain no stain.

A day or two after removal to fresh water, in tadpoles stained for one or two hours in a 1 to 10,000 solution, the mesenchjTne cells begin to show traces of stain. The dye is present as small granules occurring singly on the cell processes. Occasionally a red halo can be found around the black pigment spot present in many of these cells. With a more intense stain the red granules in the mesenchyme cells make their appearance much sooner, and they are then more numerous as well as darker in color and may be seen in the cell bodies clustered about the base of the processes as well as along the processes themselves (fig. l,B).

In tadpoles stained with neutral red, a few red granules may occasionally be found in the endothelial cells of the blood vessels. Such graimles are small and round and occur close to the nuclei, only one or two to each nucleus. The difference in the appearance of the practically unstained blood capillaries and the lymphatics with their bright red, coarsely granular patches is very striking.

No stain was found in the nerves of the tadpole's tail nor in any of the cells inside the blood vessels or lymphatics. \\'ith the exception of the branching system beneath th(> skin, which

'2'M\ ELEANOR LINTON CLARK AND ELIOT R. CLARK

appears to be in a class by itself, the living cells of the tadpole's tail can be divided into three classes with respect to their manner of staining with noutral rod:

1. ("ells which show no granules stained with neutral red. Those are nerve cells, blood cells inside the vessels, and some of the wanderinp; colls.

2. Cells which contain small red granules, regular in shape and of fairly even distribution, after staining with neutral red. These are the epidennal cells and the endothelial cells of the blood vessels. An increase in the strength of the stain has no effect on the size, shape, or number of the stained granules in this type of cell.

3. Cells which show large accumulations of red granules after staining with neutral red. These are the endothelial cells of the Ijinphatics, certain large wandering cells, and the stellate connective-tissue cells.

^^'ith these three tj-pes of cells, the neutral rod granules increase in number and size with an increase in the strength of the stain or merely with the passage of time after the larva has been transferred to fresh water.

BISMARCK BROWN

Bismarck browii is a well-known basic azo stain, soluble in lipoids, which has frequently been used as a vital dye. In the present experiments, Bismarck brown was found to be verj^ similar to neutral red in its action, although it stained somewhat

Fig. 1 Sketches from the tail of a living frog larva (RaiKi pipiciis) which had been stained with neutral red, I to 10,000, for two hours, and then tran.sferrcd to fre.sh water. The sketches were made three days later. A, Lymphatic capillary. The perinuclear areas contained red in the form of granules, fl, Mesenchyme cells with red granules in certain of the processes. C, Wandering cell containing red granules and red 'bodies,' of various sizes. Enlargement = approximately X 740.

Fig. 2 Sketches from the tail of a living larva, stained with trypan blue, 1 to 1600, for four days. Sketch made a few minutes after transferring to chloretone solution. A, Lymphatic capillary with blue granular slain in the perinuclear areas. B, Mesenchyme cells with blue granules on many of the processes. C, Wandering cells containing blue granules of various sizes. Knlargemcnt = X 710.

2.SS KLEANOR LINTON CLAHK AM) KLIOT H. cr-ARK

loss intensely tliaii a neutral red solution of the same strenjitli. The clearest |>ietnres were ohtaiin-il in plaeiiifi a tadpole in a 1 to Kt.HOO solution of the ilye. leavir.^ it there for three or four hours anil then removiiif^ it to fresh water. As in the case of r.eutral red, tadjioles stained overni^iht in a 1 to "JOO.dOO solution were much more deeply stained.

Like neutral red, this slain penetrates the skin rai)idly and, after a few mir.utes, is visible in the epidennal cells in the fonn of round firjinules, usually one to each ceil. In the refjion heI'eath the epidermis, there is i;o trace of the system which shows up after neutral retl, but here the stain is deposited as numerous rods and needles. Fischel ('01) has figured these structures, both inside of cells and between them, and does not he.sitate to call them crystals. One-half to one hour later, cells in the anterior of the tail fin l)egin to take up the stain. The ])erinuclcar areas of the lymphatics and some of tlie hniji' wandering cells first show the dye in large amounts, and a few brown granules may be detecteil in the walls of some of the blood ves.sels. A day after a tadpole, stained in this way, has been trarsft rred to fresh water, brown gi-anules can be observed on the processes of the mesenchyme cells. The crystals in the ei)iilennis fade and disappear after a day in fresh water. On the other hand, the stained granules in the epidennal cells remain ad those in the lymjjhatic endothelium, ui the waTulering cells, and in the connective-tissue cells become more numerous and promnient.

.\s in the case of neutral red, lymphatics stauied with liismarck brown show the dye only, in the area around the nucleus. Tiie stain is present as brown or black granules of diiT<'rent sizes and the space between the gi'anules is coioretl a yellowish brown. The wall of the lymphatic, in the neighborhood of these stained areas, stands out more distinctly than the rest of the endothelium, as if it had been outlined with pen and ink. ^^"ith a very faint stain, obtained by leaving the tadpoK* for half an hour in the dye solution, this outlining of the lymphatic wall and a very few brown gramdes in the perinuclear areas are the only elTects of the staining. With a 1 to .JOUO concentration of the dye or after staining for several hours in a weaker solution, the granules

VITAL STAINING IN THE TADPOLE's TAIL 230

ill llic ixTiiiiicIcar aroasof tlic lynipliatic Ix'coino larger ami iiioro iiiiiiicrous ai;ci often appear in cIuiiiijs.

The staining of the large waiiderinK cells is similar to that (leserihed for neutral red. Hut, in addition to the small brown P'aiuiles, largo refract ile gloimles with regular outlines which took a scarlet or brown stain, were jiresent in some of the wandering cells.

No stained granules were observed in the nerve cells nor in the blood cells insiih' the vessels. It will be seen from the above description that, aside from the characteristic deposit of crystals ill the subepideniial region and the failure to stain the branching system in this region, liismarck brown stains the same structures as neutral red and in a practically identical manner.

TRVPAX BLUE

Trypan blue is an acid azo dye belonging to the group of bci zidiiie dyes containing trypan red and pyrrhol blue, which have arou.sed so much interest recently because of their action as vital stains. Tryi)an red, first used by I-^hrlich as a cure for trypanisomiasis, was discovered incidentally to be a true vital stain for the tissues of the host. Nicolle and Me.snil ('()()) discovered trypan blue and found it to be e<[ually effective, and BoutTard ('0(5) investigated its staining ])ro])erties. Later, (Joldmann ('()!() made a more extensive study of the effects of staining with jiyrrhol blue and described various cells which stained witli the dye and to which he gave the name 'pyrrhol cells.' Evans and Sduilemann ('14, 'lo) used trypan blue chiefly for their investigations of vital staining. All of these workers used manunalian material for their studies.

Wislocki ('16, '17), in studying the effect of trypan blue on amphibians and fish, found, in addition to cells corresponding to those of mammals in their ability to store this dye — namely, the KuptTer cells of the liver, groups of mononuclear cells in the mestMiterv and omentum, and the cells of the convoluted tubules of the kidney a marked vital staining of the eiiithelium of the gills, which he considered to be an evidence of an excretory action, and a staining of the lining and contents of the alimentary

240 KLEANOU LINTON Cl.AUK AM) KLIOT R. CLARK

ciiiial. wliicli :ii)p»>:ii('(l to rciircsciil flic i)lac(' of al)S()rplioii of tilt' <!>(•. And, ill addition, lie describes the Iynii)hatic system as staining in its entirety and in a hrilliaiit manner witli trypan blue. In the tail of the tadi)olc. where liver, alimentary canal, ki(hu\v, and gills are all absent, he states that trypan blue is a specific vital stain for the lymphatic endothelinm, sinc(> no other cells show a trace of the dye. He states that the dye is present in the fonii of jiramiles in the i)erinuclear areas of tlie lymphatics.

In the ])rcsent exiicrimcnts, tadpoles, some of them newly hatched and others two or three weeks old, were i)laced in solutions of trypan blue in tap water. A dilution of 1 to KidO was the one most frequently employed, this being the average of the various strengths recommentled by Wislocki. In contrast to neutral red and Bismarck brown, this dye is absorbed very slowly, tadjioles showing no trace of the dye after remaining twentyfour hours in the stahi. At the end of the second day, a l)lue stain was visible in the lym])hatic eiidolhelium. and this Ix-caiue more marked with every flay that the tadjiole remained in the stain, until, at the end of a week, the lymi)hatics were vividly blue, as described by Wislocki.

If a tadpole is removed to fresh water after a week in the dye solution, the vital stain is not only retained by the cells which have taken it up, but . as in the case of neutral red, it becomes even more intense. In fact, the most rapid staining with trypan blue was obtained by placing a larva overnight in a 1 to tiUO solution and then transferring it to fresh water. Six or seven hours later, a definite stain in the lymphatics of the tail could be detected.

No staining of the epidermal ceils takes i)lace witli try])aii blue. As already stated, the perinuclear areas of the lymi)liatic take the stain, and the blue color is present in the fonii of granules of different sizes and a pale blue tinge between tlie granules. In ad<lition, black and brown pigment granules are also present (fig. 2, -4). The stained areas resemble closely those jircsent after staining with neutral red and Bismarck brown. In older test this shiiilarity of action, a tadpole which had been stained for a week in trypan blue, was selected and a lymjihatic capillary drawn with the camera lucida. The larva was then placed in a

o

VITAL STAINING LV THE TADPOLE's TAIL 241

solution of iioutral rod, 1 to oOOO, for twenty niinutos, and apain placed in tho observation chaiuber and the same region located and the lymphatic again drawn. The neutral red had replaced the blue stain, and the red areas corresponded exactly in size, shape, and position to those previously dra^\^l.

In addition to the deep stain of the lymphatic endothelium, some of the large wandering cells in the subcutaneous tissue of the tail were observed to contain an abundance of blue granules (fig. 2, C). These cells are similar to those which stain with neutral red and Bismarck brown. The stained wandering cells can be seen two or three days after placing a tadpole in the dye solution, or as soon as the stain becomes visible in the lymphatics.

After four to six days in a solution of this strength, small blue granules make their appearance on the processes of the mesench^^ne cells (fig. 2, B). At first such granules are somewhat pale and hard to identify without the aid of the oil-immersion lens. A few days later, they become darker and they continue to increase in number and in intensitj' of staining, so that, after one or two weeks in the dye solution, these blue granules in the mesenchyme cells are visible with the low power of the microscope and are as conspicuous as those present after staining with neutral red. The distribution of trypan blue granules is the .same as in the case of the other two dyes, namely, on the cell processes and in the cell body near the base of the processes.

A few small but undeniably blue granules were noticed near the nuclei of some of the blood vessels.

No blue stain was observed in nerves nor in the blood cells insitle the vessels. There were always a number of wandering cells present in the tissue spaces which failed to stain.

.Vside from its slower action and its failure to stain any of the cells of the epidennis, trypan blue was found to stain the same structures and in the same manner as neutral red and Bismarck brown.

The total lack of stain in the epidennis pennits one to obtain a clearer picture of the deeper structures anil, for this reason, trypan blue is the most satisfactory of these tliree stains as an

242 ELEANOR LINTON CLARK AND ELIOT R. CLARK

aid in tlic study of livinp; Ijaiiphatics. It has also the great advantage that it can be preserved after fixation and tlie effect of tlio staining studioil in ponnaiient preparations. However, if, as freijuently happens, a rapid stain of the hvuig j>^nphatie is desired, it is useful to know that a similar result can be obtained within a few minutes by placing the tadpole in a solution of neutral red or Bismarck bro^\^^. The present observations show that trypan i)lue is not a specific stain for the lynij)liatics of the tadpole's tail, since some of the wandering cells and all of the mesenchyjne cells were found to possess the power of storing the dye. For this reason it can scarcely be u.scd as a means of distinguishing the origin of different tj'pes of cells.

The relatively slow action of trypan blue, together with the fact that none of the stain is deposited in the epidennis, would lead one, on theoretical grounds, to accept Wislocki's hypothesis that the dye is absorbed through the alimentary canal. If this were true, the dye would necessarily reach the tail through the general circulation, as in the case of injections of dye into the peritoneal cavity. To test this point, the blood hearts were removed in a number of newly hatched tadpoles before the circulation had commenced, and, a day later, the larvae were placed in a 1 to 1(300 solution of the dye. Tliree days later, the htiiphatics and some of the large wandering cells showed the typical collection of blue granules and, a few days afterward, blue graimles appeared in the connective-tissue cells. The stain appeared as soon as in the control specimens, was equally intense, and was pre.sent in the same locations. Therefore, the conclusion seems warranted that tryi)an blue jioiict rates the skin of Amjjhiljian larvae, as do neutral red and Bismarck brown, and that, in all probability, its slower action is due to its lower rate of difTusion.

METACHROMATIC STAINING wnil TUVPAN BLUE

Von Mollendorf ('15) has shown, by dialysis experiments, that tr>'pan blue is composed of two dyes — a red substance which is highly difTusihlo and a blue substance which is less diffusihle

VITAL STAINING IN THK TADPOLE'S TAIL 243

and onliiiurily masks the red eleinent. Sdmlemann Tlo) and Evai s and Schulemann ('15) have described a metachromatic staining with certain vital dyos, among them Congo rubin, in which rod vacuoles were observed to contain blue granules. Schulemann fountl by experimentation that Congo rubin would change to blue either with tlie adilition of acid or with an alteration in the electrolytes, caused by adding certain salts. He concluded that the second explanation, nameh', a change in the physical state of the dye, was the true one for this phenomenon of metachromatic staining.

In the course of the present experiments a number of cases of metachromatic staining with trypan blue were encountered and we were unable to prevent or to produce this type of staining at will. The first example was noted in a tadpole which had remaii cd in a 1 to ItiOO solution of trypan blue for six days. On the third day in the stain, the lymphatic endothelium and certain wardering cells were seen to contain blue granules, and on the following two days the stain became a more brilliant blue. But, three daj-s later, when this tadpole was observed under the micro.scope, the perinuclear areas of the lymphatic and the stained wandering cells were seen to contain pink granules as well as purple and blue ones. During the next few days the red stain became more conspicuous and finally replaced the blue entirely. In this specimen, the stained granules in the mesenchyme cells were red when first noted.

.Vfter this peculiar experience, particular pains were taken with the cleanliness of dishes, and dye solutions were watched with especial care to prevent an^' accidental contamination. However, some days later, another tailpole. which had been placed in an 'old' ilye solution (one made up a week previously) showed red coloration of lymphatics and wandering cells after three days in the stain, or as soon as any stain became visible. As in the former case, the gills, alimentary canal, aid certain injured areas in the tail, where a diffuse stain was present, all remained blue. For some tune we believe that the fact that the dye solution had been allowed to stand so long in this case was the reason for tlie change in color, but a week later two more

244 ELEANOR UXTOX CI.ARK AXD ELIOT R. CLARK

tadpoles, which had been Hviiig in a freshly made up solution, showi'd a violet coloration of thcMr lymphatics, which changed to pink soon after removal of the larvae to fresh water. During all this time, manj' tadpoles had been examined which showed the typical blue coloration of lymjihatics, wapdering cells, and mesenchjnne cells.

VITAL STAINING IN THE TADPOI.e's TAIL 245

In tlio casos in wliich rod staining rfsiiltcd from the uso of (ryimn blue, the picture proseiitcil rcseinljled closely that seen wlien blue staining was obtained, save for the difference in color. Thus, no stain appeared for two or three days; then the Ijniijihatif endothelium and some of the wandering cells showed the red color in the form of granules and, a few days later, n-d granules made their appearance on the processes of the connectivetissue cells (fig. 4). The graiuiles in the l>^nphatic endothelium, however, were often finer than those which were present in the case of the typical blue staining or after the use of neutral red. In the wandering cells, blue and purple granules were often visible in addition to the red ones. In the mesenchyme cells, a red halo around the black pigment spot often showed before anj' stain appeared elsewhere. The red granules on the processes of the mesenchjniie cells were definitely noticeable a day or two earlier than in the case of the blue stain. This was probably due to the greater ease with which red granules can be distinguished from the unstained elements of living cells. As with the blue stain, an occasional stained granule was observed in the walls of some of the blood vessels. The only marked tlifference in the case of the tadpoles showing this metachromatic staining was noticed in some of the superficial pigment cells which were 'seen on several occasions to contain a few red granules. This was never noted in larvae staining normally with trypan blue.

VITAL SIAIMNC AND INJECTED KAT

In a former paper (E. R. and 10. L. Clark, '17), we showed that small globules of fat injected into the subcutaneous tissue of the tadpole's tail were absorbed through the activity of l(>ucocytes and lymphatics. We also mentioned that, in such tadpoles which had been stained with neutral red, the stained leucocytes in the vicinity of the old globules were especially conspicuous.

This experiment of injecting fat into the transparent tail fin and then staining the tadpole intra vitam was repeated. The two stains used were neutral red and trypan blue. Some of the large

240 KI.KANOn LINTON CLAllK AND lOMOT R. C'l.AItK

\vftiul(M-iiig cells wliich ohaiicod lo lie in the n(>inlil)()rh()()(l of tiic olive oil at the timoof iiijoction inowd toward the f!;l<>'"d<' and flattoiicd out on its surface. Numerous leucocytes, ininratiiif? from near-by blood vessels, crowded around the jjeriphery of the fat. All of these colls surrounding the olive oil became pigmented with finely divided fat, and most of them also became loaded with tiie colorinji matter, red or blue as the case mipht be. .\s in the nonnal specimens, the lymjjhatic enilothelium and certain large wandering cells scattered at other points in the tail, took up the stain brilliantly. Witli botli neutral red and trypan blue, certain of the migi-ated leucocytes which had reached the oil globule and which there proceeded to ingest the fat in the form of tine brown pigment granules, never showed the slightest trace of the dye. Also, certain wandering cells, present in the tissue near tlie site of injection, were not attracted towaul the olive oil and showed neither brown jiigmcnt nor stain. These cells, which surroundetl the oil glol)ule and took part in the fat absor|)tion, when stained with gentian violet, a nuclear stain, were all shown to possess a single round nucleus.

.\nitsclikow ('14) has shown that the Kupffer cells of the liver, the reticulo-endothelial cells of the spleen, bone marrow, and iymjili glands, and the macrophages of the connective tissue — those cells which are kno^^^l to possess the i)ower of storing colloidal metals and colloidal vital dyes - also have the jiower of cholesterin fixation after feeding with fat. On the other hand, Fiessinger and Marie ('09) have demonstrated lipase in the lymphocytes. The present observations show that many of the cells whicli take up injected fat also stain brilliantly with vital dyes. In this instance, the colloidal ilye, tryi)an blue, and the 7nore highly diffusible neutral red produce identical results. In addition, certain other leucocytes (possibly lymjihocytes) which are also attracted toward the fat, do not stain with these dyes. .\n<l still other wandering cells show no reaction either toward the fat or the dyes.

The three dyes, neutral red, Hismarck brown, and trypan blue, were used almost exclusively in these observations, because they provetl to be true vital dyes, because of the similarity of their

VITAL STAINING IN THE TADPOLE's TAIL 247

action and because they stained the Ij-mphatic endothelium with especial distinctness. The other stains tried did not prove so satisfactorj' from tliese points of view, and the result of their use will be reported very briefly.

GENTIAN VIOLET

Russell ('14) reports that this anilin dye, which is effectual in killing bacteria in strengths of 1 to 1,000,000, at the same time proved to be a true vital stain for frog tissue, in cultures, staining the whole culture and the nuclei in particular and not preventing further growth of the tissue He found that cultures made from adult frog tissue would grow and show the stain in dilutions of 1 to 1000 up to 1 to 20,000.

Tadpoles placed for an hour in gentian violet — 1 to 10,000 — were violet in color and, on examining such a larva in the observation chamber, all the cells of the tail were found to be beautifully stained, the cytoplasm a pale lavender tinge and the nuclei a deeper violet. In addition to this unifonn lavender color, many small black granules stood out clearly in the cytoplasm. Active Brownian movement of these granules could be observed with the oil-Lnmiersion lens. However, we were unable to obtain a true vital stain for the cells of the tadpole's tail with this dye, for lar^■ae stained in this manner were invariably dead and macerated on the following day. !>taining for one hour in dilutions of 1 to 20,000, in which case the resulting stain was much fainter, also proved fatal. Tadpoles, stained for fifteen minutes in a 1 to 20,000 solution, survived for over twenty-four hours, but showed only the faintest trace of color. Strengths of 1 to 50,000 and 1 to 100,000 for one or two hours were tried, but the tadpoles failed to show any stain and they li\'ed for only a few (lays afterward.

.Although proving toxic in strengths necessary for obtaining a satisfactory stain, gentian violet proved to be of value as a nuclear stain in those cases in which it was possible to sacrifice the tadpole. The use of gentian violet to demonstrate the character of the nuclei of those leucocytes which collect around the globules of injected fat has been mentioned.

TUB ANATOMICAL nBCORD, VOL. 15, NO. A

248 ELEANOR LINTON CLARK AND ELIOT R. CLARK

METIIYLEN BLUE

This dye, which EhrUch ('8G) showed to be a vital stain for the nerves of the frog, has been used by many investigators. In addition to its esjM'cial afrmity for nerves, niothylon bhie has been described (Fischel, 01) as staining granules in many cells, especially the pigment cells in the skin of .Vjnphibia, certain granules in the epidennal cells, and the mucin content of the Ix^ydig cells. Arnold {'99) mentions blue granules in nerve cells, mast cells, and leucocytes after staining .Vnii)hil)ian tissues with this dye. Elu-lich, Arnold, and Fischel all obtained double staining effects with methylen blue and neutral red, certain granules staining with one tlye and others with the other.

Po.ssibly the strengths of this staui tried hi the present experiments were too weak, but no very striking results were obtained by the use of methylen blue as a vital stain for the tails of tadpoles. After one hour in a 1 to 10,UU0 solution of the dye, certain of the large superficial pigment cells of the tail showed a greenish-blue coloration of their granules. This staining effect has been descriljed by Fischel. No staui was noted in any other type of cell with the exception of certain small leucocj-les within the blood vessels, which were stained a bright diffuse blue. Possibly better results could be obtained bj- more extensive investigation.

'°4*t^ DISCUSSION

■ 'q:e. It is obviousi:|j|v difficult to foniudate any one theorj' of vital

staining which Wi jjl adequately explain the action of all vital dyes. Thus, Fischei ^,"s ('01) statement that onlj' the basic dyes are taken up by living ,-. tissue is contradicted by the striking results obtained l)y staining j„ viv„ ^vitli pyrrhol blue, trypan blue, aiid other acid benzidine d> .(.j^^ 'fi,^. lij„,i,l theory of vital staining, brought forward by Ove,j ^q^ ('00), has also been shown to be inadequate as a general expla „fition, in view of the action of this same class of dyes, all of whici jj ^j.^ insoluble in lipoids. And the assertion of many authors that^ jj^pyj-g staining and nuclear staining are always evidence of the; (j^ath of the cell so stained or at

VITAL STAINING IN THK TADPOLE's TAIL 249

least of injury to it, lias bocii sliowii to bo untrue in tho case of Kcnliau violet, which HusscU ('14) has shown to be capable of acting as both a diffuse protoplasmic and a nuclear stain.

At the present time, the two chief theories tlealing with the action of vital dyes arc: 1) Tlie chemical theory, advanced bv Flhrlich ('04, '09), that vital staininp; is evidence of a true chemical union between some part of the dye molecule and an element in the cell, known as the chemo-receptor. This explanation is sujjposcd to apply to all vital tlyes. 2) The physical or phagocyte, theory, of Evans and Schulemann ('14, '15), that the colloidal dyes, such as trj-pan blue and lithium caniiine, are taken into the cells by a process correspondinp; to phagoc^iosis and are housed in the fonn of chemically unchanged dye granules in the cytoplasmic vacuoles of certain types of cells. These authors do not include the more higlily diffusible lipoid-soluble dyes, such as neutral red, Bismarck brown, and methylen blue, in the category of vital dyes which are phagocytized in this manner.

That the lipoid-soluble dyes stain only the prefonned cell granules is the claim of most investigators. The affinity of methylen blue for the Nissl botlies of the nerve cells and of neutral red for the graTmles of mast cells are well-known examples of this kind of vital staining. That different granules are revealed by the use of different vital stains is also evident. The mitochondria, which stain specifically with Janus green, differ from certain larger granules which stain with neutral red, in certain eggs and in tissue cultures (M. R. l^wis, '17). Many authors have demonstrated two sets of graimles in the same cells by a double stain of neutral red and methylen blue. However, it is apparent that even the highly diffusible dyes camiot be cla.ssified rigidly as stains which are dejjosited in the prefonned cell structures only. Thus Arnold ('00) and Fischel ('01), both ardent advocates of the theory that vital dyes stain only prefonned granules, give descri])tions of the brilliant staining of tlie mucin content of the Leydig colls with neutral roil, rischol speaks of certain patches of neutral red in the gills of salamanders which he considers to be unclianged chemically, anil Arnold's description of reutral red granules of various shapes, some of them

250 ELEANOR LINTON CI^RK AND ELIOT R. CLARK

sharp-cornered, which occur in clumps or masses in certain ceils, tallies exactly with the description of the dye concretions which accumulate in the pjTrhol cells. It mipht also be mentioned that the exact nature of these 'prefoniied granules' has not yet been discovered and, in this connection, Plato's ('00) observations are of interest Ho studied the behavior of the cell vacuoles in livinp; vorticella and found that it was the contents of these vacuoles which stained with neutral red. He also found that bacteria, spermatozoa, and red blood corpuscles, inside of leucocytes, wore resolved into minute pjanules which stained with neutral rod and wliich were indistinguishable from preformed granules. Stole ('02), in studying the effects of vital staining on amoebae, found that the contents of the vacuoles were the granules which stained with neutral red.

Thus it is possible that the more highly diffusible dyes may stain cell contents or secretions or may be stored as unchanged deposits of the dye, in addition to staining the preformed cell structures. On the otlior hand, there is evidence that the higher colloidal dj-es may occasionally stain the preformed cell elements. M. R. Lewis ('17) notes that the cell granules which stain with neutral red, and which can be seen in unstained tissue cultures, also stain with pyrrhol blue. Kiyono ('14) mentions that the epithelial cells of the kidney, adrenal, and hypophysis, which are not otherwise phagocytic, take up the benzidine dyes. Evans and Schulemann ('15), while admiting that the cells referred to by Kiyono are stained by trypan blue and Uthium carmine, claim that the type of staining in these cells, which is characterized by round droplets with regular outlines, is totally different from the true phagocytic staining of the macrophages where the dye is present as clumps of dark blue granules.

In the present observations, there appears to he evidence for both varieties of staining reaction on the part of the two highly diffusible dyes emploj'ed and also of the colloidal dye. And there is a conspicuous difference between those cells which show only a few round granules after staining (such as the epidonnal cells with neutral rinl and Bismarck browni and the endothelial cells of the blooil vessels with all three dyes) and those cells which take

VITAL STAINING IN THE TADPOLE'S TAIL 251

up tho (lyos in large quantities. In the first type of cell, the granules do not increase in size or number with an increase in the intensity of the stain, while in the l>Tnphatic endothelium, wandering cells, and inesenchjnno cells tlie granules increase in number and size after an intense or prolonged staining with anj' of the three dyes. Since the work of Evans and Schulemann renders it highly probable that intense granular staining with trypan blue is evidence of a form of phagoc>'tosis on the part of cells disjilaying this reaction, it seems natural to assume that an identical response of the same cells toward neutral red and Bismarck brown may also receive the same explanati<in.

That the Ijnnphatics of the tadpole's tail possess a marked phagocytic power was sho\\'n by one of the authors (E. R. Clark, '09) in observations of the manner in which h-mphatic sprouts grew toward red blood cells, extruded into the ti-ssues, sent out processes toward them, and actively took them in. A recent observation, made in connection with injections of suspensions of carbon and carmine granules into the subcutaneous tissue of the tadpole's tail, is also of interest here. On certain occasions, when the suspensions were injected directly into the lumen of a lymphatic capillary, we noted that the granules of carbon or carmine soon made their appearance in the perinuclear areas of the lymphatic wall — the same regions which are tilled with dye granules after vital staining. Wislocki's ('16) observation that lymphatic endothelium shows a strong avidity for trypan blue led him to fonnulate the h\-pothesis that the phagocj-tic \\inphatic of Amphibia represents an intermediate stage in the evolution of the pjTrhol cell, since only specialized portions of the circulatory endothelium of adult mammals retain this property, and that, in early stages of ilevelopment, the whole vascular endothelium might possess this power of phagocj'tosis.

The mononuclear wandering cells of the tadpole's tail, which stored the granules of trypan blue and neutral red to such a noticeal)le extent, un(loul>tedly belo.ig to that class of cells which have been shown by various authors to react to xital stains of the benzidine group— the pyrrhol cells of (lokbnann ('09), the macrophages of Evans ('15) and the histiocytes of Kiyono ('14V

2'>2 ELEANOR LINTON CLARK AND ELIOT U. CLARK

Those saiiip colls hin c been shown l)y Ribbert ('04), Schlect ('07), Pari ('10), and AschofT and Kiyiino ('13) to take up lithiiun eannino in a similar manner. They correspond to the descriptions given by Ponfick ('09), Hoffmann and Langerhans ('09), and Siebel ('80) of those cells which take up particles of cinnabar. They are also the same cells which possess the power of storing fat, according to Anitschkow ('14), and which Kiyono ('14) has described a.s taking up colloidal silver. Metchnikoff ('92, '05) divideil the mobile phagocytes, without much regaril for their origin or morphological differences, into: 1) the macrophages, which react chiefly toward particulate matter and cell debris, and 2) the microphages, including the polymorphonuclear leucocytes, which show an especial a\idity toward bacteria. The large wandering cells, which store vital dyes, have recently been called macrophages by Evans ('15) who ahgns them with the macrophages of jNIetchnikoff as phagocytes of foreign particles. They have been assumed to be of tissue and endothelial origm (Aschoff and Kiyono, and Evans) and only rarelj' to occur in the blood stream.

That the division of the mobile phagocytes into two classes, made by Metchnikoff, is not a rigid one in all cases has been demonstrated by a number of observations. Rosenthal ('14) found, after injecting living non-virulent cocci into the blood stream, that the Kupffer cells of the liver and the reticulo-endothelial cells of the spleen (cells classified as macrophages) took up the t)rganisms more rapidly than did the polymorphonuclear leucocytes. Similarly, Bartlett and Ozaki ('17) have shown that after injections of micrococcus aureus into the blood stream, 75 per cent of the organisms present in the liver and spleen were contained in wandering cells and in endothelial cells and onlj' 25 per cent in the polymorphonviclear leucocytes, although in the lungs those percentages were reversed. And F. .V. Evans ('14) found that while the polymorphs failed to stain after injections of filtered solutions of lithium cannine, they took up the dye I)articles after injections of unliltered solutions, the jnononuclear macrophages staining in both cases. Moreover, Downiey ('17) has shown that the polymorphonuclear leucocytes will stain in a

VITAL STAINING IN THE TADPOLE's TAIL 253

tj'pical iiiiiimer wth trj^ian blue when they are outside the blood vessels or within a vessel which has been isolated from the circulation. 'The expcriniciits with vital stains and injected fat, reported here, appeared to show that some of the leucocytes which were unstained while circulating became filled with dye granules after leaving the vessels. However, the ob.servations also showed that not all of the wandering cells nor all of the migrated leucocjtes of the tadpole's tail would store the graimles of these vital dyes.

The present observations place the stellate connect ive-ti.s.sue cells of the tadpole's tail in the same category' with the Ijinphatic endothelium and certain of the large wandering cells with regard to their mode of response toward these three vital dyes, although in no ea.'^e was the accumulation of granules so great as in these two latter cell types. Most writers state that only the clasmatocytes or resting wandering cells of the connective tissue stain with the colloidal dyes. However, Evans and Schulemann ('14) mention briefly the finding of small blue granules in connectivetissue cells of the fibrol)la.st type after staining with trypan blue. Moreover, Kiyono ('14) found a few silver spots deposited in the fibroblasts after injections of collargol. That the mesench>ine cells pos.sess a definite power of phagocytizing jjarticles of carbon and carmine injected into the tadpole's tail has been .shown by the authors (see article Anat. Rec. Oct. 1918).

It is interesting to note that the three types of cell which display this especial avidity for storing vital dyes are itlentical with those which possess the power of ingesting particles of carbon and cannine. The similarity of the picture found in these experiments and in those recorded in the preceiling aitide offers a strong argimient for the phagocytic theorj- of vital staining in the case of the.se cells.

.SITMMARY

Neutral red, Bismarck brown and trypan blue are true vital stains which can be used to advantage in the study of the living cells and tissues of the transparent tails of .\mphibian larvae.

254 ELEANOll LINTON CLAKK AND ICLIOT R. CLARK

'rh(> inorr higlily dilTusiblo dyes, neutral nnl and Bismarck brown, stain the cells nuirh more rapidly than the colloidal dye, trjpan hhie. Hut trypan blue has the atlvantagc that' it can be preserved in pennanent prejjarations.

\»-utral reil and Bismarck brown stain small, roRular. and apparently prefonncd granules in the cells of the epidennis. In addition, the contents of a richly branching subcpidennal system are brilliantly .stained with neutral red. Trypan blue does not stain any of the cells of the epidermis.

All three dyes stain an occasional and probably preformed granule in the walls of certain blood vessels.

Neutral red, Bismarck lirown, and tnpati blue are all deposited in a similar manner as large accunmlations of dye granules in the perinuclear areas of the Ijinphatics, in certain large mononuclear wandering cells and leucocytes and, to a less degree, on the proce.s.ses of the stellate connective-tissue cells. The physiological relation.ship shown by this reaction toward these vital dyes on the part of lymphatics, wandering cells, and mesenchyme cells is probably due to their common property of phagocytosis.

niBLIOGRAPHY

Anitschow, N. 1914 L'ber vitale Fiirbung" und Cholesterinspeicheruni; im

Orpnnismus. Mediz. Klinik, No. 11, p. 465. Arnold, .1. ISOO fbcr GranuKifiirbunn lebcnder und uberlebender Lcukocy ten. Viroliow's Arch. f. .\n.it. u. Phy.«iol., vol. 157, p. 424.

1900 tiber GranulafiirbunR lebender und iiberlebendcr Gewebe.

Virchow's Arch., vol. 1.59, p. 101. AscHOFF and Kiyono 1913 Zur Frafie dor grossen Mononuclearen. Folia

Haom. Archiv., vol. 15, p. 383. Baktlett and Ozaki 1917 Phagocyto.sis in vivo under variou.s condition.s.

Jour. Med. lies., vol. 37, p. 139. BonPFARD. G. 1906 Injection des couleurs de benzidine aux animaux norinaux.

.Annates de I'lnst. Pasteur, vol. 20, p. 539. Clark, E. R. 1909 Observations on living growing lymphatics in the tail of

the frog larva. .Anat. Uec, vol. 3, Xo. 4. p. 183.

1912 Further oK-Jcr vat ions on living growing lynii)hatics: their relation to the mesenchyme cells. Am. Jour. .\nat., vol. 13. No. 3, p. 351. Clark, E. U. anh E. L. 1917 A study of the reaction of lymphatic endothelium

and of leucocvles Inward injcrled fat. .Am. Jour. .\nal.. vol. 21. No.

3, p. 421.

VITAL STAINING IN THE TADPOLE's TAIL 255

Downey, II. 1'.)I7 Ucactions of blood and tittsuc cells to arid colloidal dyes

under experimental conditions. Anat. Uec, vol. 12, No. 4, p. -i'lO. EnRLicn, P. 1886 Uber die Melhylen Blau Reaction der lehenden Nerven substanz. Biol. Centr., vol. G. p. 214.

1S94 Cber Neutralrot. Allg. Mi-d. Centr. Zeit., vol. 63. p. 20.

1!H)4 Cicsammclte .\rbeil zu IminiinitatHforschung. Berlin.

190'.) Bcitriige /.ur experimentcUe Pathologic und Chemotherapie.

I>cip/.iK. Evans, F. A. 1916 Expcrinionlal .itiuiy of the mononuclear cells of the blood

and tissues. Arch, for Int. Med., vol. 18, p. 692. Evans, H. M., and Schulemann 1914 The action of vital .stains belonging to

the benzidine group. Science, N. S., vol. 39, No. 1004, p. 443.

1915 Uber Natur und Gcnese der durch saure FarbstofTe entstehenden

Vitalfiirbiingsgranula. Folia Hacm. Arch., vol. 19, p. 207. Eva.\8, H. M. 191.5 Tho macrophages of mammals. Amer. Jour, of Physiol.,

vol. 37, p. 243. FiacHEL, A. 1901 I'ntersuchungen uber vitale Fiirbung. Anat. Hefte, vol. 16,

p. 415. GoLDMANN, E. E. 1909 Die aussere und innere Sckretion des gesunden und

kranken Organismus in Lichtc der vitalcn Fiirbung. Beitr. zur Klin.

Chir., vol. 64, p. 192. Hoffmann and Lanoerhans 1869 Uber den Verbleibdes in die Circulation

cingeftihrten Zinnobcrs. Virchow's .\rch., vol. 48, p. 303. KlYONO, K. 1913 See A.schofT.

1914 Zur frage der histiozytiiren Blutzellcn. Folia Hacm. Arch.,

vol. IS. p. 149. Lewis, M. R. 1917 The effect of certain vital stains upon the development of

the eggs of Ccrebratulus lacteus, Kchinorachnius parma. and Lophius

pi.scatorius. Anat. Rec, vol. 13, No. 1, p. 21. Masuda, N. 1911 Unlcrsuchungen iiber die Zellfunction mit Hilfc der vitalcn

Fiirbung. Zeit. f. Exp. Path. u. Ther.. vol. 9, p. 250. Metchnikoff, E. 1883 Untorsuchungen iibcr die mesodermalen Phagocyten

einiger Wirbelthicre. Biol. Centr., vol. 3, p. 560.

1892 LcQons sur la pathologic compari'c do rintlammalion. Paris. V. MOllen'oorf. W. 1915 Die Dispersitiit dor Farbstoffe: ihrc Beziehung zu

Ausscheidung und Spcicherung in die Niere. Anat. Hefte, vol. .")3,

No. 1, p. 89. NicoLLE AND Mksnil 1906 Traitcmcnt des trypanosomiasis par les "couleurs

de benzidine." Annales de I'Inst. de Pasteur, vol. 20, p. 417." Overton, E. 1900 Studicn iibcr die Aufnahme der Anilinfarben durch die

lebende Zelle. Jahr. f. Wiss. Bot.. vol. 34. p. 669. Pahi, O. .\. 1910 Tber die V'erwendbnrkeit vitaler Karmineinspritzungen fiir

die palhologi.sche .\natomie. Zeit. f. Path., vol. 4. No. 1, ]>. 1. Plato, ,J. 1000 t'ber die "vitale ' Fiirbbarkeit der Phagocyten des Meiischen

und einiger >Saugethiere mit Neutralrot h. .\rch. f. mikr. .\n:it., vol.

56, p. S68. PoNFicK. K. IStiO Studi<'n iiber die Schicksale kiirnigcr FarbstofTe ini Organismus. Virchow's .Vrchiv., vol. 48, p. 1.

•2.)b KLEANOK LINTON CLAHK AND ELIOT R. CLARK

HiiiiiF.iiT, H. 1004 Dip AhiichpidiiiiK intnivcnoH injizicrtcn gcl6stpn Karmina in

lion Opwebon. Zcit. f. AHr. I'hysiol., vol. 4, p. 201. RcElCK.K 1904 Zur Kriine der l"iirl)l)iiikoit dcr Icbenden Substanz. Zeit. f.

Alln. I'liysinl., vol. 4. p. 141. Rosenthal, W. 1014 Phaijozytose vcrsuche ira Ticrkorper. \'orh. dcr Deut.

Path. Ges.. 17. JalirR.. p. L'8(). RnssEL^, D. G. 1914 The t-lTort of gpntiun villet on protozoa and on tissues

growing in vitro, with especial reference to the nucleus. Jour. Exp.

Mod., vol. 20, p. .54.5. SciiLECT, IT. 1907 Expcrimenfclle Untersuchungen iibcr die Resorption und

die Ausschcidung dcs Lithionkarmins unter physiologischen und

pathologischcn Bcdingungen. Zieg. Beitr. zur Path. Anat., vol, 40,

p. 312. ScHCLEM.\.N.v, \V. 1912 Chemische Constitution and Vitalfarbungsvermogen.

Zeit f. Exp, Path. u. Ther., vol. 11, p. 307.

1914 See Evans, H. M.

101,5 t'ber Mctachromasie bei Vitalfarbstoffen. Zeit. f. Exp. Path.

u. Ther., vol. 17, p. 401. SiEBEL, W. 1886 Uber die schicksale von Fremdkorpen in der Hlutbahn.

Virchow's Arch., vol. 104, p. 514. Stolc, a. 1902 t'bcr das Verhalten des Neutralrots im lebendigen Protoplasma.

Zeit. f. Allg. Physiol., vol. 1, p. 209. WiSLOCKl, G. B. 1016 The staining of amphibian larvae with benzidine dyes,

with especial reference to the behavior of the lymphatic endothelium. ■

.'Vmer. Jour, of Physiol., vol. 42, No. 1, p. 124.

1917. The action of vital dyes in teleosts. .\nat. Rec, vol. 12, No, 4,

p. 415.

Resumido por cl autor. Edward Phelps AUis, Jr.

Sobre el origen de la hiomandfbula de los Teleostomos.

Trmnilatlon by Dr. Jo!^ Nonidez, Columbia University

ACTBOllB ABSTRACT Or thih papck

BMt'ZD DT TBK IIIIILIOOItAPIIIC HEIlVICB

ON THE ORIGIN OF THE liVuMA.XDIJiULA Ul" THE

TELEOSTO.MI

EDWARD PHELPS ALLIS, JR. Menlon, France

OXE FIGURE

In a work published in 1914, I came to the conclusion that there must be, in fishes, "a prijnarily somewhat independent mass of mesoderm ' cells lying lateral to the neurocranium and dors;il to the dorsal ends of the niundibular and premandibiilar arches, in the position of the pharyngeal elements of the branchial arches, which pharyngeal elements are wanting, as independent structures, in the mandibular and premandibular arches of all fishes." These cells were assumed to be capable of chondrification and to have given rise both to the ascending and otic processes of the palatoquadrate of the Dipneusti, .\mphibia and Roptilia, and to the lateral wall of the trigemino-facialis ch;imber of fishes and manmials. Similar cells related to the hyal arch were said to have possibly given rise to some portion of the otic capsule, and its derivative the operculum, and probably also to the toleostean hyomandibula.

In a later work, published in 1915, I came to the conclusion that the cartilages derived from the mesoderm cells abo\'e referred to had, in all probability, their serial homologues in the extrabranchials and mterarcual cartilages of the branchial arches of the S('iachii, the posterior articular head of the telcostean hyomandibula being derived from the dorsal extrabranchial of the hyal ;irch and the anterior articular head from the intenu-cuhi cartilage between that arch and the mandibuhu- arch. The sjinplectic was said to probabl>- be a primarily independent cartilag<', and probably an hypertrojihied middle one or ones of the branchial rays of th(> nutndibuhir arch. The single articular

2')S EDWARD PHELPS ALUS, JR.

liciiti of the liydiimiulibulu of tlu- C'hondrostci was said to apparontly coiTcsjiond to the antorior articular head of the toU'ostpan liyoinaiitiilnila. The hyoiiiaiidiliula of Polyptcrus was left largely out of consideration, but it was said that the suprapharynpobranchials of van Wijhe's ('82) descriptions of that fish and certain others of the danoidei were quite certainly repn>s<'nted in the extraliranchials of the Selachii, the suprapharyngobranchials accordingly being serial honiologues of the posterior articuhir head of the hyomandibula.

Since the jniblication of the work last above referred to, I have had occasion to examine the branchial arches in Polypterus, and I not only find that the suprapharyngobranchials of van Wijhe's descriptions are simply the epibranchials of their respective arches, but that there are, in each arch, cartilages that (|uite certainh' represent the special niesodenn cells that were assumed, in the two works above referred to, to have given rise, in the hyal arch, to the hyomandibula, and in the mandiljular arch to the ascending and otic processes of the palatociuadrute.

Van Wijhe, in the work above referred to, described, in the dorsjil half of the first branchial arch of Polypterus, a small cartilage that he considered to represent the epibranchial of the arch, imd two bones that he called the supra- ami infrajjharyngobranchials, the infrapharj-ngobranchial apparently being considered by him to correspond to the typical selachian jiharyngobranchial, and the suprapharyngobranchial to be a fifth element of a complete and typical branchial arch. The epibranchial is said to be abnost completely concealed in a ligiuiient that envelops both it and the inf^aphar}^lgobranchial, and that has its insertion on what \'an Wijhe considered to be a jiart of the jirootic covered by the thin lateral edge of the parasphenoid. The suprajihar>aigobranchial is said to be relatively large, to abut against a cartilaginous jjortion of the lateral wall of the neurocranium, and to have its ili.stal portion ileeply grooved to lodge the efferent arterj' of the arch. In the second and third branchial arches there is said to be no epil)ranchial, and it is said that the ujiper ends of the sui)ra- anil infraijharyngobranchials of those arches may be fused to form a short tube' which encloses the efTerent

HYOMANDIBULA OF THE TELEOSTOMI 259

artery <if the ;ircli. In tho rorrosponflinp; p;irt of the fourth iireh there is suitl to be a sjriiill i)hiiryiifi;iil)r:iuchi;il, but in the H^ure given it is md ex-lettered as tui infnipharyngobninchiiil.

In a 75-nuii. specunen of Polypterus senegahis that I have exjunined in serial transverse sections, there is no trace of tlie independent so-called epibranchial cartilage described by van Wijhe in the first branchial arch, and I also find no trace of it Lo adult specunens of Polypterus bichir and Polypterus ornatipinnis. In the first branchial arch of the 7o-nun. specijnen, the supraand infrapharjnigobranchials of van Wijhe's descriptions are found as independent cartilages, and they are certainly simply, respectively, the nonnal epibranchial and pharjTigobranchial of the arch. The epibranchial articulates by its distal end with the ceratobranchial of its arch, and by the anterior comer of its proximal end with the pharyngobranchial, and the posterior corner of its proximal end has been prolonged to form a stout process which has acquired articular relations with the lateral wall of the bulla acustica,there lying ventralto thevenajugularisandthetruncus facialis. The phar^^lgol)ranchial articulates with the epibranchial, as above described, and, running anteromesially and somewhat ventrally, enters the angle between the lateral and ventraj (horizontal) plates of the ascending process of the parasphenoid, and there has its attachment. In its course it lies unbedded in the lateral surface of the thjinus, dorsal to a stout ligament that extends from the angle of the ascending process of the parasphenoid to the dorsal end of the ceratobranchial of the first branchial arch, this ligiunent being the one that is said by van Wijhe to envelop his epibranchial and infrapharjnigobranchial. .V groove on the external siu'face of the epibranchial (suprapharyngobranchial of \an ^^'ijhe), between it and the phar%nigobranchial (infrai)haryngobranchial of van Wijhe), lodges the efferent artery of the arch.

In the adult specimens of both Polypterus bichir and Polypterus ornatipinnis, I find strictly suuihir conditions; but the epibranchial and pharjiigobnuichial have apparently fused with each other, and each has luidergone extensive ossification, the two so-fonned bones being in contact at their distal ends ami

2()0 EDWARD PHELPS ALUS, JR.

there uiuiiovably connected with each other. The process on the posterior comer f)f the proxunal end of the epibranchial has been completely ossifiod, oxcoptinK tho art icvihvr cap by which if articulates with the lateral wall of the bulla acustica, and this process of the bone is strictly similar to that shown by van Wijhe in his figures of Amia and I^pidosteus, and by me in my fiRiires of Anua and Scomber (.\llis, '97, '03). In Amia I did not find the sujirapharjiigobranchial described by van Wijhe in that fish. l)ut in Scomber, in a corresponding position, I found an independent piece of cartilage that articulated with the pharjTigobranchial of the second branchial arch and that 1 called a suprapharjnigobnmchial.

In the second and third branchial arches of all my specunens of P()K7)terus, the 75-nmi. one as well as the adults, the epibranchial and pharyngobranchial of each arch are completely fused with each other, and the anterior (lateral) and posterior (mesial) edges of the so-formed piece have been produced dorsally so that they touch, or fuse with each other, dorsal to the efTerent artery of the arch, thus enclosing it in a short tube. The nerve of the arch runs posteriorly dorsal to this tube, not traversing it.

Each of the first three branchial arches of this fish is furnished with two rows of branchiae, each row supported by cartilaginous branchial rays the bases of which have fused to form a jiractically continuous bar of cartilage, as shown in the accompanymg figure. The rays thus form a comb-shaped structure the base of which is arched in a curve that corresponds approximately to that of the branchial bar of the arch when the mouth is opened and the branchial chamber expanded. When tlie mouth is closed and the branchial chamber contracted, the levator muscles have pulled the distal (ventral) end of the epibranchial >ipward, and that element and the pharjaigobranchial arc then directed ventro-antero-mesially at a marked angle to the ceratobranchial. The comb-shaped structures formed by the branchial rays of each arch can not midergo a corresponding change of fonn, because of the relative rigidity of their basal liars, and the dorsiil portions of those bars, the parts related to the epibranch

HYOMANDIBULA OF TIIK TKLEO.STOMI

L'OI

iiil urul ph;irvnn'>l>r;iiichi;il of oacli uicli, project (lorso-untcrniiicsially, Imt jiiorc iiicsially, iiiid less unlcriorly, tluin the tlnrsiil pritiuii- (it ilic lir.iiicliial liai'. this leaviiifj; ;i wide space l)et\veeii tlifjiiscl\('s uiul liiut i)(irtii)ii of the hraucliial l)ai'. 'i'lic bjisal burs of tlie rays, projectiiifr dorsally, lie external to the U'vator nuisclcs of the arch, and their dorsal ends, which lie dorsal to the vena jiifiularis, arc there attaclied by connective tissues.

Fig. 1 Lateral view of the posterior portion of the ncurocraiiium of Polypterus, showiiiR the first t)raiicliial arch in place, with the related anterior series of eartilagiiious branchial rays, but the posterior branchial arches antl the hyal arch removed. X 2. br., branchial rays of first branchial arch; chr., ceratol)ranchial ; cbr., epibranchial ; Jr., facialis foramen; </., proove for vena jugularis; phr., pharyngobranchial ; ps., para»phenoid.

'I'he nerve and efferent artery nf the aicli now pass ventral to the anterior one of the.-;e two l)ranchial-ray bars, and then onward ix'tween the two bars onto the external surface of the ceratobranchial of the arch.

In Ainia, Lepido.steus and Polyodon cartilaginous branchial rays sijnilar to those of PolyptiM-iis are foinid. and th(>ir i)ases are in contact with each other, but not so completely fused as in Polypterus; ami in those fishes, alsr), the branchial-ray bars project dorsal to the epibraiu-hial and pharniRobranchial of the arch

THI: ANATOMICAL KECORn, VOL 15, NO. 5

•JCi'J EDWARD PHELPS ALUS, JR.

to wliicli thoy arc related. 'I'liere is jiccordiiiKly every reason to helievo that sijuilar conditions existed in the iiiunediate ancestors of tlics(> fishes, and tliat in tlioso fishes hrunchial rays, caj)al)le of fusing with eacii otlier at tiieir bases, were found also in the hyal and inandihular arches. The dorsal ends of the !)ranchial-ray bars of the latter arches would then lie close to the l)uljiinp: auditory portion of the neurocranium, dorsal to the vena junularis, and h(>nce in a position to form, in the Iiyal arch, a hyo)nandil)ula with one or two articular heads, and in tin* inan(lil)ular arch the ascendin of the head of niy specimen, this edge of the accessory hj'ojuandibula is covered with bone, this being as van ^^'ijhe ('82) found it in the specimen described and figured by him. A stout ligament always extends from the head of the accessory h3'omandii)ula to the doi.sal edge of the opercular process of the hyomandibula, and lies po.sterior to the nervus hyoideus facialis; this ligament and the accessory liyomaiulibula thus ([uite certainly representing the posterior articular head of the teleosteaii hyomandibula. The efTerent artery of the arch lies postero-intemal to

HYOMANDIBULA OF THE TELEOSTOMI 203

lliis ligiijiiont, thus coiTcspoiitliiifi, in its rclHtiniis to tlic l)r;uiclii;il rays, to thf posterior offoroiit artory of the Soljirhii. Tho position of the IK r\us jnandiljularis facialis, anterior to the iuitcrior rrticular head of tlic liyonian(lil)nla, is douhtloss due to this nerve liavLiig sej)arated from tiie nerviis lij'oideiis shortly after the triuicus facialis issued from its foramen, thus pennitting it to slip over the florsal end of the anterior hranehial-ray har before that bar had aecjuired articulation with the cranial wall. The ventral end of the hyomandiijula presents two angles, or processes, one of which articulates with the interliyal and is apparently fonned by the posterior branchial-ray bar of the arch, while the (jther articulates with the (juadrate, fonns the so-called syniplectic process of the hyoinandibula, and is doubtless formed by the anterior branchial-ray bar. The interliyal must then represent the coalesced and relatively greatly reduced epal and pharyngeal elejiients of the arch.

Ill the Holostei and Teleostei the conditions are sinxihir to these in Polyjiterus excepting in tliat the posterior articular head (.f the hyojuandibula is more fully developed, and in that the nervus mantlibularis facialis does not separate from the nervus hyoiileus until after tlie truncus facialis has pa.ssed between the two heads of the hyomandibula.

In the Cliondnistei, the j)osterior articular head of the hyomandibula is wholly wantuig, this indicating that the posterior branchial-ray liar has more or less completely aborted, and, douiitless in correlation with this, the interhyal has ac(}uired articulation with the s_\inplectic which jiiust be either a detached portion of the anterior branchial-ray bar or l)e derived, as suggested in my earlier work, from braiiclii;il rays of the mandibular arcli.

In the Plagiostomi, the hyomandibula articulates witli the cranial wall ventral to the vena jugularis, and is formed, in the Selachii, by the epihyal, laul in the Hatoiilei by the jiharyngohyal (.\llis, '1')). There is in these fishes but a single row of branchial rav's, a posterior one, and it is founil in the hyal as well as in the branchial arches. Associated with these rays there are so-called dorsal and ventral extrabranchials, which are currently consiilered to be sunply nuHlitied dorsal and ventral ones of the

204 EDWAHD PHELPS ALMS, JR.

hrancliijil rays actuiilly rtiund in tlioc lislics. liiaiis ('06), howovor, cnnsich'i's them tn hclmi}; tn an indciMMidcnt calcfion' of skeletal elements, for, in ejMl)ry<is of Heptanehus, he found them lyinn not only at a consi(leral)le distanee from the hranehial rays, hut also at right juigles to those rays and parallel to the inner hranehial hars. These relations to the branchial rays at onee suggest a hranehial-ray bar that has been develo|)e(l either in relati<in to tliose dorsal and ventral ones of the posterior rowthat were prijnarily related to the pharyngeal antl hyal elements iif the areh. If the extrabranehials have this latter origin, which seems probable, then the dorsal one would be of similar origin to that here ascribed to the posterior articular head of the tele(stean hyoinandibula. The conclusions arrived at in my earlier works, and briefly stateil hi the openuig paragra])hs of the present article, would then have to be moilifieil sunply by the substitution of the "anterior branchial-ray bar of the hyal arch" in jilace of "an interarcual cartilage that lay between that arch and the mandibular arch," and the sjiuplectic would be derived from the anterior branchial-ray bar instead of Iroju the branchial niys of the mandibular ardi.

The conditions here fountl in recent Teleostomi and Plagios(cnii could evidentlj^ not be derived the one from the dtlui- williout reversion to a type from which they both nuist have descended, and as there are no indications of any such reversion having taken i)lace, the separation of it he two lines here indicated must have taken place in very early geological times, for even in the crossopterygian Tristichopterus, rejuains of which are found in lower Devonian rocks, the hyomandibula nuist have articulated with the neurocraniiun dorsal to the vena jugularis, for Trafjuair (75) says that the posterior margin of the jKilato-nspensory apparatus, "apparently corresponding to the hyoniandibular element," gives articulation externally to the preojM'rcular cheek-plate, and is itself connected ilorsally with the sfiuiunosal region of the cranium: each of which conditions indicates that the hyomandibula lay external to the vena jugularis and articulated with the cranium dorsiil to that vein.

I'bUU df Carnol^fl. Mcnion, Prftnc«  April 13, 1«I8

HYOMANDinULA OF THE TELEOSTOMI 20')

LITHUATIHK CITICD

Allis, K. p., .III. IS'.l" 'Ihc crniii:!! iniisclos, and craiii;!! .'iiid (irst spinal norvr.s

ill Aiiiia calva. Jour. .Morph., vol. 1'2, I'o.ston.

I'.Xy.l Till' .skull, aiul llit> cranial ami first spinal inusclcs and ncrvos

in Sconilicr scoinhrr. Jour. Morpli., vol. IH, L;in<-s.

Ii)14 'I'lu! pituitary fo.ssa and triufniino-fac-ialis chambpr in Ccratodus

forslcri. .\iiat. Anz., Ud. 40, Jena.

litl.'i Tlic homolo|;ii-s of tin- liyoinandiltula of llic nnatliostonio fislip.s.

.lour. Morph.. vol. L'(>. I'liilada. IJrai's, II. liKM) (M)cr den iMiiltryonalcn Kirriicnapparal von lleplandius.

Anal. All/., Hd. Ji), .lona. Tii.\<iiAiii. H. II. 1S70 On the cranial osteology of Polyplcrus. Joiirii. .Viial.

and I'liysio!.. vol. ,'>. London.

1875 Structure and affinities of Tristichoptcrus alatus. Trans. I{oy;d

Soc, vol. 27. Kdin. VVijiiK, J. W. VAN l,S,Si (*ber da.s Viscpralskolcl und die Xorvcn dos Kopfes der

(■anoidcii und von C'cratodus. Nicdcrl. .\rcliiv fiir Zool.. Hd. ."), II. 3.

Leiden.

THE ANATOMICAL RECORD. VOL. IS. NO. tt JANVAHY. 1910

Hesuiiiido por el iuitor, Hicluinl K. Scaininon.

Sohre el dcsjirrollo y Hna estructura del cuerpo adiposo bucal.

Trftntlation by Dr. 3o»6 F. Nonidei, Columbia University

AfTIIOH H AfWlHAf.T Of THW PAPKR IIMIl'BD BT TIIC niDLIOaKAPIIIC HRRVIcr. JANCART

## ON THE DEVELOPMENT AND FINER STRUCTURE OF THE CORPUS ADIPOSUM BUCCAE

HICIIARD E. SCAMMOX Insiiluie of Anatomy, University of Minnesota

NINE FIOrRES

The corpus adiposum buccae or sucking pad is a specialized and sharply circiinisfrihod mass of adipose tissue which lies in the cheek partially wedged between the masseter and buccinator muscles and covered externally by the superficial fascia of the face and the zygomatic muscle. Posteriorly, it is connected by a stalk with a much larger fat mass, termed by Forster ('04) the corpus ailiposum malae, which is located between the temporal and the i)terygoid nuiscles and which possesses a superficial process extending over the outer surface of the temporal muscle beneath the temporal fascia. The sucking pad was apparently first mentioned bj* Heister in 1732, who, thinking it was glandular in character, termed it the glandula molares. Winslow, about twenty years later, again descril)ed the structure as a glantl and wrote of a series of small

' This study was carried out with the aid of a grant from the Research Fund of the University of Minnesota.

- The body has received many names. Besides the term applied to it by Heister, under a misconception of its nature, the structure has also been called the boulo (jraisseu.se, boule de Bichat, Wangenfettpfropf, Wangenfctlpolstor, Saugpol.ster, sucking pad, and sucking cu.><hion. It is not clear that the B. N. .\. term, corpus adiposum buccae, which I have employed here, wa,s originally intended for this particular fat mass; in fact, it is more probable that this expression was meant to indicate the entire mass of which the corpus adiposum malae fonns the main body. However, most modern authors have used the B. N. .\. term in the narrow sense of the sucking pad proper, and to avoid further Bynoiiymily I have followed their example. Berg ('ID, in his classification of the fat nia.><ses of the body, places the corpus adiposum buccae in the category of inleriniiscular fat masses together with the adipose tissue between the layers of the temporal fascia and the orbital fat.

267

268 HICIIARD E. SCAMMON

ducts which passed fnmi it tliioiifili the buccinator muscle to open into the oral cavity near the hist molar tootli. Bichat recognized the true fatty nature of the sucking pad and referred to it in his Anatomic (leni'-ralc in 1801. He is sometimes cited as the discoverer of the body. Bichat's remarks on the sucking pad are very brief and are purely incidental to a discussion on the presence of adipose ti.ssue in early life. It is ([uite likely that the true nature of the body was known to anatomists before this time, although the examination of a large amount of the literature of the eighteenth century dealing with the anatomy of the fetus and child has failed to reveal any descriptions beyond those already mentioned.

The body as seen in the adidt was figured by Burns in 1S21, but it is not clear from this author's description that he regarded it as a normal structure. In 1852, Gehewe, in a Latin thesis, gave an excellent account of its gross anatomy and described its development in so far as it could be seen with the naked eye. Since this time the gross form and relations of the body have been figured and described by several authors, the most complete accounts being those of Ranke ('84), Lafite-Dupont ('00), Forster ('04), and Shattock ('09).

The phylogeny of the sucking pad has been studied in detail by Forster ('04). He finds that the entire mass of the corpus adiposum malae of the higher Primates is derived from the extra-orbital fat pad of the lemurs, which, in turn, is formed from an outgrowth of the periorbital fat mass of lower mammals. The corpus adiposum buccae, or facial extension of the corpus adiposum malae, is developed in the Primates as the orbital gland disappears and the muscles of mastication undergo partial regression.

LehndorfT ('07) investigated the chemical composition of the sucking i)ad and found it richer in the fats of high melting point (palmitic and stearic acids) and poorer in oleic acid than the general superficial fat. .^hattock (09), however, is of the opinion that the difTerence between the two is too slight to be of any great significance.

The function of the sucking pad has been discussed at length by Hanke ("84), Forster ("04), Lehndorff ('07), and Eisler ('12).

Very little has been written on the development and finer structure of the sucking pad. (lehewe ()2) found the first traces of the body in fetuses of the third month and noticed its gradual increase in size up ti) the time of birth. His studies were made entirely by niacn)sf()])ic metliods. R()l)iii and (limbert ('tj4) described the structure as appearing about tlie sixtieth day of fetal life as a number of clusters of small fat-cells. They found that the later growth of the body took place by the formation of new clusters as well as by the increase in the size of the earlier ones. Lafite-Duixmt ('()0) described the botly in a fetus 12 cm. in length as consisting of a dense mass of mucous connective ti.ssue, the fibers of which were arranged in the vertical plane of the face. This mass contained a few clusters of leucocytes. In a fetus of five months this body was transformed into a mass of adipose tissue and the embryonic mucous connective tissue had entirely disapjieared. This transformation began in the central and lower jjart of the organ. Ranke ("84) figured and described the finer structure of the su(!king pad in the late fetus and the new-bom. He found it to consist of numerous lobules of unilocular fat-cells separated by broad septa of connective tissue. The whole body was surrounded by a definite capsule of fibrous connective tissue as well. A large number of blood-vessels ramified upon the outer surface of this cai)sule and their branches penetrated it to break up into terminal plexu.><es around the fat cells of the lobules. This description was confirmed by Shattock ('09), who also noted that the sucking pad was present in the fourth month of fetal life. Berg I'll) mentions that the body is in a fetus 10 cm. in length, although no fat-cells were observed at this stage.

DEVELOPMENT

The time of formation of the sucking pad. like that of most of the fat masses of the l)oily, is suliject to some variation, l)Ut the region which it will occupy later is clearly marked out in fetuses 4 or o cm. in total (crown-heel) length. At this time the lateral walls of the buccal cavitv, which hitherto have been somewhat coiiipri'ssc'ii from sitlo to side, coiuiuonct' to tliickcn considerably with the lateral extension of the developing maxillae, so that a broad band of tissue intervenes between the epithelium lining the oral cavity and the skin covering the chock. The margins of this mass arc already occupied l)y sheets of developing muscle — by the anlage of the l)uccinator medially, and by the facial portion of the sphinct(>r colli laterally. These nuiscular sheets thus form the side walls of a region which is quadrilateral in frontal section and which is bounded by the ma.xilla above and by the mandible and the masseter muscle below. This region is closed anteriorly by the approach of the anterior part of the buccinator nuiscle and the oral portion of the spliinctcr colli, but posteriorly it becomes continuous with the pterygoid region and through it with the orbit which is as yet incompletely enclosed by its bony walls.

The region thus outlined may be termed for convenience the buccal space. It is filled with a delicate mesenchyma which is looser meshed than that of the face generally. In this mesenchyma are embedded tlie parotid duct and a coarse plexus of veins. The duct passes through the facial portion of the sphincter colli, crosses transversely through the buccal space, and, after piercing the buccinator muscle, opens into the oral cavity. The venous plexus arises from the large veins at the base of the orbit and passes obliquely downward through the space. It drains in part into the facial and in part into the internal maxillary vein. The radicles of this plexus, which are of extremely irregular caliber, are surrounded by a mass of loose-meshed mesenchyma, which, however, has not difTerentiated sufficiently as j'et to be termed preadipose tissue. A frontal section of the cheek of a fetus of this stage is shown in figure 1.

.\ definitive anlage of the sucking pad is generally found in fetuses from ti to 8 cm. in total length, although sometimes it does not appear until a little later. By this time the buccal space has become somewhat narrowed through the growth of the muscles of mastication, and the individual muscles which are formed from the facial portion of the sphincter colli are clearly (litTcrontiatetl. The parotid duct pursues the same course through the space as it does in younger fetuses, but a definite connective-tissue sheath is now beginning to form around it. The molar glands are clearly difforontiatod, but

Fig. 1 Frontal section of the face of a human fetus, 60 mm. in total length, showing the region of the future sucking pad. Epithelial structures are represented in solid black, mesenchyma in stipple, muscle by short parallel lines or by coarse stipple, bone by close vertical ruUng. and blood-vessels in solid outline. P.p.. parotid duct; iU.?)., anlage of buccinator muscle: ^ftt., mandible; M.f., aniagen of facial muscles; M.m., anlage of niassetcr muscle; .Uj., maxilla; O.m., oral epithelium; V'., venous plexus.

have not pierced the buccinator muscle. In two specimens of this stage which I have examined the orbital inclusifin was located just lateral to the buccinator muscle and anterior to the internal pterygoid (fig. 2, O.i.).

By this time the arrangcinont of the veins in this region is ronsiderably modified. The upper part of the plexus is differentiated into several trunks which eoniiect with the inferior \eins <if the orl)it al)ove, while the lower jjart forms vessels which drain into the facial vein below. These lower trunks represent the vena ophthalinofacialis of (Jurwitsch and Sesemann or the vena facialis profunda of Tronch authors. The middle j)art of the (iriniual jilexus connects posteriorly with the pterygoid i)lcxus. It is l)roken up anteriorly into a number of small venules which anastomose freely. The sucking pad is in the process of formation around these venules, 'i'he peripheiy of the nuiss is slightly differentiated into a capsule which is indicated more by the direction of the fibers forming it than by a condensation of the tissue. Within this capsule the mesenchyma is wide me.shed and delicate except immediately around the venules, where it is somewhat condensed, forming thin sheaths about the vessels. Mixed with the preadij)ose tissue are a considerable number of you^ig Ijlood-cells. These maj' be the result of an accidental extravasation from the smaller vessels into the tissue of the sucking pad, but I have observed them in three of the four specimens of this stage which I have examined, and apparently the}- were also seen by Lafite-Dupont in a. somewhat okler specimen. None of the epithelial structures which penetrate the buccal space lie in the immediate region of the anlage of the sucking i)ad at this time. Figure 2 is a drawing of a transverse section of the left cheek and neighboring structures of a fetus 7 cm. in total length and illustrates most of the important features of the .sucking pad at this stage.

In fetuses from 12 to 1(3 cm. in total length the corpus adipo.sum buccae has approached its final form and young fat lobules are conunencing to rei)lace the preadijiose tissue seen in earlier stages. The ma.ss now tills the outer part of the buccal space. It is surrounded by a definite capsule of developing fibrous tissue. Within this capsule the organ consists of a me.shwork of fibers of young connective tis.sue in which are embedded a few developing fat lobules and the plexus of veins already described. The fat lobules are confined almost entirely to the peripliery of the anterior part of tlie organ. They consist of preadipose tissue and true fat cells. The latter, which are quite small, are found mainly in the centers of the lobules. The duct of the parotid gland comes in contact with the capsule of

Fig. 2 Tran.svorso soetion of the left cheek of a huiiinn fetus 7 cm. in total length. Method of drawing .similar to that employed in figure 1. C. corpu.s adi|>osum buccae; D.p., parotid duct; C. aniagcn of molar glands; .U./».. buccinator mu.scle; Md., ramus of mandible; W./., facial musculature and fa,scia; M.m., masscter muscle; A/. p., internal pterygoid muscle; O.i'., orbital inclusion; O.m.. oral mucous membrane.

are now embedded in the substance of the buccinator muscle, but the}' do not come in direct contact with the cai)sule of the sucking path Small fat lobules are in the process of formation, both external to the facial musculature and fascia and also in the portion of the buccal si)ace which is not occupied l>v the sucking pad and by ei)ithelial and vascular structures. The condition of flic sucking pad at this stage is illustrated by tigure 3, a frontal section passing through the extreme anterior part of the l)n(ly in a fetus 1") cm. in total l(>ngth.

Fig. 3 Frontal section of the left check of a human fetus 15 cm. in total length. The section pns.se8 through the extreme anterior part of the sucking pad. C, corpus adipoijum buccae; />./).. jiarotid duct; F.v., facial vein; 0., molar gland.s; .1/./)., buccinator Inuscle; ^f(l., mandible; .'/./., facial muscles and fascia; .\tx.. maxilla; O.m.. oral mucous membrane.

After the sucking pad has reached the stage just described, it grows rather rapidly. It expands outward and also backward over the superficial surface of the masseter muscle and contributes rrmsidorably tn the roundod form of tho fhook which is so noticeable in huiiuui fetuses of the latter half of iiitra-uteriiie life, in this expansion the capsule of the body is carried outward towards the facial muscles and fascia, and the broad band of mesenchymal tissue which formerly separated these structures is rcduccil to a narrow sheet which contains a rich plexus of veins and a few small fat lobules. The medial portion of the capsule is also pressed inward towards the buccinator muscle, l)ut an intermediate strip of mesenchymal tissue, which contains the bodies of the molar glands, still persists in this position. As in earlier stages, the parotid duct and the molar glands lie entirely outside the capsule of the sucking pad. They now po.ssess definite mesenchymal envelojies which are independent of it. These relations are shown in figure 4, a drawing of a transverse section of the right cheek of a fetus 17.5 cm. in total length.

The finer structure of the sucking pad during this period of rapid growth is somewhat variable. As was pointed out in the description of the preceding stage, the formation of fat lobules takes place first at the periphery of the body and particularly in its anterior part; from this region the process extends inward and backward first into the center of the body and then into the stalk which connects it with the corpus adiposum malae. The lobules are always formed around the first branches of the venous plexus. P'igure 4 shows a stage at which the peripheral lobulation of the body is well under way. while the central portion of the mass contains almost no differentiated adipose tissue. Thus the early expansion of the sucking pad is not depenilent upon the formation of fat lobules, but upon the growth of the mesenchymal meshwork in which they will appear later. .\s the lobules are developed the connective tissue between them is reduced to the form of broad septa. It seems probable that the formation of new lobules is completed, in the majority of cases at least, by the end of the fifth fetal month. I estimate that the body contains from 2oU to lioO lobules at this time.

The blood supply of the sucking pad can best be studied at this period while the lobules are still separated by broad connect ive-ti.sj>ue sejita. The arterioles which supjily the body enter

Fig. 4 Krontnl section of tlio right rliook of a liutnnn fetus 17.5 cm. in total length. The upecimon was ittaincil with scarlet red and the coioroil fat droplets are represented in solid black in the drawing. C.c, capsule of corpus adiposum buccne; D.p., parotid duct; F.v., facial vein; C, molar glands; M.b., buccinator muscle; ^f./., facial muscles and fascia; M.m., massetcr muscle; O.m., oral mucous membrane.

its capsvile from all directions, ami end, after passing alonn tlic septa, by breaking up into capillary plexuses among the fat-cells of the Jobules. The veins of the body are much more conspicuous than the arteries. They arise in the lobules and pass into the septa where they unite anil finally form vessels of the third or fourth order. These vessels pass through the capsule and drain into the larger veins in the surrounding areolar tissue. Eventually most of the blood from the sucking pad is drained into the oi)hthalmofacial and facial veins. The vessels of the sucking pad are shown in figure 5 — a frontal section of the body of an injected fetus 1S7 mm. in total length.

The subsequent changes in the body to tiie time of birth consist mainly in the expansion of the individual fat lobules and the reduction in thickness of the septa which separate them. With these changes the blood-ve.ssels become much less prominent. The chronology of these later changes is subject to considerable variation, being apparently more dependent upon the nourishment of the fetus than upon its age. In some instances the fat lobules expand rapidly at an early period, so that at six months they are closely pressed against one another and are irregularly hexagonal or pentagonal in outline when seen in section. The connective-tissue septa in these cases are reduced to slender strands composed of flattened cells and fibers. In other cases this process may not take place until much later — sometimes not before the last month of fetal life. It is possible that the difficulty in suckling experienced by some premature and ill-developed infants may be due in part to the incomplete development of the sucking pad.

As has been stated, the formation of new fat lobules in the sucking pad generally ceases by the end of the fifth fetal month and the later growth of the body is due to the increase in the size of the lobules. At first this increase is brought about in part by the fonnation of new fat-cells at the periphery of the lobules and in j)art by the enlargement of the fat droplets already present. The formation of new fat-cells generally ceases in the seventh fetal month, and thereafter, as a rule, growth takes place only liy the enlargement of tlic fat -cells which are already formed.

Fin. 5 A frontal section of the suckinn; pad of a human fetus IS. 7 cm. iu total len|(th. The veins of the .specimen have been injected and are represented in solid black in the drawing. The fat-cells are represented by small circles.

Fig. 6 A portion of ;i transverse section of the left check of a human fetus 32 cm. in total length. The .section \va.M .stained with scarlet red and the colored fat droplets arc represented in soli) also thought that the body became smaller with age. Ballantyne ('91) agrees with Gehewe that the body persists in maturity, as also does Shattock ('09). The survival of the structure during the wasting diseases of infancj- is a conunon clinical observation and has been commented upon by Ranke ('84), Lehndorff ('07), and others. As has been stated, Allen Burns ('21) was apparently the first observer to figure the structure accurately in the adult. Modern treatises on adult

Fig. 7 Frontal section of a portion of the face of a very well-developed and nourished new-born infant weighing 4050 grams. C, corpus adiposum buccae; D.p., parotid duct and accessory parotid glands; M.b., buccinator muscle; M.p., platysma muscle and fascia; M.z., zygomatic muscle; V.}., facial vein. X 2J.

human anatomy usually give little or to description of the body and sometimes use the term corpus adiposum buccae for the general fat mass of the cheek and not for the sucking pad proper. However, the body is briefly descrilied in connection with the mouth by Jonnesco in Poirier and Charpy's Traits d'Anatomie and in detail by Eisler in Bardeleben's Handbuch.

Fig. 8 A section passing through the skin, superficial fascia, and outer part of the suckini! pad of a woll-devclopcd and nourished new-born infant weighing nearly 4000 grains.

The body in the adult may occupy the fossa bounilcd l)y the massetcr, the buccinator, the zygomatic, and the platysma and risorius muscles, or it may extend forward and outward over the external surface of the mas.seter. The parotid duct, as in the fetus and the infant, may either pass cranial to the body or may lie in a dcei) groove on its superficial surface. Figure 9 shows several sketches of the body in adult cadavers. Figure 9, C, is of an individual who died in an advanced stage of phthisis.

Frozen .sections of sucking pads of adults show practically the same structure as that seen in the infant.

PHVLOGENETIC Sir.NiriC.VNCE

It has been suggested that the corpus adiposum buccae of the higlicr Primates represents the framework of the orbital gland wliicli is so well developed in the Carnivora and of which the molar glands of man are a vestige. This view was first advanced, 1 think, by Latite-Dupont COO). In my opinion, neither the jihylogenetic studies of Forster on the sucking pad nor my observations on the development of this structure support this hypothesis.

Forstor finds that the sucking pad is a specialized portion of a fat mass which takes its origin from tlie extra-orl)ital fat body of the lemurs and which only secondarily enters the buccal region in the higher Primates. It is thus a body from another area

Fig. 9 Four sketches of dissections of the sucking pail in .idull c.idavers. C, corpus udiposum buccae.

which invades the region of the orl)ital gland and fills the space formerly occupied bj- that organ, but it is in no sen.se the remains of it.

The sucking pad, in its development, is built up aroimd a venous plexus and not around any element of the orbitoparotid gland comjjlex. The epithelial elements of this complex in man (the parotid duct, tho nrhitul inclusion, and the molar glands) do enter the area whicli I have termed the buccal space, but they do not enter the territory which is later to be incorporated in the sucking pad and they do not pierce the capsule of this structure after it is difTcrcntiated. In fact, the molar glands, which are considered to be the vestiges of the orbital gland, do not enter the buccal space at all until long after the sucking pad has been differentiated and a definite capsule has formeil around it.

In Carmalt's paper ('13) on the anatomy of the adult salivary glands in man the statement is made that "The molar glands, when i)resent, are for the most part embedded in the entomasseteric fat mass of the 'sucking pad.' " \Miile the.se glands lie in the loose adipose and areolar tissue of this region which fills the space between the corpus adiposum buccae and the muscles on either side of it, I have not observed them penetrating the capsule of the sucking pad proper, and the relation between them and the sucking pad is one of juxtaposition only. I think, therefore, that it maj- be safely concluded that while the sucking pad replaces the orbital gland in position in the higher Primates and in man, it is not to be regarded as a vestige of that structure.

SUMMARY

1 . The corpus adiposum buccae is a sharply circumscribed mass of fat lobules which are formed around the radicles of the middle part of the venous plexus which connects the orWtal veins with the superficial veins of the face. It is differentiated within a fairly well-marked area of the cheek which may be termed the buccal space.

2. The general region in which the sucking pad arises is mapped out in fetuses 4 or 5 cm. in total length and a definitely encapsulated area is well markeil in fetuses S to 10 cm. in length. Fat-cells appear at this stage or a little later. They are arranged in lobules which are first found in the periphery of the anterior part of the body.

3. The body prows rapidly after the encapsulated area has been established. Most of this early growth is due to the expansion of the enclosed mesenchymal and preadipose tissue and not to the growth of fat-cells.

4. The later growth of the body is due to an increase in its fat content. This is brought about: a) by the increase in the number of fat lobules; h) by the formation of new fat -cells, and c) by the growth of the individual fat-cells. The formation of fat lobules generally ceases by the end of the fifth fetal month. The formation of new fat -cells ceases at a variable time in later fetal life, generally in the sixth or seventh fetal month, but sometimes not before the last fetal month.

5. The finer structin-e of .the fully developed corpus adiposum buccae does not differ from that of ordinary superficial adipose tissue except that the interlol)ular septa are somewhat narrower and arc arranged radially in the body.

6. The body persists in adult life in the large majority of cases. The presence of the sucking pad in later life is apparently not dependent on nutrition, as it may be found well developed on one side of the face and absent on the other in the same individual. It is also foimd well develojiod in individuals dead of wasting disease.

7. Observations on the development of the sucking pad in man offer no sujiport to the theory that the body represents the remains of the orbital salivary gland. The sucking pad is developed quite independently of the parotid duct, the molar glands, and the orbital inclusion, and these structures never penetrate it. The molar glands do not approach the area in which the .sucking pad is formed until some time after that structure is well established.

nrBMOORAI'IlY

Maf.i.anty.nk. a. X. ISOl All inlroduc-tioii to the diHcases of infancy. Edin biirR. Beru, W . I!)ll f'bcr (lie Anlanf uiul KiilwickcliuiK des Fettgcwebcs bcim

Mcii.schen. ZcilHchr. f. Morphol. u. Antliropol., Bd. 13. lii( MAT, F. M. X. 1801 Anatomic g6n6rale, appIiquC'o a la phyHiologie et a la

mi'-decinc. Pari.s. BuR.N's, A. 1821 Observations on the chirurgical anatomy of the head and

neck. Edinburg. Car.malt, C. 1913 A contribution to the anatomy of the human adult salivary

kIuiuLs. Studies in cancer and allied subjcct.s conducted under the

Cicorue Crocker special research fund at Columbia University, vol. 4. ElSLER. P. li»12 Die Muskcln des Stammes. (Hd. 1, 2. Abt., 1. T.. Handb. d.

.\nat. d. Mcnschen, K. v. Bardeleben.) FoRSTER, A. 1904 t'ber die morphologische BedeutungdesWangenfettpfropfes.

Arch. f. .\nat. u. Ent. FuTAMrRA 1906 VbeT die Entwicklung dcr Facialismuskulatur des Menschcn.

Anat. Hefte, Bd. 30. Gehewe, W. 1852 De corpusculo quodam adiposo in hominum genis obvio.

Diss. Dorpat. GcRwiTscH, M. 1883 (*ber .\nastomosen zwischen den Gesichts- und Orbi talvenen. .\rch. f. Ophthalmol.. Bd. 24. Heister, L. 1732 Compendium anatomicum. Xorimbergae. JoNNESco, T. 1901 Tube digestif. (Poirier et Charpy. Trait6 d'Anatomte

Humaine, T. 3, Fa.sc. 1, 2. fid.) Lapite-Dupont 1900 La gland infra-orbitaire et la boule graisseuse de Bichat.

Bibl. Anat., T. 8. Lehndorpf, H. 1907 Uber das Wangenfettpolster der Sauglinge. Jahrb. f.

Kinderheilk.. Bd. 66. Ranke, 11. 1884 Kin .Saugpolstcr in dcr menschlichen Backe. .\rch. f. path.

Anat.. Bd. 97. RoBi.v, C, ET GiMBERT 1864 De la boule graisseuse de Hichat. Compt. Rend.

Soc. Biol.. Paris, T. 16. Sesemann, E. 1869 Die Orbitalvencn des Menschcn und ihrc Zusammcnhang

mit den obcrflachlichen Venen des Kopfcs. Arch. f. Anat. u. Physiol. Shattock. S. G. 1909 On normal tumour-like formations of fat in man and

the lower animals. Proc. Roy. Soc. Med., Rept. for Session 1908-1909,

Patholog. Sect. Verneuil 1857 Une nouvclle bourse sereuse de la face. Bull. Soc. .\nat. Paris,

Sc'r. 2, T. 2. WiNSLOW, J. B. 1753 Expositio anatomica structurae corporis humani. Fran cofurti et Lipsiae.

Resuniido por d autor, Frank Blair Hanson.

Orifioios neurales en la escdpula del cerdo. Una relaci6n peculiar

existente entre la rajna dorsal de varios nervios espinales

y la supraescdpula del cerdo.

Kn los pmbriones y adultos del cerdo existe una supraescdpula pornianonte, de gran taniano. Las rainas dorsales o ramas postcriores priniaria.s de los cuatro o cinco primeros nervios espinales pa.san a trav^s de los orificios neurales de la supraeseapula no osificada. Estos orificios neurales, que nunoa so han encontrado en otros manii'feros, existen constantemente en todas las cspecies de la familia Suidae, con la sola exeepci6n de los pecaris. La presencia de estos ner\nos en el cartflago estti producida por el desarrollo del precartilago, ciue crece sobre y alrededor de ellos antes de la aparici6n del cartflago. Los nervios mencionados no pa.san nunca por fuera de la escdpula en ningiin periodo de la vida del cerdo.

TralulBtion by Dr. Joe^F. Nonidci, Columbia University

ALTBOft'A ADOTIIA'T or TBW PiPER UWf CO UX TBR UlllLIOOH.tPllIC MEItVIC K, DECKMBCR 23

## NERVE FORAMINA IN THE PIG SCAPULA A PECULIAR RELATION EXISTING BETWEEN THE DORSALIS BRANCH OF SEVERAL SPINAL NERVES AND THE SUPRASCAPULA IN THE PIG

FRANK BLAIR HANSQN Department of Zoology, Washington University

TWENTY-ONE FIGURES

T. INTRODUCTION

Several j'^ears ago Prof. J. Sterling Kingsley ('00), in studj'ing •sagittal sections of a pig embryo, found the scapula in several sections to be completely segmented into five parts. Upon giving the matter closer attention, it was determined that four nerves passed through the cartilaginous suprascapula and that in this particular section the foramina of all four nerves had been cut through as shown in figure 1.

This fact was noted in a paper read before the American Morjihological Society of New Haven, December 27, 1S99. The following (luotation from Professor Kingsley 's paper is a condensed report of the main findings of that paper as published in Science, N. S., vol. 11, p. 167, and is, so far as I am aware, the only reference to be found in the literature bearing upon this matter.

In pml)iyo pigs 18 to GO mm. limg, the dorsal crest of tlie scapula presents four foramina throunh wliiih pa.ss dorsal ner\'es, arising from tlie second to fifth tlioracic ganglia, and passing directly to the skin. Thi se were regiirded as possibly iniiicating tiuit the scapula was made u)) of metamerie parts, and it was pointed out that these results were in full accord with the recent studies of Hoik upon the uuiscles of the shoulder girdle. They might lie interpreted as adverse to (lOgenhaur's views ,is to the origin of the girdles.

»9

2W FRANK BLAIR HANSON

I'pon the suggestion of I'rofessor Kiugsley, the author of this paner undertook to deteniiine several points in regard to the history of these nerves. In the first place, which nerves are lh(>y: second, how do they got into the cartilage, and, third liow do they get out again? It was also the author's purpose to discover if a similar condition he general throughout the nianinialian series.

Concerning this last point all the evidence is negative. I have looked through series of serial sections, dissected embryos, f)r examined the skeletons of the opossum, mouse, rabbit, seal, l)at, manatee, cat, sheep, and man. These forms represent most of the major groups of mammals, but in none is there any indication of the condition as found in the pig and described in the following pages. In his monograph on the shoulder-girdle, Parker ('68) pictures the scapulae of representatives of all the orders of mammals, many of them with a well-developed cartilaginous suprascapula. But in no case throughout the whole series is there anj' liint of the passage of nerves through this region. However, Parker does not give any figures of the pig scapula; his onh' reference to this form being three drawings of the pig sternum. Had the condition as in the pig obtained in other mammals he could hardly have failed to notice it, as in all the larger pig embryos .the foramina are plainly visible to the naked eye.

Flower ( '85) gives a figure of the scapula of the red deer with a large suprascapula, also descriptions of the scapula in the horse, hippopotamus, tapir, hyrax, and elephant among the I'ngidates. He makes no mention of nerve foramina, yet could hardly have handled these bones without seeing them, had they been present..

Since the last two paragraphs were written the author has had tlie privilege of examining .several huntlretl skeletons of mammals in the U. S. National Museum at Washington, D. C. It was observed that I'ngulates in general are possessed of a suprascapula in the adult condition, sometimes but feebly developed. However, of all the skeletons rexiewcd in tiie nuiseuni, there was none out.side the familv Suidae with nerve foramina. Of

NERVE FORAMINA IN THE PIG SCAPULA 291

the Reneni of the Suichie the following were examined: Tayassu of Honduras, Sus barbatus of Borneo, 8us barbatus of West Borneo, Babirussa of Celebes, and the peccaries. The peccaries alone of all the genera above mentioned were lacking in respect to nerve foramina in the suprascapula. Taj'assu, Babirussa, and Sus barbatus are so essentially like the domestic pig in this respect that a separate description is unnecessarj'. In view of the foregoing facts, this author is prepared to say with considerable emphasis that nerve ff)ramina in the scapula are limited to the family Suidae, the peccaries alone excepteil.

While these nerves were not found i)assing through the scapula in other mammals, even in so closely related forms as the sheep and the peccary, they were always present in the pig. More than fifty pigs have been examined, ranging in size from an embryo 18 mm. in length to an old hog weighing 4.50 pounds. Nerve exits through the suprascapula, varying in number from two to five, were found in every specimen studied. The author believes this to be a normal and constant condition in the pig, but apparently lacking in the other groups of mammals.

My thanks are due to Prof. J. Sterling Kingsley for the initial suggestion; to the authorities of the U. S. National Museum for supplying valuable material, and to Miss Bertha Uhlemeyer for assistance with the recon.>;t ructions and drawings.

11, MA'IKKIAI, AND METHODS

Pig embryos of 18 mm. in length to birth, scapulae from pigs of a few days after birth to young atlult life, and one scapula from an over-sized hog of 450 pounds weight were used in this work. The smaller embryos were cut in transverse sections, camera-lucida drawings were made and the parts reconstructed in wax. For the larger embryos and postnatal specimens it was possible by gross dissection to trace the nerves directly from the spinal cord through the foramina in the suprascapula and to their distribution in the skin of the scapular region. Figure 3 is such a dissection, the specimen being approximately one week old. Figure 2 is a reconstruction in wax of the spinal cord,

292 FRANK BLAIR HANSON

spinal nerves, and the upper part of the scapula of an embryo 37 nun. in Icnuth.

III. IDEXTIF-ICATION OF THE NERVES

In several i>f the series of sections the nerves could he traced from the foramina in the suprascainihi back to the spinal cord. Figures 2 and 3 indicate clearly that the nerves are the dorsalis or primary posterior branches of spinal nerves.

I'igiire 2 is from a reconstruction in wax of a 37-mm. embryo indicating the structures under discussion. The two nerves, after passing through their scapular openings, divide and ramify over the skin of this region. From a study of the series of sections from which figure 2 was made and also from the gross dissection of nerves as shown in figure 3, it is demonstrated that these are the internal or cutaneous branches of spinal nerves, using the nomenclature of the human anatomists.

In determining which spinal nerves were affected, the method of human anatomy (Cunningham, '15) was adopted, i.e., of counting the first nerve behind the first true rib us the first thoracic sjiinal nerve. In the model from which figure 2 was drawn there are two nerves passing through the cartilage, and it was determined by the above-described method that these are the third and fourth thoracic spinal nerves. In the series of a 47-mm. pig (fig. 4) there are found three nerves going through the suprascapula at approximately the same level. These are the second, the third, and the fourth spinal nerves. In a 72-mm. eml)ryo there were four nerves in the cartilage, the fourth probably being, though not positively identified, the first spinal nerve. M least in other specimens, figure 3 being an example, the first spinal nerve seems to take this course. Kiugsley in the quotation given above says the nerves arise from the second to the fifth ganglia.

It .should bo noted here that in successive stages of growth there is an increasing numlx-r of n('r\'es present in the cartilage of the scapula. Thus in 25-mm. and 37-nun. embryos there are two of these nerves, the third and fourth spinals as shown

NERVE FORAMINA IN THE PIG SCAPULA 293

in fipincs 2, 4, and 5; while in a 47-mm. pig there are three, and in the 72-nun. embryos four are present.

Figures 4 and 5 are drawings from wax models of 27-mm; and 47-nim. pigs, respectively, and show the position of the foramina in the future suprascapula. .Vs these two figures are drawn to the same scale, they show well the interstitial growth carrying the nerves with it. Figure 5 is interesting as two nerves are firmly embedded in cartilage, while a third is just at the edge of the i)rocartilaginous or growing end of the scapula. Figure 6 is a camera-lucida drawing from a transverse section of the 37-mm. stage showing the passage of one of the nerves through the cartilage of the shoulder-blade.

It shf)uld be stated, however, that the progressive increase in the number of nerves paralleling the growth of the embryo as noted above does not hold true for the series of nine young and adult scapulae represented bj- figures 13 to 21, inclusive. These show great variation in the number and position of the nerve openings, and will be described in some detail later on.

IV. ENTRANCE OF THE NERVES INTO THE SCAPULA

The question as to how these nerves came to be in the suprascapula is easily answered. It is a well-known fact that the nervous system is one of the earliest to be laid down in the embryo. In an 18-mm. pig the scapula is still in the precartilage stage, while the central and jieripheral nervous system is a well-established structure. Figure 7 is a camera drawing of a transverse section through an 18-mm. stage of the pig. The nervous system is here seen to be in an advanced stage, while the patch of loose mesenchymatous material is the anlage of the future scapula. In this figure is also seen a spinal nerve in close proximity to the procartilaginous scapular anlage. Figures S and '.) are designed to show how the jirocartilage in its growth .surrouiuls and invests the nerves which lie in its path.

In figure 8 there is shown a transverse section of a 2o-mm. eniliryo in which one end of the scapula is clearly cartilaginous, while at the other end the procartilage may be seen forming

2*.»4 FRANK BLAIR HANSON

around a sjjinal iutvo. As tlic scapula grows it gradually includes additional nerves, thus explaining why the dififerent stages have a varying nuiuher of nerves, from one in the smallest embryo to five in the full-term fetus. Figure 9 is a camera drawing from a transverse section showing the scapula, and, lying just above it and bent around it in a \'-shaped manner, one of tlie dorsalis l^ranches of a spinal norvo. In a little later stage of development a patch of procartilage will appear dorsal to the nerve as in figure 8, and the gradual union of the two pieces of developing cartilage around the nerve will result in a nerve foramen. Figure 10 is a drawing of the scapula of a sheep embryo .5 inches in length, and is introduced here, as is also figure 11, to show how essentialh- similar these scapulae of other I'ngulates are to that of the pig, except in this one particular, that the scapula is never at any time perforated by spinal nerves.

V. POST-NATAL SCAPULAE

From general knowledge and considerations our first idea was that this could onlj' be a transient phase in the embryonic development. Therefore, one of the iirohlems set for solution was concerning the manner of the exit of these nerves from the scapula.

As alreaily stated, the work was begun upon a 2.5-mm. embryo in which two nerves pa.ss through the scapula. From this a study was made of embryos of increasing size until the full-term fetus was reached. However, there was no 'transient phase,' but, much to our surprise, a steady increase from one nerve in the .smallest embrj'o to hve in the fetus just prior to birth.

This carried the problem over from prenatal to postnatal life. For this part of the work we were fortunate enough to secure a series of pig scapulae from animals ranging in age from a few days to that of the before-mentioned over-sized hog. The exact ages of some of these scapulae are unknown, but figures 13 and 14 are from young pigs of the same litter and are about one week old. Figure 20 is from an annual ten months old, and figure 21 is that of the old boar.

NERVE FORAMINA IN THE PIG SCAPULA 295

This series of figures (13 to 21) show only the suprascapular region, and are taken from a series of drawings of the entire scapulae, which are being incorporated into an investigation now under way on the development of the scapula, coracoid, ami sternum in the pig, the mouse, and other mammal*, to which paper this present one might be said to be a foot-note.

Figures 13 to 21 .show conclusively that there is no exit of nerves from the suprascapula at any time in the life historj'. The suprascai)ula of an old hog (fig. 21) shows no indication of ossification and maintains the same relative proportion to the scapula as obtains in the smallest specimens examined. The presence of these nerves is correlated directly with the existence of the cartilaginous .supra.scapula as a permanent structure. In forms lacking a suprascapula the spinal nerves of the scapular region nin in a dorsal direction on the inner side of the scapula until they gain its upper l)order, then, turning laterally over the edge of the bone, they descend ventrally on its outer surface a short distance, and then ramify out into their muscular and cutaneous branches.

In the pig we could obtain this same condition by removing the suprascapula. If this were done it would be seen that the nerves are perfectly regular and normal and subscribe exactly to those described just above for animals without the su])rascapula. The unusual relation here between scapula and spinal nerves is due primarily not to any shifting or abnormality in the nerves themselves, but rather to the upbuilding of a large and permanent cartilaginous suprascapula arountl ami above them.

But little more need be said concerning figures 13 to 21. That they show considerable variation in the position and number of foramina is apparent at once. This latter point is easily cleared up by referring back to figures 2 and 3. Figure 3 was obtained by gross dissc'ction of a pig one week old and shows the spinal nerve dividing some distance before it reaches the suprascapula. Figure 2 shows two spinal nerves passing in an undivided condition through the cartilage, but separating into two branches each immediatelv bevond. The niimber of

290 FRANK BLAIR HANSON

foramina in any specimen, then, depends upon two things: first, the nmnber of spinal nerves involved, and, second, whether they l)ranrh before or after passing through the foramina.

Figure 1.') with its Hve nerve exits may be interpreted as having bnyiches of three spinal nerves in the cartilage. One of these, however, divided into three rami at a point medial to the scapula. The procartilage was then laid down around them, giving us. with the other two undivided nerves, the number five, that we would expect. This contention is also borne out by the relative sizes of the foramina; there are three small ones for the branching spinal nerve and two large ones for the undivided nerves. The number five is met with twice in the material at hand, the other being in a fetus, and is the largest number observed. From this, as the figures show, we have all numbers down to one.

VI. FOS.^ILS

In addition to searching for these nerves among the contemporary relatives of the pig, it occurred to me that an examination of fossil remains might prove instructive and would open up the possibility of establishing another link in the phylogenetic relationships of the mammals. To this end a careful examination was made of books containing plates of fossil mammals. These included, first of all, that monumental work of C'oj)c on the "Fossil Manuuals of the Tertiary.'" .\lso such texts as Osbom's "Age of Mammals" and others were gone through. In none of them, however, was there any indication of nerve foramina. But this is perhaps, after all, npt strange, for the nerve openings, when present, are always in cartilage, and this would not likely be preserved with the bonj* scapula.

VII. CONCLUSIONS

The observations made in the course of this investigation seem to justify the following conclusions:

1. These nerve foramina are not present in other manuuals.

2. They arc always found in the domestic pig, also in all other genera of the Suidae, the peccaries alone excepted.

NERVE FORAMINA I\ THE PIG SCAPULA 297

3. The number of nerves differs, due to variation in branching and also to the size of the embryo.

4. The dorsaUs or posterior priniary branches of thefirst four or five spinal nerves are the ones involved.

5. The nerves are normal in their course and directly comparable to the same nerves in other mammals after the removal of the suprascapula.

6. The presence of the nerves in the suprascapula is brought about by the developing procartilage, which envelops the nerves.

7. The nerves ne\er pass out of the scapula at any time in the life history of the pig.

8. The suprascapula in the pig never ossifies, but maintains the same relative proportion to the scapula throughout life.

LITER.\TURE CITED

Cope, E. D. 1884 The Vertebrata of the Tertiary formations of the West.

Vol. 3. Report U. S. Geological Survey of the Territories. CnNNiiJGH.\M, D. J. 191.5 Te.xt-book of human anatomy. Edinburgh. Flower, \V. H. 1885 Osteology of the Mammalia. London. Ki.vosLEV, J. S. 1900 The foramina of the scapula. Science, X. S., vol. 11,

p. 167. Parker, W. K. 1868 The shoulder-girdle and sternum. London. Ray

Society.

niC AN.tTOUICAL RKCORO, VOL. IS, NO. 6

PLATE 1

EXPLANATION OF FIGURES

1 Sagittal section of pig embryo. All four foramina cut through in same seotion.

2 From a wax model of a 37-mm. pig. Only upper part of scapula is shown. These are the third and fourth spinal nerves.

3 Gross dissection of a pip one week old. This is the first spinal nerve, and its doraalis branch could be traced distinctly from the ganglion, on through the Buprascapula, and to its distribution in the skin.

4 Upper and posterior end of wax model of 47-nim. jjig.

5 Supra.scapular part of wax model of scapula of :J7-mra. pig. Two foramina are entirely formed in the cartilage, while the procartilage is enclosing a third spinal nerve. This should be compared with figure 4. a larger embryo, where the third nerve is now surrounded by cartilage.

anpn, anterior branch spinal nerve sfc, skin

cr, coraooid process spc, spinal cord

dspn, dorsalis branch spinal nerve spg, spinal ganglion

gl}, glenoid fossa spn, spinal nerve

n/, nerve foramina ' ssc, suprascapula

sr. scapula

208

NERVE FOKAMINA INTHE PIG 8CAPDLA

rXANK Dl. \Ilt IfAN'HON'

PLATE I

SPN'

PLATE 2

EXPLANATION OF FIOrRES

6 Transverse section through suprascapula of 37-mm. pig, showing passage of nerve through foramen.

7 Camera drawing of transverse section of IS-nim. pig. Scapula is entirely procartilaginous, and in its growth is about to enclose a spinal nerve.

8 This is a transverse section through suprascapular region of a 35-mm. pig, showing cartilage at one end and precartilage at the other. It demonstrates very well how these nerves are surrounded \>\ the growing cartilage.

9 Another transverse section of a 27-nim. pig to show relation of nerve and suprascapula.

10 Scapula of sheep embryo 5 inches long. Dotted line indicates what will be upper limit of bony scapula.

r, cartilage pre, procartilage

cl, centrum spn, spinal nerve

nich, notochord ssc, suprascapula

3C0

NERVE FORAMINA IN THE PIG SCAPULA

FRANK OLAIlt IIANHON

PLATE 2

S PN

N T CH

CT

SPN

PRC

ssc->

301

PLATE 3

EXPLANATION OF FIGtTRES

11 Scapula of red deer showing well-developed suprascapula, but without foramina. Modified after Flower.

12 Oblique section of 27-mni. pig with foramen partly cut through.

13 Fetus approaching full term. Cartilage extends as a continuous band from suprascapula along upper margin of spine to its highest point. Three nerves pass through the cartilage in this specimen, which are undivided as in figure 2.

14 Full-term fetus showing four foramina. It is probable that these are two branches each of two spinal nerves, which divided on the medial side of the scapula. See also figure 3.

15 Pig one week old. Has five foramina, the largest number found. The three small ones in a cluster represent the rami of one spinal nerve.

16 Same age as figure 15, but is quite different as to the arrangement and size of foramina.

17 Pig several months old. This and figure 18 show the only two postnatal speeimen.s with but two foramina.

18 Older than in figure 17. Same number of foramina, but a difference in position.

msc, mesoscapula (spine) sc, scapula

n/, nerve foramina spn, spinal nerve

psc, prescapula ssc, suprascapula

302

NERVK FORAMINA IN THE PIG SC-APLLA

FRANK BI.AIH IIANIIOM

PLATE 3

nr

_ . , NF

303

PLATE 4

EXPLANATION OF FIGURES

19 Pig Hearing young adult life. Three large foramina, indicating undivided spinal nerves.

'JO Youngadult pig between ten months and one year old, with three foramina. 21 This .scapula is from an old boar (age unknown) which weighed 450 pounds. The suprascapula is still cartilaginous and three nerve openings are present. nf, nerve foramina ssc, suprascapula

sc, scapula

304

NERVE FORAMINA IN THE PIG SCAPULA

KIIANK ULAIH HANSON

PLATE t

NF

Kesumido por la autora. Florence May Alsop.

Kl efecto de las tempemturus anormales sobre el desarroUo del sistenia nervioso del enibri6n de gallina.

TnnaUlion by Dr. Joa^ F. Nonida, Columbia UDiveraity

ArrnoR s AiiarnAfT or tub papek imvzo ur

TUB BIULIOOHAPHIC HKHVICE. OltrEUDBH Zl

THE EFFECT OF ABNORMAL TEMPERATURES UPON

THE DEVELOPIXr, XERVOI'S SYSTEM IX

THE CHICK EMBRYOS'

FLORENCE MAY ALSOP Kansas State Agricultural College, Manhattan, Kansas

THIRTEEN FIGURES

CONTEXTS

I. Introduction ,307

1 1 . Discussion of literature 308

A. Methods of producing abnormalities in animals 308

1 . Abnormal heat '. 308

2. Hybridization 308

3. Effect of clicmieals 308

4. Centrifugal force 309

III. Causes of abnormalities in eggs 309

A. Abnormal ovary 309

B. Abnormal oviduct 309

C. Abnormal ovary and oviduct 310

IV. Determination of the age of chick embryos 310

A. Number of somites 310

B. Size of embryo 311

C. I^ength of time of incubation 312

V. Method of incubation 312

VI. Method of killing and fixing 313

VII. Abnormalities produced by heat 314

A. Effect of low temperatures upon development 314

B. Effect of high temperatures upon development 317

\I 1 1. Results 319

IX. Conclusions 321

Bibliography 322

Explanation of plates 326

I. INTRODUCTION

The \v(irk (lc.-<orihe(l in this paper \va.s taken up as a result of finding abnormal chick embryos among the slides used in the

'Contribution from the Zoological Laboratory, Kansas State .Agricultural College.

307

30S FLORENCE MAY ALSOP

oiiibrvology lalxiratoiy classes.^ Although the cause of these almonnal euil>rvos was not known, yet they suggested the possibility of trying to protkiee abnormal embryos by exposing the eggs to various degrees of tonipcraturc. In the disrussion which follows the writer has shown liuit an abnormal amount of heat may cause deformities in the nervous system of chick embrj-os and also that difforent kinds of abnormalities were produced by apparently the same condition as well as by different conditions.

II. DISCUSSION OF LITER.\TURE

Abnormalities in developing embryos have been produced in many ways. Some scientists have used an abnormal amount of heat, others ha\T produced abnormalities l)y ilifforent chemicals, while still others have produced abnormal embryos by hybridization and centrifugal force.

.1. Methods of producing abnormalities in animals

1. Abnormal heal. Among those who produced abnormalities by temperature variations is Cireeley ('01), who found that Ijy lowering the temperature of stentor certain well-defined structural changes took place which were not necessarily incidental to tlie permanent suspension of the vital functions of the cell.

King ('03) also has shown that excessive heat causes abnormalities in the toads' eggs and hastens development.

i. Hi/bridization. Loeb ('15) used low temperatures and heterogenous hybridization to produce blind fish embryos. Immediately after fertilization he put the eggs into a temperature of 0' to 2°C. and produced from 20 to 30 per cent abnormal embryos.

Newman ('17) al.so produced monsters through hybridization.

S. Effect of chemicals. Fere ('99) used the fumes of alcohol and ether and obtained many abnormal embryos. In an incu ' I wish to express my indebtedness and appreciation to Dr. Mary T. Ilarnian for the use of the slides colleeted l)y her and also for her criticism of this paper. I al.so dive acknowledgement to Dr. U. K. Naboura for his interest and cncouraKement in Ihis work.

ABNORMAL TEMPERATURES ON CHICK EMBRYOS 3()!»

bator set over the ventilator from the chciiiistrj' laboratory the eggs contained many abnormal chicks due to the fumes of chemicals.

Koese f'12) used narcotics as an agent in producing abnormalities in the development of hens' eggs.

4. Centrifugal force. Centrifugal force is another factor employed by some to produce abnormalities. Ranta and Clortner ('14) produced accessory appendages in the amphibian larvae through the action of centrifugal force. Also Conklin Cll) describes abnormal results obtained by the action of centrifugal force upf)n the organization and development of the eggs of fresh-water pulmonates.

III. CAUSES OF ABNORMALITIES IN EGGS

Eggs that are abnormal at the time of lajnng probably play an important part in the development of abnormal embryos, and a certain per cent of abnormal chicks found under apparently normal external conditions are probably due to conditions through which the egg passed before it was laid. These abnormalities in the eggs may be caused by several factors, according to Parker COG).

A. Abnormal ovary

The ovary may be abnormal or diseased. .\n injury may cause a breaking away of the egg from its follicle before it has ripened.

B. Abnormal oindiict

\n abnormal oviduct may retain an egg until the second yolk is broken away from the follicle. In this case both yolks are soinctiinos encasetl in one shell. Schimiacher ('%) explains this condition by an antiperistalsis in the oviduct. The egg retained in the oviduct has developed more than is normal at the time of laying, and the cooling of an egg at the time of the formation of the neural plates produces a large per cent of abnormalities, as will be iliscussed later.

310 FLORENCE MAY ALSOP

C. Abnormal ovary and oviduct

An al)ii()rmal ovary and nviduot are sometimes present and may produce al)normal eggs.

Eycleshymer ('()7) by careful calculations has found that the t<>iiipcrature of the hen during the first week of incubation is from three to four degrees iiigher than- that of the eggs under her, and if an egg is retained in an environment of 102° to 104°F. when the temperature of the eggs under a hen is found to be 98° to 1()0°F. it is very prol^able that the four degi-ees of extra heat will cause abnormal development. Especially was this fovmd to be true in the first twentj'-four hours of development of those eggs used in this experiment. The embryo was very su.'<ceptible to a change of a few degrees of temperature at the time when the neural tube was closing, and more so at the time when the nervous system was beginning to form.

The physiological zero, according to Lillie ('08), is that temperature below which the blastoderm undergoes no development whatever. It has been shown by Edwards ('02) that a small amount of development will take place at 21°C., and therefore he places the physiological zero between 20° and 21°C., although 28°C. is accepted by many authors as the degree of temperature below which no development takes place. Therefore, if 20°C". be accepted as the physiological zero, abnormal development or growth may be caused by variations in the time of development in the hen, or by development that may take place in warm weather after the egg is laid and before it is placed under incubation.

IV. DETERMINATION OF THE AGE OF CHICK EMBRYOS

A. Number of somites

The age of chick embryos is generally determined bj' the number of somites found in the earlj' stages of development. This method is used l)y Lillie and others up to the end of the fourth or sixth day of incubation, but after this time the number of somites doe.s not increase, and embryos are classified according to length.

ABNORMAL TEMPERATURES ON CHICK EMBRYOS 311

B. Size of embnjo

Thoro wcro many difFir-ultics invf)lved in trying to dcterniine the ago <tf tlie cnihrjos b\- the greatest rervieal-caudal length. The cranial and cervical flexures appeared at dififerent stages of development in the embryos which had been incubated for the same length of time. An egg kept in the incubator for twenty-four hours under high temperatures often developed both head flexures, while another left in the incubator fortyeight liours in temperatures Ix-low normal had in a large per cent of instances formed onh' the cranial flexure. In a small per cent of those eggs that had been incubated for forty-eight hours below 102°F. no flexure was formed. But in these embryos the tissue appeared to be dead, as it did not stain properly.

.\bnormal flexures were present in many of the embrj'os. Probaiily the most noticeable was a bending backwards of the trunk, forming a convexity on the ventral side of the chick, . which is the exact reverse of the normal development. Even the tail bud in a small number of these abnormal flexures was turned dorsally instead of ventrally.

.Vnother condition which made it impossible to determine the age of the chicks by body length was the variation in size of the embryos which were otherwise equally developed, i.e., the numlier of somites was the same, the flexures were e(iually developed, and all had been kept in the incubator under the same degree of heat and during the same length of time. Still some were found to be 50 per cent or more larger than others. This might have been due to heredity, as it is found frequently in eggs that iiave been incubated in normal temperatures.

Another great hindrance to cla.ssifying the age of embryos in this experiment by either of these methods was the partial development of certain parts, for example, only one nerual plate tieveloped in some embryos, and this development in most instances was abnormal. One side of the brain developed more than the other in a few of the embryos, while in others no brain was formeil.

.'UJ FLORENCE MAY ALSOP

( '. Length of time of incubalioyi

The length of time of incubation is not generally used as a l)asis of classification because under the best of conditions the variations in development is sufficient to prevent close grading. Jut in this paper the length of time of incubation is the only method employed to classify the age of the embryos. The •omite method cannot be applied because many of the chicks do not develop somites at all. Tliis was found to be true of those eggs which were incubated at .low temperatures. A few embryos showed no somites after having been in the incubator twent^'-eight hours. On the contrary, a few embryos developed accessor}' somites. A second row was formed lateral to and alternating with those somites of each of the first two rows of .'^omites.

These conditions all made it difficult to use any method in determining the age of the embryos produced under abnormal temperatures. Therefore, in speaking of a twenty-four- or forty-eight-hour chick in this work the writer is referring to an' embryo obtained from an egg wliich has been in the incubator twenty-four or forty-eight hours, respectively, regardless of its degree of development.

V. METHOD OF INCUBATION

The eggs used were obtained from the Poultry Department of the Kansas State Agricultural College. Each egg was marked with the number of the hen that laid it. Also the number of her mate was recorded. These records were kept in case the history of a particular chick was needed to help solve or verify certain deformities that might appear in some of the specimens, and to determine whether or not the cause of certain tj'pes of abnormalities wa.s in the parent stock or was caused by the method of incubation. Such data were not necessary, howe\'er, because the large per cent of abTiormalities was in all probability due to tlif method of incubating tlie eggs.

Three hundred and three eggs were incubated for this experiment. This does not include those abnormal embryos collected

ABNORMAL TEMPERATURES ON CHICK EMnRYOS 313

by Dortdr Iliirman nor those incuhatod ami used as a control or check upon the results (thtained here.

Mr. Hoyd, a student in the college, incubated one hundred and eighty-six eggs under normal conditions. He was making eml)ryology slides for the Zoolog\' Department, and the eggs used in his work were incubated at the same time and under the same conditions, with the exception of temperature, as those used in my work. Data were kept upon the abnormal chicks fouml in these eggs which were incubated at normal heat, and these results serx-ed as a control and were compared with those obtained by running the eggs at abnormal temperatures.

The eggs were all incubated in a wooden incubator, which was ciuite satisfactory, as the wooden frame was not susceptible, to any great extent, to a change in the temperature of the atmosphere.

The heat was furnished by a kerosene lamp. It was easily kept under control, x'arjing not more than a degree from what wa.s desired.

The incubators were kept in the ba.sement of the incubating building of the poultry farm of the college. The temperature here was as cool and constant as it could be found at this time of the year, which was during the months of .June and July.

The eggs were collected e\ery hour of the day. The date and hour of laying were marked upon each shell. In no instance had the eggs been laid more than twentj'-four hours before they were put into the incubator.

No eggs were incubated longer (luin three days, and therefore this would limit the nervous system to the early embryonic brain and neural tube. The deformities described are those found only in the brain and neural tube.

VI. MKTIIOD ol- KILLING AND FIXING

The embryos wctc killed and partly fixed in the egg shell. One side of the shell was cut away; the albumen was poured out and a few drops of 10 per cent nitric acid was poured upon the embryo to kill it and partially fix it. The specimen was then cut away from the blastoderm, taken out of the shell, and the

THE ANATOMK At KKCORD, VOL 15 .NO. d

^]A FLORENCE MAY ALSOP

vitrllinn inoinhrano and adhering yolk matorial were washed off with water. Tlie embryos were now transferred to Houin's fhiiil for further fixation, .\fter one or two hours the embryos were phieed in 70 per cent alcohol, which was changed several times during each of the next two or three days. Finallj', when the picric acid was removed, the specimens for whole mounts wore stained in alum carmine and mounted in balsam. Some of these embryos after being examined as whole mounts were dissolved off the slides, embedded in paraffin, and sectioned.

All of those embryos incubated for seventy-two hours were stained in borax carmine for .sectioning. These were cleared in zylol and mounted in balsam.

VII. ABNORMALITIES PUODUCED BY HEAT

The discussion which follows takes up the different kinds of abnormalities and the per cent of abnormalities which were produced in the nervous system of chick embryos. Abnormal temperatures were the onlj'^ factor taken into consideration in producing these results.

^4. The effect of low temperatures upon development

Different variations of temperature were applied to the eggs with different results. Low temperatures were employed first. By low temperatures is meant any degree of heat lietween n4°F. and 1()2°F. (102° to 104°F. is the temperature generally considered as normal in incubating eggs.) No eggs were incubated below 94°F., because the per cent of embryos that died increased, and it did not prove satisfactory to run the incubator at a temperature below 94°F.

Edwards ('02) describes a condition observed by Warj'nski in which the embryo is made abnormal as a result of low temperature. He says,

The yolk when cooled rises and presses the hlastoderiu aniiinst tlie vit«'lline nieinhrane, to which it sticks. If this happens during the first two clays, while the embrj'o is unprotected by the amnion, the pressure cau.ses an arrest of devclopineiit and consequent nialfonna

ABNORMAL TEMPERATURES ON CHICK EMBRYOS 315

tions. Tlic cxatt charactor of those, and tlic n-Kion of the cinbn'o in whii-h they occur, cannot he predicted, since it is a matter of chance as to the part (jf tlie hhvstoderni which will adhere to the vitelline nienibrune.

The above condition was not found in any of the embryos produced in this experiment. The vitelline membrane washed off easily with no part of the blastoderm adhering to it.

The abnormality which occurred most frocjuently as a result of incubatirif^ eggs at low temperatures was the lack of folding in of the neunil plate into the neural tube for some distance above the liriniilivc knot. The neural folds in the anterior region generally formed, although in some cases abnormally, and these folds extended posteriorly about as far as the first somites. This cfindition was very noticeable in those embryos which had been incubated only twenty-four hours or less (figs. 1 and 2). Also below the region of the primitive knot the plate failed to develop for a short distance. But posterior to this place the folds, in most instances, developed normally.

.\t the primitive knot not only was the tube formed, but extra thickenings of the walls and extra cells, apparently of ectodermal origin, nearly filled the central canal in some embryos and clo.-ing it completely in others. In still other specimens the extra tissue filled the canal in such a way that two or three neural canals were found in one neural tube. This condition was best studied in the transverse sections of the forty-eight- to seventy-two-hour chicks (figs. 11 and 12), although it was easily traced developing in the eighteen-hour chicks. The abnormality appeared here as a mass of cells developing in the region of the primitive knot. It stained more heavily than the suiToundin'g cells and was easilj' followed through the series of older embryos.

In the twent\'-four-hour specimens two distinct folils were beginning to form on either side of the primitive node (fig. 1). The cells at this point were apparently able to resist the lack of heat and proceetled with development, while the cells of the neural i)late for a ilistance anterior aiul posterior to this region di(.l not nmltiply so rapiilly, and conscciucutly the neural folds

316 FLORENCE MAY ALSOP

here were not so far developed as those in the rcpion <if the primitive node.

In the tliirty-six-hour chicks tlic iicunil tuhc had, in most of the embryos, (frown together nearly tlu> entire U'ngth of the tube. Hut that part of the neural fold in whieh development first started stained darker tlian that anterior or posteri(»r to it, showing that the tissues were thicker and that more cells had l)een produced in this part of the neural tube than elsewhere.

The abnormalities in the forty-eight-hour chicks did not show .so distinctly as in the embryos that had been incubated a shorter length of time. Of the fifty specimens examined as whole mounts, thirty-seven appeared normal. Twelve of these .seemingly normal chicks were embedded in paraffin and sectioned. The result showed eleven abnormal and one normal tube. The central canal in all eleven was either closed or partly closed with the thickening of the tube wall. The abnormality in all cases was more distinctly shown in the lumbar region than elsewhere. The canal in some specimens contained loose tissue the entire length of the tube below the hind brain.

Of the thirty embryos incubated seventy-two hours, all but one seemed normal. .\11 of the twenty-nine sectioned transversely. One was destroyed in the process of cutting. Of the remaining twenty-eight, twenty-three had developed an abnornud neural tidje, or nearly S3 per cent were almormal. This jier cent would have been larger, 1 think, if those embryos which had died before the seventy-two hour stage of development was reached could have been included. Nearly 23 per cent were unable to resist the low temperature or were too almormal to live three days in a temjKTature which ranged from four to seven degrees below that which is found in the natural" incubation of the hen.

Another abnormality jiroduced by low temperature was the curved j)rimitive streak (fig. 3). This was seen in the early stages of growth and developed into a tube that was abnormal in curvature (fig. 4). In the majority of these embryos the posterior third of the tube turned off to the left side instead of extending in a straight line with the anterior two thirds. This

ABNORMAL TEMPERATURES ON CHICK EMBRYOS .'5 1 7

condition was found more often in the embryo helow the age of twenty-eiKht hours than in the older chicks; although it was found in one forty-seven-hour specimen which was no further developed than a nomial twenty-four-hour chick should he (fig. 4).

A third condition which occurred in tlie development of emhryos under low teniperatines was that of one neural plate forming into a neural fold l)efore the other began development (fig. 7). This lack of development of the neural fold appeared more often in the younger embryos than in tliose older. The other neural plate finally developed after a longer period of incubation, for in but a small number of forty-eight-hour chicks did one neural fold show a development in advance of the other.

/?. The effect of high feniperntiire upon development

Embyros incubated with excessive heat developed different kinds of deformities than those described above. The amount of heat used here varied between UH" to 1()8°F. No eggs were incubated at a higher temperature than 108°F. The excessive heat at this point caused nearly 25 per cent to die before the end of the period of incubation.

A large per cent of the abnormalities appeared in the brain region. The most conspicuous of these was a constriction of the neural tube below the optic vesicles. The extra amount of heat seemed to attect the rate of growth of some i)arts of the brain region more than other parts. The optic vesicles and mi(il)rain region developed more rapidly than that ]iart of the V)rain l)etwcen them, hence the constriction or folchng in followed (fig. 6). The neural tube posterior to the brain developed uniformly- in most embryos, with the excejition of a few wluch showed ti»e extra (leveloi)nH'nt again in the luml)ar region. This resembled very nuich tlie abnormality found in the same place in those embryos developed with low temperatures.

The higher temperatures had an effect ujKin the somites of the embryos wliich was the reverse of that caused iiy the lower degrees of heat. Nearly 4 per cent of the chicks incul)ated at

31S FLORENCE MAY ALSOP

104° to 108°F. developed extra somites lateral to the ordinary somites (fig. 8), while in embryos produced below 101 °F. the mmiher of somites was diminished, or in some of the twentyfmir-hour chicks they did not form at all. In ail the embryos l)\il line, where the epus had lieen incubated longer than twentyfour hours, the somites could l)e distinfiuished. In the one exception the egg had been incubated forty-.seven hours under low temperature. The neural tube was normallj' developed as far as could be discerned in the whole mount, Ijut no somites could be counted.

In none of the embryos examined could the 'accessory optic vesicles' described bj' Locy ('97) be found. He describes them as follows:

Th(MV exists in the hraiii walls of the chick and .\cantliias serial (lifTerciitiatinns of epithelium, that take the form of vesicles, closely connected with the optic vesicles, and therefore called accessory optic vesicles. These structures arc vcrj- transitorj- — extending over a period of three hours in the chick — and tlicv disappear before the true lirain vesicles arise with which they might otiierwisc become confu.<ed. Their existence supports the hyi)othesis that the vertebrate eyes an; segmental and that the ancestors of vertebrates were primitively nniltiplc-cyed inasmuch, the optic vesicles arise before the brain vesicles, the primitive relationship of the former is not the diverticula from the latter. This contlition is secondary.

The question as to what time in the early development is the endjryo most susceptible to an abnormal temperatiu-e was worked upon to a certain extent, but owing to the small number of eggs used in .some instances the result could not be as conclusive as it would have been if a larger number of eggs had been used.

Eggs are put into the incubator at a low temperature, and the heat was increased several degrees for different lengths of time. Others were placed in the incubator at a certain temjjerature and the heat was lowered several degrees. Still other eggs were incubated at a nearly constant temperature. A few of the eggs were put into the incubator at a low degree of heat, the temperature was raised and tiien allowed to decrease.

ABNORMAL TEMPERATURES ON CHICK EMBRYOS

319

VIII. RE.SL-LTS

Tho foUowiuK table shows that a variation f)f a few degrees of heat pro(hiceil a large per cent of aljnornialities as long as all variations wore below normal.

TatiU nhoiririfi effect of tow lemperalures upon developing chick embryos

TIUF

NI'UnER

IN INI r

or EUfiH

DATOR

USED

/72

2S

\T2

1

47-48

.->o

4S

12

27-32

42

26

10

24

25

24

8

22

6

18

15

KINDS or uors-ra

Transverse

Whole

Whole

Transverse section

Whole

Whole

Whole

Transverse section

Whole Whole

RANQK OP TCUPBHATrRC

96-99-96

96-99-96

9,5-101

95-101

9.5-101.5-97

98-96 / 95 5-1001 \ 102. 5-99/

95.5-1001

102.5-99/

98-96.5

97-96 5

NrUBBR

OP NORMAL BUnRYOS

O

37

1

21

1

NrUltER

OP \n.NonuAL

EUBRrOH

231

1/

13

11

21 10 24

3 slow

PER rmsT

op AHNORUAUTIES

83

26 92 50 100 96

87.5

50

All slow in development

The abnormalities produced in tlie twenty-four-hour chicks did not grow out to any noticeable degree in the seventy-twohour embryos. Or, in other words, the seventy-two-hour chicks did not outgrow the alinormalities produced in them at the twenty-four-hour stage. In the above table the per cent of abnormalities is somewhat less in tho sevonty-two-hour chicks than in the younger embrj'os. But this does not take into consideration those embryos which were dead when the shell was opened. .\11 of these that were in a condition to be examined for stnicturo at all wore abnormal. Those apparently deformed conditions may have been caused by the degeneration of the cells. Yet the eggs must have been abnormal or deformed which caused the embryos to die under the lack of heat which was resisted to a certain extent by tliose embryos which were alive when examineil.

The eggs incubated for twenty-seven to thirty-two hours are grouped together in tho tal>le. None of those embryos were sectioned, but were examined as whole mounts. The per cent

320

FLORENCE M.\Y ALSOP

of jibnornial clurks was the same as the per cent (if noriiial ehicks. But, as was shown in the case of tlio fort y-cijjiht -hour embryos, the per cent wouhl no doubt prove nuidi greater if these specimens had l)een sectioned.

No very satisfactory results were obtain(>d from incubating eggs less than twenty-four hoiu's under low temperatures. The ejnbryos hail not ileveloped sufficiently to conclude whether they were normal or abnormal. Most of them showed a very low degree of development. A slight dilTerentintion of cells in the primitive streak had taken place.

Table showing effect of high lemperalures upon developing embryos

TIMB IN

ISITIIA.TOH

Nl'UBEK OF I008 rSKD

KINIIS OP

Monsis

KANGE or TCMPERATt'RE

N-rMBER OP NORMAL EMBRYOS

NI-MIIER OP

ABNORMAL

EMBRYOS

PERCENT OF ABNORMALITIES

48 28 24 22

18 16 14 11

Whole Whole Whole Whole

105-107

103-lOS

10.5-107.5

107-107.5

1

3 2

17 16 11

9

44 100 7982

Total

6

52

90

Most of the embryos in this table were farther advanced in their develojmient than an embryo ordinarily is on being in the incubator the lengths of time specified above. For example, the twenty-four-hour chicks were developed fully as much as a thirty-six hour embryo would naturally be under normal temperature (Compare figs. 5 and 7).

The result of Mr. Boyd's work showed that out oi one hundred eighty-six eggs incubated fifteen were abnormal. But during his work the light in the incuixitor lamji acciilentally l)urned out, producing out of the eighteen eggs being incubated four abnormal emi)ryos. Excepting this set of eggs, there were one hundred .seventy-one eggs, eleven of which were abnormal. Thus according to his work less than 6.5 per cent of the eggs produced abnormal chicks under apparently normal artificial conditions.

One interesting condition noted in the abnormal embryos which were jiroduced under seemingly normal conditions, was that the deformities in these chicks wcic dissimilar to those

AHNORMAL TEMPERATIRES ON CHICK ?:MHRYOS

321

lirodiK'cd !)>• the variations in tcMiijx'raturo. The ai)iiomialitios foiiinl in -Mr. Boyil's cnil^jos were on tiio whole niufh like sonie of those noticed by Doctor Harman in the laboraton' slides. A few were monsters which were different from any of those found in the laboratory or produced by artificial means.

The al)normalities produced by excessive heat were located in somewhat different parts of the embryos than those produced by a limited amount of heat. This can be shown quite clearly in the table below.

LOCATION or ABNORMAUTT

LOW

TEMPERA TIRE

HIGH

TEMPERA TCRE

TOTAL

IN LOW

TEUPERA TL'RE

TOTAL

IN IIIGU

TEUI>CnA TURE

PER < (NT

1 N LOW

TEUPERA TURE

PER rtXT

IN IIIOII

TEUPERA TIBE

Brain only

83 22

6 12 35

22 105

41 47

17 83

40

Neural tube only

54

Hent and tube together. . . .

Some embryos had an abnormal brain only. In others the abnormalities were in the neural tube. While still others possessed an abnormal brain and neural tube. The formation of the neural folds into a tube that was abnormal occurred more frequently than any other condition. .\s shown in the table above, only six conditions of abnormality were located in the head region alone. Ninety-five embryos were abnonnal in the neural tube. In fifty-seven of the embryos both the brain and the neural tulie were affected.

IX. CON'CLUSIONS

1. Excessive heat and a limited amount of heat produced death in many chick embryos and various forms of abnormalities in the nervous system of others.

2. Excessive temperatures ha.stencd the development of embryos, while low temperatures retardetl their rate of growth.

3. The seventy-two-hour chicks did not outgrow any of the abnormalities produced in them at an earlier stage of development.

4. Temp(>ratures l)etween 103° and 10S°F. produced 1)0 per cent abnormal embryos. Of the.se abnormalities 40 per cent were in the head region, 54 per cent were in the neural tube.

322 FLORENCE MAY AI.SOP

">. In tlioso ogRs iiicul)ati'il at 04° to 101°F. 07 per cciit were nl)norinal; 17 per cent of these abnormalities were in the brain repion and S3 per cent were in the neural tube.

G. Incubatinp crrs at normal temperature pro(hire(l nearly G.o per cent al)normal embryos. ^lany of these abnormalities were different from those deformities produced under abnormal temperatures.

ijihlioc;raphy

.\llk\, W.m. F. I'.tlG .'^lu(lics on the spinnl cord and medulla of Cyclostoincs

with special reference to the formation and expansion of the roof

plate and the flattening of the spinal cord. Jour. Comp. Xeur., vol. 20,

no. 1. B.\LFoin, F. M. 1873 On the disappearance of the primitive groove in the

embryo chick. (Juar. Jour. Micr. Sc. vol. 13. B.WTA, .VRTHrii M., AND GoKT.SKli. Uos.s .AlKEN 1914 The production of accessory appendages and other abnormalities in amphibian larvae

through the action of centrifugal force. Proc. .Soc. Exp. Biol, and

Alod., vol. 9, no. 7.

1915 Accessory appendages and other abnormalities produced in

amphibian larvae through the action of centrifugal force. Jour. Exp.

Zool.. vol. 18, no. 3. Beckwith, T. D.. and IIarbon, G. D. 1914 The poor hatching of normal

eggs. Science, X. ,S., vol. 40, no. 1024. Bkown, M. C. 1910 Freak eggs. Poultry Digest, vol. 4, no. 11. Co.NKLiN, E. O. 1911 The effects of centrifugal force upon the organization

and development of the eggs of fresh water pulmonates. Jour. Exp.

Zool.. vol. 9, no. 2. Edwards, C. L. 1902 The physiological zero and the index of development for

the egg of the domestic fowl. Callus domesticus. Am. Jour. Phys.,

vol. 6. no. 6. EvcLK.snvMER, A. C. 1897 Some observations and experiments on the natural

and artificial incubation of the eggs of the common fowl. Biol. Bull.,

vol. 12. no. 6. Fint. Ch. 1894 Xote sur I'influence de la temp<'"rat\ire sur I'incubatiori de

I'oeuf de |)oule. Jour, de I'anatomie et de la physiologic. Paris,

T. 30.

1899 The influence de repos sur les effects de I'exposition prdalable

aux vapeurs d'alcool avent I'incubation de I'oeuf de poule. Compt.

rend. Soc. de biol., T. G.

1910 Uemar(|ues sur I'incubation ties ocufs de poule prives de leur

ci)()uclle. Compt. rend. .Soc. dvbiol., T. 52. Glaser. Otto 1913 On the origin of double-yolked eggs. Biol. Bull., vol. 24,

no. 3.

1910 The theory of autonomous folding in embryogenesis. Science,

X. S.. vol. 44, no. 1130.

ABNORMAL TEMPERATIKES ON CHICK KMHRVOS 323

Glaser, Otto 1917 On the ini-clianisin of inorpholoKiciil difTercntiation in the

norvKiiM syHtcm. II. The relation between compression and the

development of a scries of vesicles. Anat. Kec, vol. 12, no. 2. GoLDF.Mtii, \. J. 1913 Studies in the production of grafted embryos. Biol.

Bull., vol. 24. no. 2. Greeley, A. \V. 1901 On the analogy between the effects of loss of water and

lowering the temperature. .\ni. Jour. I'hys., vol. 0. no. 2. HAnniTT, Chas. \V. 1S99 Some interesting egg monstrosities. Zool. Bull.,

vol. 2.

1912 Double eggs. Am, Nat., vol. 46, no. 549. Hakma\, Maky T. 191S Abnormalities in the chick embryo. Science, X. S.,

vol. 47. HooE, M. A. 1915 The influence of temperature on the development of a

Mendelian character. Jour. Exyj. Zool., vol. 18, no. 2. HruBAUD, M. E. 190.S .Some experiments on the order of succession of the

somites of the chick. .\m. Nat., vol. 42, no. 499. KiLSEY, H. 1911 Subdivision of the spinal canal in the lumbar region of

chick embryos. Melbourne I'roc. K. Soc, Vict., vol. 24. Ki.NG, II. D. 190.3 The efToct of the heat on the development of the toad's

egg. Biol. Hull., vol. 5. no. 4. La.ndman, Otto 190S An open cleft in an embryonic eye of a chick of eight

days. Anat. Anz., Jena, Bd. 32. I.KCAiLLOX, A. 1910 Influence de la temperature sur la segmentation et la

degcnerescencc de I'oeuf non feconde de la poule. Paris C. U. Soc.

Biol., T. 68. I.KWis. F. T. 1907 .Specific characters in early embryos. .Am. Xat., vol. 41,

no. 4S9. l.ii.t.iK, Fha.nk R. 1903 E.xperiment on the amnion and the production of

aiiaraniote embryo of the chick. Biol. Bull., vol. 5, no. 2.

1904 The development of defective embryos and the power of regeneration. Biol. Bull., vol. 7, no. 1.

1908 The development of the chick. An introduction to embryology.

Henry Holt & Co., New York. Ldcv, W. i\. 1897 .\cccssory optic vesicles in the chick embryo. Anat. Anz.,

Bd. 14. LiiKit, jAcgi'Ks. 191.") The blindness of the cave fauna and the artificial production of blind ti.sh embryos by heterogeneous hybridization and low

temperatures. Biol. Bull., vol. 29, no. 1. Mali., F. U. 190S A study of the causes underlying the origin of human

monsters. Jour. Morph., vol. 19, no. 1. Ml WiioHTKH, J. E., AND WHIPPLE. \. O. 1912 The development of the blastoderm of the chick in vitro. Anat Uec. vol. 6, no. 3. Mo.NTitosE, T. Bi'HKows 1911 The growth of tissues of the chick embryo

outside the animal body witlvspecial reference to the nervous system.

.lour. Exp. Zool.. vol. 10. no. 1. Nkwman. II, II. 1917 On the producliim of iniinsti'r.< by hybridizatiiui. Biol.

Hull., vol. 32, no. 5.

324 ■ FLORENTK MAY ALSOP

O'DoNooiu'E. ('. n. li'lO Throe examples of diiplirily iti chirk embryos with

n case of ovum in ovo. Annt. Anz.. Jcnii. Hcl. 37. Oliver, K. K. 1911 On the dispiiireniont of tlic optic lobes during the de vehip""""' "f •'"" brain of the fowl. Melbourne Proc. R. Soc, \"ict.,

vol. -.M. P.\KKKn, C'l. H. ISIOG Double hens' egRs. Am. Nut., vol. -10. no. 469. P.\T()\, Steward 1011 Experiment on developing chickens' eggs. .lour. Exp.

Zool.. vol. 11, no. 4. Patterso.v, J. Thos. 1909 Note on accessory cleavage in the hen's egg.

Science, N. S., vol. 29, no. 715. Peebles, Flore.vce 1S9.S Some rxperinients on the primitive streak of the

chick. Arch. f. Entw. Morh.. Hd. 7. •

190.'? A preliminury note on the position of the primitive streak and

its relation to the chick embryo. Biol. Hull., vol. 4, no. 4.

1904 The location of the chick embryo upon the lilijslodcrm. Jcur. Exp. Zool.. vol. 1, no. 3.

Peter, K. 1905 t*ber individuelle Variationcn in der tierischen Entwicklung. Verh. Ges. deutch. Xaturf.. Bd. 76.

1905 Der Grad der Beschleunigung tierischer Entwicklung (lurch erhohte Tenipcratur. Verh. Ges. dciitch. Xaturf., Bd. 70.

Pick, E. \V. 1911 Egg abmu-malitics. Poultry World, vol. 7.

R'EESE, A. M. 1912 The effect of narcotics on the development of the hen's egg. Science, N. S., vol. 35, no. 903.

ScHAPER, Alfred 1897 Die friihesten difTerenzierungsvorgiinge in centralnervensystem. Arch. f. Entw. Mech., Bd. 5.

Schumacher, S. 1896 Ein Ei im Ei. Zool. Anz., Bd. 19.

Stockard, C. R. 1914 The artificial production of eye abnormalities in the chick embryo. Anat. Rec, vol. S. no. 2.

Whit.man, C. D. 1883 A rare form of the blastoderm of the chick and its bearing on the question of the formation of the vertebrate embryo. Jour. Micr. Soc. vol. '^3.

Vd.siiida, Sadao 1910 Ijo kerhai no ni rei (on two cases of abnormally developed chick embryos). Dobuts. Z. Tokyo, vol. 22.

PLATES

All figures werp drawn at table level with a no. 5 eye-piece and a l()-mni. objective, also with the aid of a camera lucida. Front lens of objective was removed for whole-mount drawings. .\11 drawings reduced one-half.

325

PLATE 1

EXPLANATION OF FIGURES

1 Twenty-four-hour embryo, incubated at 95.5°F. to 98.5°F. Broken lines fhow plane of sections in fig. 2. a. tliroUKh ojjtic vesicles showing folds failing to unite; (>. through neural plates back of fifth somite, no neural folds present; r, through primitive node region, neural folds formed; (/, through neural plate posterior to primitive node region, no neural folds ])resent; c. through tube forming in posterior region;/, through posterior region.

2 Twenty-eight-hour chick incubated at OC.oT. to 98.5°F. Letters corresponding to those in fig. 1 indicate sections through these regions.

3 Twenty-four-hour chick with curved primitive streak at a. Incubated at 10.5°F. to 107..5°F.

4 Forty-seven-hour chick with curved neural tube at a. Folds failed to form at b. Incubated at 95°F. to 97°F.

32C>

ABNORMAL TKMPKRvVTURES OX CRICK EMBRYOS

rLUHCNCB HIT AJAOP

PLATE 1

<?&K

r

Si,

u.

327

PLATE 2

EXPLANATION OF FIGURES

5 Twenty-four-hour chick. Incubated at 103.5°F. to 108°F. Development equal to that of normal thirty-six-hour chick.

6 Twenty-eight-hour embryo incubated with temperature at 103.5°F. at beginning of incubation, raised to 108°F. at end of fifth hour. Constriction formed below optic vesicles at a.

7. IVenty-fiiur-hour chick. Incubated at 102.5°F. to 99°F. a, one neural fold forming in brain region; U. abnormal development of notochord.

8 Twenty-eight-hour embryo incul)atcd at 103. o^F. to 108°F. a, accessory somites; 6, abnormal brain; r, posterior limit of neural folds.

328

AHNOn-MAI, TKMPKIUTURKS ON CHICK EMBKYOS rLOHEN'C u\T »moi>

PLATE 2

■:^h

i\>..

3.29

i

8

TBE ANATOMU Al. RECORD, VOL. 15, \n. A

I'LATE 3

EXPLANATION OF FIGURES

9 Twenty-nine-hour embryo incubated at 100.5°F. to 101. 5°F. Xeural tube in brain region not closed. No tube formed in remainder of body.

10 Twenty-fwo-hour embryo incubated at lOyT. to 107. 5°F. Neural tube formed only in brain rcRion.

11 Transverse section of scventy-two-hour chick through posterior part of neural tube. Incubated at 97.5°F. to 96°F. Tube showing three central canals at a.

12 Transverse section of same embryo, a, two central canals present in neural tube.

13 Forty-eight-hour chick incubated at 96.5°F. to 97°F. a, extra tissue in lumbar region.

330

ABN'ORMAL TEMPERATfRES ON CHICK EMBRYOS

»L'KII:\fr WAT ALAUP

PLATE 3

%^

\ I

K^

16^

12

13

381

PROCEEDIXnS OF TIIK AMKRIfAX SOCIETY OF ZUUl.OdlSTS

SIXTEENTH ANNUAL MEETING

'I'lic American Society of Zoologists held its Sixteenth Annual Meeting jointly with Section F of the American Association for the Advancomont of Science and in affiliation with the American Society of Naturalists and the Ecological Society of America, December 2Ch 27 and 28, 191S, in Oilman Hall, Johns Hopkins University, Baltimore, Maryland.

Election of Members

At the session for transacting business, held at 5 o'clock on Friday, December 27, Pr(>sidcnt George Lefe\Te and VicePresident L. L. Woodruff being absent, William Patten was appointed chairman for the session. The following persons, having been recommended bj' the Executive Committee to the Society for election to membership, were duly elected:

Baker, Arthur Challe.v, B.S.A.. Ph.D. (University of Pennsylvania, EntomoloRical Assistant. Bureau of Entomology. Washington, D. C.

Dki^viler. Samcel Kandall, Ph.B., Ph.D. (Yale'), Instructor in Anatomy. Yale .Medical School, .{nalomicat Laboratory, Yah Medical .School, Xcw Haven, Conn.

Hu.vT. Harrison Uandai.i,, H..'*., Ph.D. (Harvard ), Assistant Professor of Zoology, West Virginia University, Morgantoim, H'. Va.

Powers, Hdwix Booth. .V.M.. M..S. (Chicago), Ph.D. (Illinois), Assistant Professor of lliology, Colorado College, Colorado Springs, Col.

Taliaferro, William Hay, B..S., Ph.D. (Johns Hopkins University\ Second Lieutenant Medical Research Division, Chemical Warfare .Service, tH Park St., Xew Haven, Conn.

Roberts, Elmer. B.S., Ph.D. (University of Illinois), .\ssociate in Genetics, rnivcrsitij of Illinoix, I'rbana. 111.

Election of Officers

The Committee on Nominations consisting of D. H. Tennent, P. Ci. Harrison and M. F. (iuycr, having reconmiended persons

334 AMEHICAN SOCIETY OF ZOOLOGISTS

for olortinn to tlic various oflicos of the Society and no other noiiiinatioiis Ix'iiig prcsenteti, the following elections were made:

For President, C. M. Child, Chicago, Illinois. To serve one year.

For Vice-President, 11. 11. Wilder, XortlKuapton. MtussucliuSGtt.s. To serve one year.

For Secretary-Treasurer, W. C. .Vllee, Lake Fore.st, Illinois. To serve three years.

For member at large of the Executive Committee, to serve five years, George Lefevre, Columbia, Missouri.

Report of the Secretary-Treasurer

On account of pressing duties Captain Caswell Grave, Chemical Warfare Service, resigned from the position of Secretary of the Society in September and W. C. .\llee was elected by the F^xecutive Committee to act in his place until the .\nnual Meeting. Captain Grave was able to continue to perform the duties of the Trea.surer and submitted the following report:

Seven ii. embers were recommended to be dropptnl for nonpajnnent of dues and two members resigned from the Society.

Financial Statement

The financial statement of the Treasurer for the year 1918 is a.s follows:

Receipts

Balance on hanil January 1. 1918 $733.85 Back dues for the year 1916, 1 at$5.00 5.00

Hack dues for the year 1916, 1 at 7.00 7 00

Back dues for the year 1917, 6 at 5.00 30.00

Back dues for the year 1917, 5 at 7.00 35.00

Back dues for the year 1917, 1 at 8.00 (foreign; S.OO

Back dues for the year 1917. 1 at 11.50 11 .50

Dues for 191S. .59 at .'.00 295.00

Dues for 191S. 1 at (i 00 (Life member) C.OO

Dues for 19I.S. 167 at 7 00 1169.00

Dues for 19IS. 1 at nO .50

Dues for 1918, 1 at 7 97 (foreign) 7 97

Dues for 1918, 10 at II 50 115.00

.\u(?u8t 1, 1918. Fifth dividend, Ind'l. Sav. & L'n Co 9.00

f)ctohpr 7, 1918, Interest at 4% on deposiU 38.93

Total $2471 75 PROCEEDINGS 335 Expendil'iriH January to December inclusive For fplctinims and trlcphonc calls .$7.43

For cxprc'SH churRcs .27

For stationery anti stamps. 33. 15

For typewriting 3.99

For typewriter ribbon 100

For printing aniiouncoments ami proKriuns 33 00

For 253 .sulwrript ions to journals I Wistar Institute) 1570.00

For Railroad fare and I'ullinan. Lake Forest to Baltimore and return 69.98

For hotel cxponsos of Sccrotary in Hidtiniorc 14.64

Total »1733.96

Balance on hand December 28, 1918 737.79

Report of the Auditing Committee

The Auditing Coinmittee reported as follows:

We have examined the books of the Treasurer and have found that the balance here given Is correct, provided that the items in credit and debit columns have been correctly entered. Data for the verification of these items have not been presented.

(Signed) S. O. M.\st,

A. RlCH.\RDS.

By motion the reports of the Treasurer and the .\uditing Committee were accepted and the incoming officers were instructed that in the future it should be their policy to pay bills of the Society by check.

At the opening of the session Saturday afternoon, December 28, the .\uditing Committee requested leave to present a new report which follows:

^^'(' have found upon reexamination of the Treasurer's books, that except for petty cash expenditures, data for the verification of all expense accounts are at hand and that the accounts are correct.

(Signed) S. O. M.\st,

A. RiCH.VRDS.

330 AMERICAN SOCIETY OF ZOOLOGISTS

Committee on Prcmedical Education

Tho Coinniittco on pro-incdical ('(hicatinn appointod in 1915 and continued in l'.)lt) was discliargcd witliout report.

Deposit of Records of (he Society

On reediest the incoming Secretary-Treasurer was instructed to deposit the recf)rds and past corrospondonee of tho Society in the fire proof vaults provided for that i)urpose b}' the Marine Biological Laboratory of Woods Hole, ^fass.

The Secretary was instructed to extend the thanks of the Society to the officials of Johns Hopkins University and to the Local Committee for the entertainment of the Society during the meetings.

Session for the Presentation and Discussion of Papers

At sessions held during the afternoon of December 26 and the morning and afternoon of December 27, IS papers were pre.sented in full and lil were read by title.

In the absence of the I'resident antl Nice-President, Bennett M. Allen, was made presiding officer of the Thursday afternoon session. William Patten, Vice-President of Section F presided at the inorniiifi and afternoon sessions on Friday.

The morning of December 27 was given over to a joint session with the Ecological Society of America when the following papers were presented:

W. J. Crozier, University of Illinois. Further Contribution upon the Natural History of Chromodorus Zebra: the question of adaptive coloration. A. S. Z. (H(>atl by title.)

iMlwin ]i. Powers, Colorado College. The Hydrogen Ion Concentration of the sea water of Puget Sound anil the Beactions of the herring (Clupea Pallasii Cuvier) to Hydrogen on concentration in sea water. E. S. .\.

The P. H. of Puget Sound in the vicinity of Friday Harl)or varies with weather comlitions, tides, depths, and locality. The

PROCEEDINGS 337

«

herring reacts positively to a Pii of 7.9 to 8.0. The reaction is positive to this Ph concentration both from a lower and a higher I'll.

H. 11. Reed, Cornell University. The Zoological ."significance of the functional fenestral plates in the ear capsule of caudate amphibia. A. S. Z. (Read by title.)

Harry C. Oberholser, National Museum. Ecological Investigations under the Federal Government (30 min.). E. S. A.

The most important ecological investigation under Federal Government auspices are carried on as a basis for other work, and are of far reaching importance. The Fish Commission studies the relation of fishes to their environment; the Forest Service that of trees; the Bureau of Plant Industry of various other plants, particularly with regard to plant diseases antl plant introductions; the liureau of .Vnimal Industry, the life history of internal animal parasites; the Bureau of Entomology, the life history of iasects in their relation to economic problems; and the Biological Sur\-e>-, the relations of animals, birds, and other animals to their environment and to each other, for the determination of the life zones of distribution.

W. H. Longley, Goucher College. The Coloration and Habit-; of West Imlian and Hawaiian reef fishes. A. S. Z.

Henry S. Pratt, Haverford College. The Distribution of the Internal Parasites of the Fi-sh and other Aquatic Vertebrates of Oneida Lake. New York. (15 min.) E. S. A. (Read by title.)

v. E. Shelford. Illinois Natural History Survey. Suggestions as to the Climograph of deciduous forest invertebrates, as illustrated by experimental data on the codling moth. (20 min.) A. S. Z. and E. S. A. (Read by title.)

W. J. Crozier, University of Illinois. On the Nature and Source of some adaptive features in the etiology of Chiton. A. S. Z. (Read by title.)

On Saturday morning, December 28, the Society held no session but met with the American Society of Naturalists to listen to their program of papers on Evolution and Genetics.

33S AMKUICAN SOCIETY OF ZOOLOGISTS

Retiring Secretary-Treasurer

At tlio opcniiifi of tho Aftornoon Session, in addition to the report of tlie Auditini!; Conmittee alrejidy ^iven, the retiring Secretary-Treasurer, Caswell CJrave, was tendered the thanks of the Society for his faithful, eflicient and loyal services. The motion to this effect was unanimously passed amid applause.

The Society then passed to the afternoon's program.

Conference between Govermuent and Laboratory Zoologists.

Sul)jcct: Methods of Securing; Better Coiiperation between Clovernment and Laboratory Zoologists in the Solution of Problems of General or National Importance. Professor C. E. McClung, presiding.

Paper on l)lans and problems of the Bureau of Entomology that can be furthered by cooperation with laboratory zoologists. Dr. L. O. Howard.

Discussion led by Professor ,1. (I. Needham, Cornell University.

Paper from the Bureau of Fisheries. Dr. Hugh Scott.

Discussion led by Professor H. B. Ward, University of Illinois.

Paper from the Bureau of Animal Industry. Dr. B. H. Ransom.

Discussion led by Professor Herbert Osborn, Ohio State University.

Paper from the Biological Survey, Dr. E. W. Nelson.

Di.scu.ssion led by ^lajor C. A. Kofoid, Fort Sam Houston.

In the absence of Major Kofoid, Professor R. K. Nabours, Manhattan, Kans., gave a short discussion of Dr. Nelson's paper.

Dr. McClung then introduced Dr. J. C. Merriam, ViceChairman of the National Research Council, who outlined the tentative plans of the Council for advancing cooperative re.search in .Vmerica and Professor McClung concluded the discussion with the explanation of the application of these plans to the problems of cooperation between Government and Laboratory Zoologists.

The proceedings of this Conference will be published in full in Science.

PROCKEDtNGS 339

LIST OF TITLES

rAIlASITOLOGY

1. On the transmission of two fowl tapeworms. James E. Ackcrt, Kanson State

Agriciiltiiriil C'ollogr. •

2. Recent discoveries cnnccrning the life history of Auraris liimhricoides. G. H.

Ransom and W. D. Foster, Bureau of Animal Industry, Wjishington, D. C.

3. The true homology of thecutioulaandsubcuticulaof trcmatodes and cestodcs.

H. S. Pratt, Huverford College.

COMPARATIVE ANATOMY

4. The metamorphosis of two species of oyclops: Cyclops signatus (C. Albirlus

Jnrine) and Ci/clops americanus Marsh. Esther F. Byrnes.

5. Tlip olfactory organs of Orthoptera. X. E. Mclndoo, Bureauof Entomology,

Washington, D. C.

GENERAL PHYSIOLOGY

6. The formation of buds 'Tethya' buds in sponges of the genus Coppatias.

\V. J. Crozier and Blanche B. Crozicr, Bermuda Biological Station for Research.

7. On the temporal relations of asexual propagation and gametic reproduction

in Coscinasterias; with a note on the function of the -Madreporite. W. J. Crozier, University of Illinois, College of Medicine.

8. The olfactory sense of lepidoptcrous larvae. X. E. Mclndoo, Bureau of

Entomology, Washington, D. C.

9. Sensory reactions of Chromodoris zebra. W. J. Crozier, Bermuda Biological

Station and L. B. .\rey. Xorthwestern University Medical School.

10. The relative stimulating efficiency of continuous and intermittent light upon

Vanesxa antiopa. William L. Dollcy, Jr., Handolph-.Macon College, .\shland, \'a.

11. The rales of COj produced by starved and fed Paramecia and their possible

relations to the rates of oxidation in the unfertilized and fertilized sea urchin egg. E. J. Lund, University of Minnesota.

12. The photoreactions of partially blinded whip-tail scorpions. Bradley M.

Patten, Western Reserve University, School of Medicine.

13. Excretion crystals in ameba. .\. \. Schaeffer, University of Tennessee.

14. The reactions an<l resistance of certain marine fishes to H. ions. C. E.

Sholford, I'niversity of Illinois.

15. A simple method for measuring the COj produced by protozoa and other small

organisms." E. J. Lund. University of .Minnesota. 10. The efTect of KCX on the rate of oxygen consumption of Planaria. George

Delwin Allen, University of Minnesota (introduced by E. J. Lund). 17. The influence of temperature and concentrations on toxicity of salts to fish.

Edwin B. Powers, Colorado College (Introduced by V. K. Sholford).

340 AMERICAN SOCIETY OF ZOOLOGISTS

ECOLOGY

IS. Further contributions upon the natural history of Chromodoris zebra; the question of adaptive coloration. \V. J. Crozier, University of Illinois, College of Medicine.

19. The zoological significance of the functional fencstral plate in the car capsule

of caudate amphibia. H. D. Reed, Cornell I'nivcrsitj-.

20. The coloration and habits of West Indian and Hawaiian reef fishes. W. 11.

Lonuley, Goucher College.

21. Suggestions as to the climograph of deciduous forest invertebrates as illus trated by experimental data on the codling moth. V. E. Shclford, Illinois Natural History Survey.

22. On the nature and source of some adaptive features in the ethology of Chiton.

W. J. Crozier, University of Illinois, College of Medicine.

EMDRYOLOGY

23. The anlagc of endoderm and mesoderm in the opossum. Carl Uartman,

University of Texas.

24. The oestrous cycle in rats. J. A. Long, University of California.

25. Result.sof extirpation of both thyroid and |)ituitary glands in tadpoles of Bufo

and Kana (.5 minutcsl, Bennett M. .Mien, University of Kansas. 20. Miscellaneous notes regarding experimental studios upon the endocrine glands of Kana and Bufo (10 minutes). Bonnet M. .\llon, University of Kansas.

27. Effect of the extirpation of the thyroid gland upon the pituitary gland in • Bufo. Mary Elizabeth Larson, University of Kansas (introduced by Bennot

M. Allen). "

EVOLUTION AND GENETICS

28. The solitary and aggregated generations in Salpidae. Ma>Tjard M. Metcalf,

Orchard Laboratory.

29. Correlation of fertility and fecundity in an inbred stock. Roscoe R. Hyde,

Indiana State Normal School and Johns Hopkins University.

30. The extent of the occurrence of sex intcrgrades in Cladocera. Arthur M.

Banta, Station for Experimental Evolution.

31. Nuclear reorganization and its relation to conjugation and inheritance in

Arcella vulgaris. H. M. MacCurdy, Alma College.

32. Several ways in which Gynandromorphs in insects may arise. T. II.

.Morgan, Columbia I'niversity.

33. Duplication. C. R. Bridges, Columbia University. (Introduced by

T. n. Morgan.)

EXHIBITS

1. Demonstration of sex intcrgrades in Cladocera. A. M. Banta, Station for Experimental Evolution.

2. Models showing typical stages in the development of the human embryo. Department of Embryology, Carnegie Institution of Washington.

ABSTRACTS

1. On the tmn>tiiiissinii iif two find tapvtvormfi. James E. Ackert, Kansas State AKriciiltural Colli-Kc.

In studyiiiK tho life cycK-s of fowl ccstodes the writer recently demonstratetiexperinientally that the house Hy, .Ui<Aca (/owf.s</c«L., may transmit to ehickeiis a tapeworm which appears to be Davaincu tetragona (Mftliii 185S) IManchartl 1801. ("hieks hatched in an incubator were reared'in a screened expei'iniental feeding house. The cement floor and walls (eighteen inches liifih) exduile all worm-like animals and the vestibule facilitates in eliminating any winped forms.

The feed of the chicks is carefully inspected and free from animal tissues excei)t occasional feedings of fresh beef. Under such conditions experimental and control chicks have been kept continuously for more than four years. Frequent intestinal examinations of control chicks (luring this period have never yielded a single parasitic wonn.

The Hies were trapped at local poultry yards in which spring chickens were found by examination to be infested with tapeworms. The live flies were immersed in tap water and permitted to dry, after which the house flies, .1/. domcstica. were sorted out. one by one, and given in small numbers to young chicks. Occasional movements of the flies indicated that any larval tapeworms in them probably were unafTected by the immersion. Between September 23, and Octolier 19, 1918, several thousand M. domcsticn were given to seventeen young chicks reared in the experimental feeding house. Four of these chicks have been examined to date. The results from chicks 2.32 and 243 examined November 1, 1918. were negative, no parasites having been found, but the intestine of Chick 235 (November 4, 1918) contained ten mature tapeworms which have th(> characteristics of Dorainea tetrayoiia. Two tapeworms, obviouslj' of this same species, were removed on November 4, 1918, from the intestine of Chick 250.

In a similar experiment (1917) the fowl cestode, Daminea cesticiUiis (Molin), was transmitted to chickens. An account of the experiment, including a description of the tapeworms transmitted, is being pul)lished in the current number of the .Journal of Parasitology-.

2. Recent discoveries concerning the life histori/ of Ascaris liimfiricoides. H. H. H.wsoM and W. D. Fostkr, Bureau of .\nimal Inilustry, Washington, D. C.

Major Stewart (I. ^^. S.) has lately reconleil the results of investigations on Ascaris liimbricoidcs which necessitate revision of former conceptions of its life history. The present writers have re|ieated and supplemented Stewart's experiments, confirming his main results but reaching different conclusions.

ui

842 AMERICAN SOCIETY OF ZOOLOGISTS

.l.srari.s- I'jrijs after iiu'iil):ilion wlu n swallowed hatch in the intestine. The larvae within a short time after hatching can he foinid in the liver and portal vein. Reaching the lungs in the circulation, they undergo considerable development within a few days. \'ia trachea and esophagus they reach the intestine and develop slowly to maturity if the atiim.al infested is a suitahle host. Otherwise (rat. mouse, guinea-pig, rahhifi they soon pass out in the feces without further deveiopiiient. and (|ui<'kly die. SK-wart's view that rats and mice act as intermediate hosts of the Axcnris of man and pig is untenahle. Partial development of the parasites in these animals is an expression of incomjilete adaotation to strange hosts, and not a phase of the normal lif(> cycle.

Asrnris and related forins may hear a causal relation to lung tfouhlcs of ohscure origin iti children, jiigs, and other young animals. The larvae can cause fatal pneumonia in pigs.

In lamhs and young goats the pig Axcori.s can develop much further than in rats. mice, etc., and may reach a stage approaching maturity. Very young |iigs appear more suseeptilile to infection than older pigs. Ascnris eggs injectcii subcutaneously will hatch, the larvae migrating to the lungs and developing thereafter as in infection per os.

S. The true homology of the cuticula and subciiticula of trematodes and

reModes. H. S. Pratt.

Most textbooks of zoology teach that the cuticula of trematodes sjid ccstodcs is homologous to that of arthropods and annelids and is secreted by the subcuticula which is thus homologous to the hypodermis. I presented facts in \'ohnne 43 of the .Vmerican Naturalist which tend to show that no such homologies exist, but that both cuticula and subcuticula belong genetically to the parenchyma and are consequentlj" mesenchymatous and not extodermal structures. I now present an additional proof of this position. A cystocercous ccrcaria, C. fuscxi, recently studied, possesses prominent wart-like protuberances on its tail. These lie outside of the layers of longitudinal and circular muscle filiei-s and subcuticular cells, and are compost d '•xelusively of the characteristic tissue forming the parenchyma of tlv worm bounded on the outer surface by the general cuticula. The s ibculicular cells thus do not enter the protulierances and cannot secrete their cuticula, which is as in all trematodes and cestodes, simply the peripheral portion of the parenchyma.

4. The metamorphosis of two species of cyclops: C. signatus, (C. albidus Jiirinr) and C. americanus Marsh. Estiier F. Byrnes. There are nine stages in the metamorphosis of both species. The ty|>ical nauplius with three i)airs of appendages molts. The second st.Mge h:is a fourth apjK iidage. The second molt produce a third nauplius with the fifth and sixth appendages indicated. The third molt produces a typical cyclops with six antemial segments. The mouthparts are present as in the adult. Hami of the swiunning feet an- unsegnientetl. The fourth molt: There arc seven antennal segments.

PROCEEDINGS 343

Haiiii i)f tljc first and second fct't are two-jointed. The third foot is uiiscuMifiiliMl !in<i the fi»iirtli indicated. Thr fifth molt: '/"hen" are nine antciwial segments. Hanii of first, second and third feet are twojointed; Thi' fourth for)t is iinseginented and the fifth foot is present. The xixth moll: There are ten antenna! .seRinents. All the feet are twojointed. The fifth foot is fully developed. The seventh molt: There are eleven antennal segments. .\ll rami are three-jointed a.s in adult. .\tidomen immature. The lujhth molt produces an atlult with seventeen antennal scKnients.

C aniericamis shows a marked irregularity in the jointing and annaturr of the feet in stages'/ and 7. This irregularly suggests an explanation of the wide variation found in the armature of the adults of the viridis type. C. signatus shows no corresponding variations.

Klongation of parts is by intercalation of segments.

During the progressive development, adult characteristics that appear early in the metamorposis undi'rgo no modification during later stages.

Duration of metamorphosis varies from several to 10 weeks.

■5. The olfdctoiii ort/iins of Ortlinptrni. X. E. McIxDOO, Bureau of En tomologj", ^\'ashington, D. ('.

This investigation is a continuation of my study on the morphologjof the olfactory pores of insects. Both sexes of twenty-one species, belonging to twenty genera and representing the six families, have been examined. The immature stages of Blatella and Melanoplus have also been studied. Olfactory pores were always present on the legs and antemiar: usually on the wings (if present), abilominal segments, cerci, head and ail the moulhparts; and sometimes on the ovipositor. Relative to the antennae, olfactt)ry pores are present on only the first and second segments: this is the first time that I have seen these organs on the antennae of adult insects, except a few at the bases of the antennae of the honeybee and of a certain weevil; nevertheless, they are common to the antennae of all larvae yet examined. They are more widely <listril)ute(l in (^rthoptera than in any other order yet studied; the mmiber of them on the wings is comparatively few. while the mouthparts are abundantly supjiliccl with them; the numlier on the antennae varies considerably, although fifty is a common number for an antenna.

In distribution and extt'rnal structure, these olfactory pores resend)le the lyriform f)rgans of spiders more than i\o the same organs in any other order yet examined. They are generally oblong, sometimes almost slit-shaped, but the eye-shaped tyix' is the most common. Some of the pore bor<lei-s are r:idially striated; this is the first time that I have observed this type of border on aiiult insects.

344 AMERICAN SOCIETY OF ZOOLOGISTS

6. Thr formation of buds ('Tethiia'-budii) in sponges of the genus Cop]mti<is. W. J. CnoziKK and Blanche B. Ckozier. Bermuda BioIdgicid Station for Hcscarcli.

A spOnRC wliich we shall describe under the name Cojipatias inillhrooki, sp. nov., has been found to produce buds very closely siinulatinp; the well-known Donatia-buds (,'Tetliya'-buds). Hud-rci)r(>duction lias not hitherto been iliscoverod in this genus, which is toxonomically !»kin to Donatia; in fact, although 4 marine sponge 'genera' have been reported to form buds, of several types, the characteristic buds of Donati.'t have occupied a relatively unique position.

Hence it is curious that C. niillbrooki inhabits ^langrove creeks harboring likewise 3 well-differentiated types of Donatia, all reproducing in the way usual for this genus, namely by means of b\ids. Each of these budding sponges, which occur in great profusion, has a more or less definite propagative season. The one species of Donatia found at Bermuda but not occurring •in the mangrove creeks — D. lyncurium, which lives imder stones on exposed shoi-es, has never been found to produce buds in this region, although this habit is exhibited by it in other regions (e.g., in the Mediterranean).

7. On the temporal relations of asexual propagation, and gametic reproduction, in Coscinasterias; irith a note on the function of the madrepore. W. .1. Crozier. University of Illinois, ("'olleg(^ of Medicine. Asexual propagation of Coscinastcrias temisjiina by spontaneous division of the body into two parts, conunonly comprising 3 and 4 rays respectively, is at a minimum during the several months preceding, and during the actual period of, gametic reproduction (Jan.-Feb., at Bermuda); and at a maximum during the simuner season midway between the sexual periods. The two methods of nniltijilication practiced by this species arc therefore supplementary in temporal incidence as well as in kind.

Thr- foiTTiation of new rays at a division-surface is frequently accompanied by the appearance of new madrepores. The number of madrepores is positively correlated with the total number of rays, in such a way as to suggest some kind of functioiuil significance attaching to this relation. It is also suggested that a deficiency in this relation might be implicated in determining the onset of self-division.

8. The olfactory sense of Lepidopterous larvae. N. E. McIndoo, Bureau

of Entomology-, Wa.shington, D. C

Since all lar\'ac are more or less selective in regard to their food, it has been assumed that they can smell, although, so far as known to the writer, no experiments have been perfonned to prove this statement, and no one has discovered olfactory- organs in them, except the ones called olfactory pores descrifn-d by the writer in the June number of thi' .lournal of Morphology.

The following larvae were used in the exp<'rinients: T(>nt caterpillars, fall webwomis, tussock-moth larvae, arniyworms and larvae of

PROCEEDINGS 346

Papilio polyxpnes. The following sources of odors einployfcl aiul the avcranc icaction time of tlie above iarvai' to them are: Oil r»f [H'|)[K'rmint, 1 }.() seconds; oil of thyme, 0.5 seeonds; oil of winternreen, 17.(5 seconds; leaves of pennyroyal, 20.1 .seconds; leaves of sfx-armint. 22.9 seconds; wild cherry leaves, 42.5 seconds; fresh grass, 19.1 seconds; honey. 51.1 .seconds; protruded glands of above Papilio larvae, 22.8 seconds; and as a control a clean and empty vial, tiO seconds (totally negative).

In making a coni|)arative study of the disposition of the olfactf»rv pores, thirty sjx'cies. i)elonging to t wentyn-ight genera and representing twenty families, were used. ()lfactory pores were invariably found on the epicraniiim, front, anteimae, all the mouthparts, trochanters, tibiae; usually on the tarsi; and sometimes on the first thoracic s<>gment, last abdominal .segment and on the last prolegs. The total number of pores varies from fifty-seven to eighty-four with sixty-nine as an average. In structure they are similar to those in most adult insects.

9. Scnsorii rcdctionx of Chromodoris zebra. W. J. Crozier, B(>rmu(la

Biological Station and L. H. Arey, Northwestern University Medical

School.

Differentiated receptive mechanisms mediating reactions to tactile, chemical, and shading stinudation, to the constant intensity of light, and jK'rhaps to heat, induce local responses through the agency of peripheral, nonsynaptic, nerve nets, which in the gill plumes and perhaps in other parts exhibit deciiled polarization. Reactions of parts distant from the site of local activation involve central, ganglionic, synaptic transmission.

The nudibranch is, probably through the eyes, positively phototropic. The bi-anchia! collar is also sensitive to light, which causes the gill plumes to be extended. The gill plumes react, variably, to shading. Sexually mature individuals are negatively geotropic. A temperature of .'il-32°( '. induces negative reactions. The 'rhinophores' are directive organs for negative rheotropi.sm in strong ciUTcnts. \'ibrations transmitteil through the water are not responded to. Chemotropie responses are imi)()rtant I'nr conjugation. Locomotion is mainly muscular, and is jiccomplished by the lateral margins of the foot which sucks locally. The i)ositive slereotropism of the anterior end of the foot is responsible for righting.

W. The relalire stimulaliiKj rfiicirncy of continuous and intennitlcnt light upon Vanefixa antiopn. \Villi.\m L. Dolley, Jr., Randolph-Macon College, Ashland, Va.

I'lU'ther investigation of the reactions of Vanessa antiopa in intermittent light shows that at cert.ain llash-fre(|uencies the stimulating effect of intermittent light is greater than that of continuous light of e(iual illumination; at other (lash-fre(|Ui-ncies it is less than that of continuous light; and at still others it is e(|ual to that of continuous light. The intermittent light used was of an illumination of 3.5 m.c. and was

THB ANATOMICAL RKCORD, VOL. I.V NO. 6

34(5 AMERICAN SOCIETY OF ZOOLOGISTS

produced by a rotatinR sectored disk witH one-fourth removed. At a lliusli-ficciuciicy of 20 per second 7 out of 10 insects reacted more stronKly to intermittent light tlian to continuous light of ('(pial illumination. At tlash-fre(|uencies of 2 and o per second 100 and 80 ])er cent respectively of the insects tested reacted less strongly to intermittent light than to continuous light. At tlash-frequencies of 10, 30, 10, 50, 00, and 100 per second most of the insects tested reacted etiually strongly to intermittent and to continuous light. These experiments consequently siii>\v that the stimulating efficiency of intermittent light depends upon the flash-frequency and that it may be greater, equal to, or less than that of continuous light.

//. The rates of COi production by starved nnd fed Paramccia and their possihle relation to the rates of oxidation in the unfertilized and fertilized sea urchin egg. E. .J. Luxd, I'niversity of Minnesota. Feeding a starving Paramecium with yeast or yolk of hen's egg increases the rate of CO2 production by the cell from two to three times, thus confirming previous published results on oxj'gcn consumption. This acceleration of the oxidations occurs in the absence of cell division. The process of cell division, as such, in all probability is not associated with any marked change in the rate of oxidations. These results are so closely parallel to the conditions obtaining in unfertilized and fertilized sea urchin eggs that it becomes highl>- jirobable that the acceleration of the oxidations subsequent to fertilization of the echinoderm egg is due to the fact that the yolk of the egg becomes available for assimilation by the living protoplasm of the egg dm-ing the act of fertilization, and in this way results in increase of speed of oxidation sunilar to that in a fed Paramecium.

12. The photoreactions of partially blinded whip-tail scorjnons. Bradley M. Pattex, Western Reserve University, School of Medicine. Reaction measurements previously made on normal whip-tail scorpions (Patten, 1917) wer(> used as a liasis of comparison for measurements made on partially blinded animals sui)jected to the same conditions of illumination. The change from the normal reaction induced by the covering of a photoreceptor was taken as anindexof theeffectivenes.s of the photoreceptor prevented from functioning.

Each of the photoreceptoi-s (median eyes, lateral eyes, and cutaneous sensitive area.s) \va.s eliminated unilaterally, and bilaterally; singly, and in combinations with other receptoi-s.

All animals in which the receptive niechanisni was left in a functionally asymmetrical condition exhibited, when subjei'ted to bilaterally balanced illumination, deflections toward the side which had been made less sensitive. The amplitudes of the deflections were proportional to the degree of unbalance which had been produced in the photorecept i ve mechanism.

Animals in which the receptive mechanism wa.s left in a symmetrical condition showed an undisturbed balance of reaction when subjected

PROCEEDINGS 347

I to the actifin of fqual opposed liphts. Under lateral or anterior illumination, ainplitiidc of the deflection was reiluced in proportion to the extent of the interference with the receptive inechanisni.

By comparing the changes from the normal reaction induced by elimination of the various photoreceptors, their relative effectiveness can be approxmated as:

median eyes: lateral eyes: cutaneous sensitive areas: 1: 1.6: 2.2.

13. Excretion crystals in Ameba. A. A. Sch.\effer, Universitj' of

Tennessee.

Nearly all species of ameba contain visible crystals in the endoplasm. In most species, if not in all, the crystals are surrounded by a vacuole; they do not lie in contact with the protoplasm. The crj'stals are nearly always optically active as indicated by t"he polariscope, though sometimes they arc not. The shape and size of crystals in amebas are of the fii-st importance in species determination. The composition of the crystals still remains uncertain. Thej' are probably an excretory product of some sort. They are not excreted to the outside and they do not seem to be dissolved once they are formed. After the crystals are onde formed they seem to remain for a long time within the ameba. Very rapidly dividing Amoeba protens have verj' few crystals while those dividing slowly have many crystals. Those that do not divide for six to ten da.vs become stufTed with crj-stals. Several individuals of Amoeba diacoide.t, a species closely related to proteus, that did not divide for thirty days liecamc so loaded up with crystals that they were quite opaque and locomotion was accomplished only with difficult}'. Crystals may thus have two possible fates: they may be dissolved afeain — for which there is no evidence, or once formed thej* remain so long as the ameba lives.

14- The reactions ami resistance of certain marine fishes to H ions. V.

E. SnELFOUD, University of Illinois.

The more sensitive marine fishes react to differences between a pH of 8.1 and 8.2 produced by the adtlition of a very small quantity of H2SO4 to sea water, at one end of a gradient tank. This takes place in a manner which indicates an ability to distinguish differences in pH of 0.025. Some species select the higher H ion concentration; others select the lower, according to their halntat relations. As the H ion concentration is increa.sed above the optimum the fishes become less able to distinguish differences; the reaction to a difference such as that lx>tween pH 7.0 and 7.2 is less definite than that indicated above. The Pacific herring reacts negatively to 0.8 part per million of sulfurous acid (HoSO.i) in a manner which indicates an ability to distinguish 0.6 part p(>r million. In this case the difference in H ion concentration is very slight, probably too slight to be distinguished. Reactions to other chemicals indicate that small amounts of other ions may predominate over small H ion concentration. .\ light increase in II ions above neutrality (pll 6.80) is fatal to herring. Other fishes are more resistant; the flat fishes are remarkably .so.

348 AMERICAN SOCIETY OF ZOOLOGISTS

/.). .1 siiiiplf milluxifor measuring the COj produced by protozoa nnd other siiuill onjtiiiismx. !•". J. Li'ND, rnivcrsity of Mimicsotii. The ii|)p:ir:itus consists of a wide iiiouthod ulass stoppi-rcd hottlc of about 100 CO. capacity, from the stopjH'r of wliidi is siispciided a small flat stciid(T dish coiitaiiiiiip llic organisms. ( )n the hottoiii of tlic bottle is placed a kimwii (|iiaiitity of weak BalOIIlj for absorbing tlu- COj from \arioiis sources in the l)ottle. With properly arranged controls the CO; prcxhiced l)y the organisms may be determined.

Tests in whieii were used known (juaiitities of Na-jCO;, showed that with careful mani|Milation one is al)le to determine to witliin about 5 p<>r cent error, a ((uantity of ("Oj equal to that set free from one milligram of \atC'(^3 by an acid. Many duplicate determinations may be made during the same period of time, and temperature accurately controlleil by immersion of the bottles in a constant temperature l)ath.

h). The effect of KCX on the rate of oiijijen consumption of I'lanaria.

TiEORGE Delwin Allen, Univcrsitv of Minnesota. (Introduced by

K. ,1. I.und.)

The oxygen consumption of Planaria was measured by the Winkler method, and it was found to l)e reiluced by ().()()n2 molecular KCX to less than 30 per cent of the normal. The amount of the i-eduction varied with the concentration of the cj^anide. Weaker concentrations, however, caused proportionately greater reduction than stronger concentrations.

The rat(> of oxygen consuniption in a cyanide solution was practically the same during periods of ditTerent length from 2 tf) 30 hours.

The action of the cyanide was I'eversible in that worms recovered their normal rate of oxygen consum|)tion rapidly an<l completely after removal from the cyanide .solutions.

Worms in a solution of 0.00007G molcculai- KCN absorbed only half as much oxygen per gram body weight as worms that were made inactive by cutting off the heads.

Cyanides reduce the rate of oxidations in Planaria, therefore, by oQ per cent or more, independently of their action on nuiscuiar and ciliary movement. •

17. The influence of temperature and concentration on the toxicity of sails to fish. Kdwi.v R. Pf)WERS. Colorado College. (Introduced bv V. E. Shelford.)

The effect of temperature on the toxicities of the chlorides of lithium and ammonium does not follow van't Hoff's rule in its entirety. The relative toxicities of lithium chloride at difTerent temperatures to the goldfish follow very closely the square root of relative standard metabolism of the goldfish as given by Krogh. .\ close approximation of the relative <leleterious efTect of obnoxious substances to fish can be determined by comparing the constants of the e(iuations of the theoretical velocity <if fatality curves of the fish when kilfed in these substances. The resist:ince of the fish to these substances can be determined bj- the

PROCEEDINGS 349

same method ainco the resistance of the fish is the reciprocal of the de leterioiis cITcct of the sutistiince on tlic fish. The rehitivc toxicities of NaCl, M(;( 'I2, CaC'lj, and lia( 'I2 to the bluiit-iiosed minnow {I'iimphales nolatus Raf.) arc not the same as the relative conductance of these salts.

18. Further contributionx upon the nntunil histori/ of Chromodoris zebrn:

the (lue.stion of atlnptive coloration. W. J. Croziku, University of

Illinois, College of Sledicine.

This species is availalile for observation, and in great numbers, during most of the year. Size and eharaefer of pigmentation vary considerably. Several significant features of the color-variation have been measured. At certain periods as many as half the intlividuals collected have been found to exhibit extensive injuries, probably inflicted by fishes; the.se injuries are rarely fatal. Less serious are the smaller injuries encountered u|)on the mantle-margin. The Rills, also, which vary in coloration, are fre(|uently found to have been bitten. The origin of such injuries through the l)il('s of fishes has been watchetl.

These facts have made |)ossibIe the quasi-(iuantitative study of a situation uiii(iue in its importance for the theory of animal coloration, surpa.ssing in critical value that known in anj' other species where of the coloration has b(H>n attentively examined. It is found, for example, that although the brilliant "yellow" element in the pigmentation of C. zebra contains from 15-65 per cent orange with 25-35 per cent yellow, in different specimens, the incidence of the several types of injuiy is in no way correlated with the intensity, or the manner of distribution, of the yellow pigment. Similar qviantitative comparisons of the fretiuencies of injury for each of the s(>veral kinds of gillcoloration, and of mantle-margin i)igmentation, lead to results of a character agreeing with this conclusion.

In the light of other pha.ses of this investigation, it must therefore be considei'ed that ('. zebra is neither invisible, nor for any reason inaccessible, to animals which might inHict damage upon it. The chief value of the facts recorded lies in their being indejiendent of any hiunan notions relative to the concealing or revealing function of the coloration of C". zebra. It is demonstrated that although variations appropriate for the natural elimination 'less adapteil' types of pigmentation is present, and although a conceivably 'selective' agent, the biting of fishes, is known to be at work, no one of the mo<les of coloration is in fact more innnune than another.

The conclusion from this study is in agreement with my other r(>searches on this topic: the coloration of C. zebra has no 'warning' significance, neither is it 'concealing:' it is not homochromic upon natural backgrounds; the animals themselves provide evidence, independent of tile investigator's ideas, which shows that an efficient repugnatoral mechanism is ])ossessed by this midibrancli. but that its coloration may not legitimately be regarded as adaptive either in its origin or in its present significance.

350 AMERICAN SOCIETY OF ZOOLOGISTS

19. The zoological significance of the functional fcnestral plate in the ear rni>nHh of caudatf amphibia. H. D. Reed, Cornell University.

The manner in which elements coinliino to form the definitive fenestral plate in the Tailed Aniphiliia suppests a division of this order into two legions each with its own iiartieular niori)ho!opic type of fenestral structure. One lepion includes the .Viiiliystoinidae, ("ryptobranchidac, Salamandra, Sircnidae, Triton and Dieniictylus. The other includes the Necturidae, Aniphiuniidac, Typhlomolgidac, Plethodontidae, and Desnuipnathidae.

The perfected apparatus could have been useful only in a terrestrial environment. This indicates that all living forms have passed through a pronounced terrestrial period and those which are now aquatic are .secondarily so. It is interpreted that others are gradually changing to an aquatic abode possessing already a long larv'al period while otliers still have never experienced a regressive radiation and exhibit in structure and the suppression of the larval stage a more perfect harmony with terrestrial existence.

20. The coloration and habits of West Indian and Hawaiian reef fishes. W. H. LONGLEY. Goucher College.

The coloration of fishes and the habitual relation they sustain to their environment are correlated upon the same terms in the West Indian region and in Hawaii. Their fixed colors, with the exception of red, repeat the dominant color notes in their surroundings, and their transient color pha.ses are demonstrably induced by the nature of the places into which thcj'- move. There is evidence too that patterns, no les.s than colors, are displayed according to sj-stem. A\'hen. for example, a fish may appear in either a cross-banded, a longitvulinally striped, or a self-colored pha.^e, there is marked tendency for the first-mentioned to appear when the individual is at rest, and one of the others when it moves, or is about to move.

In displaying their colors and patterns as indicated scores of species of fishes eonfomi. it .seems safe to say, to a natural system of camouflage, whose principles are capable of experimental demonstration, for the simple reason that the creatures possess the ability to respond visibly to the tests to which they may be subjected. Appreciation and formulation of these principles would place naval camouflage, for example, upon a scientific rather than an empirical basis.

The new ob.ser\ation that some fishes ciiange their coloration as they rise vertically, and leave the bottom .'uid its influence, supi)lements the knowledge that they comiiionly change their apjiearance as they move horizontally from surroundings of one sort to those of another. ]")ifTerences in positi(Hi of comparatively few inches may be followed habitually in some species by definite changes in coloration. It is not inqirobable then that colors or patterns appearing in some species during the breeding season alone do not differ in function from those displayed at other times by the same species under different conditions. Ry as much as this is true, "nuptial coloration" is an index of changed location for a

PROCEEDINGS 351

period, and ixfcpt tliat it may be cvoi^ed by interna! changes dependent upon the sexual cycle may bear no more intimate connection with the process of reproduction.

Pictures will be shown illustrating some characteristic differences in habit on the part of fishes, the extent and character f)f their color changes in nature, and the limited possibility of securing i)ictures at present showing their surroundings in their natural colors.

21. SttggeslioiiK as to the climograph of drci^litous forent inrertehratex as iUuslratrd hi/ ixperimenial data on the codling moth. V. E. Suei^ford," Illinois Natural History' Survey.

The climograi)h (a graphic expression of the relation of an animal to temperature and humidity or evaporation in combination) of animals belonging to different climates may be expected to show characteristic differences. 'Hie results on the codling moth show a wider range of humidities which give successful emergence of pupae, at lower than at higher temperatures. Temperatures above 89.5° F. retard development: teiiiiieratures below 58° F. give proportionately more rapid development than temperatures above 58° F. The shortest pupal life for any constant temperature is usually at the lowest evaporatitm or the highest humidity, and the time at the higher evaporations and lower humidities is usually less than the maximum. Air movement may modify the lengtli of the pupal stage 20 per cent. In general the climograph is oblicjue in the same manner as that for man as shown by Taylor.

22. On the nature and source of some adaptive features in the etliolngj/ of Chitofi. W. J. Crozier, University of Illinois, College of Medicine. A discussion of some progressive modifications in the habits of Chiton

tuberculatus as its age advances, involving: advantageous adjustments in the matter of food supply and the operations of feeding: certain features of coloration and ai)pearance: a probably significant degree of assortive fecundation with resj^ect to size (age); and the mechanism whiMcby the fertilization of the eggs of older females is insured. With particular reference to the way in which these aspects of the life of chiton are interconnected, and their dependence upon the heliotropism of these animals as influenced directly by environmental disturbances.

23. The anlagc of entoderm and of mesoderm in the opo.'^sum. C.kkl H.\nTM.\N, The I'niversity of Texas.

In blastocysts of about C)0 cells (about 24 hours) certain cells in the fonnative half of the egg grow in size, round u]i and migrate into the cavity of the vesicle. These are the entoderm mother cells and form the anlage of the ento<lenn. They nuiltiply and flatten out against the inner surface of the embryoni(' ectodenn.

When the vesicle has attained a diameter of 1.5-1.8 mm., (he mesoderm begins to proliferate (beginning of the sixth day of gestation, days before parturition!) The axis of the embrj-onic area is indicated before the appearance of the mesoderm by the thimung of an eccen

352 AMKHKAN SOCIKTV OK ZOOLOGISTS

trically (= posteriorly) situated patch of ectoderm (seen as a light field hy tnmsmilti'il linlit). The first incsodcriiial coils can he rcconiiizcd with certainty in the opossum cuK because the enihryonic area in lliis form consists at this stane of a single layer of cells. The first mesodermal cells migrate down out of the ectoderm in a roundish group in the mid-sagittal plane of the embryonic area behind the lifiht field just mentioned. The uroup soon elouRates and theanlageof the primitive streak is indicated. Their oi-ifjin is strikingly similar to that of the entod( rm: both nerm layers ari.se by nn'tiration of cells from the midifferenliated su|)erficial layer. The pre|)aralions sliow that the entoderm makes no contribution to either the jirimitive streak or the head proce.ss.

The paper is illustrated by a series of photographs of the eggs in the living state, of .surface views and of sections.

54- The oestroux ci/cle in rats. J. A. Long, University of California.

The length of the cycle averages very nearly five days. It is marke<l by changes in the vaginal and uterine nuicosae and by the liberation of eggs from mature follicles. In the vagina at the end of the dioestrum the nuicosa thicki'us greatly as the result of mitoses (stage 0) : the outer cr'Us become stripped ofT exposing a cornitied layer which is dry and histerle.ss (stage 1); the cornified cells become loosened to form a slight amount of chee.sy .substance (stage 2); stage 3 is marked by the ailvent of leucocytes, the disappearance of the cornified cells and the desquamation of the deeper non-cornified cells which together with the leucocytes characterize the dioestrum. The musoca is now moist and glistening.

The uterus, besides exhibiting changes in its mucosa, during stage and the first part of stage 1 becomes greatly distended by the secretion of clear fluid lin which sperm become very active) which diminishes toward the end of stage 1. Copulation takes place during stage 1. Ovulation occurs at the end of stage 2 or at the Ix-giiming of stage 3. The uterine mucosa is regenerated at least in part by mitosis of its cells.

Suckling may delay the .second ovulation following parturition about 40 days. The first ovulation follows the op<'ning of the vagina by about a day or two. During the first few weeks following puberty the cycle is lf)nger, 9 to 17 days.

The cycles following infertile coiiulations are usually 10 to 19 days long. Stimulation of the cervix of the uterus by merely in.serting a glass rod during stage 1 [irolongs the next cycle to 1 1 to 19 days! It is suggested that the vaginal plug acts in this mechanical way.

£6. ResulLs of extirpation of both thyroid and pituitary (jlandx in tadpoles of bnfo and rana. (5 minutes.) Benxett M. Allen, University of K.'insas.

Tadpoles from which the first beginning of both thyroid and pituitary glands had been extirpated, developed in jirecisely the same manner as <lo those from which the pituitary glands alone have been re

PROCEEDINGS 353

moved. iMght arc still liviriR eight months after removal of these glands. Tlicy show the same eolor changes (jbserved in tadpole.s from which the pituitary gland has alone hcen removed. The ilcvelopment of the hind liinl)s takes place at the same rate and to the same degree as in tadpoles from which either the thyroid or pituitary gland alone has been removed. The germ glands develop in proportion to the size of the body.

26. Miscellaneous notes regarding experimental studies upon the endocerine glands of rana and bufo. (10 minutes.) Bknxet M. Allen', University of Kan.sas.

1. Numerous tad|)oles upon which removal of the thyroid gland had been attempted, metamorphosed tardily and at an abnormally great size. In these, more or le.ss imperfect thyroid glands were found. One giant thyroidless Hana pi]iiens tadpole transformed, one year after o])eration. into an unusually large frog (31.1 mm. body length") 27.9 per cent longer than the average length (24.3 nun.) of ten newly metamorphosed controls.

2. Two Bufo larvae and one Hana, all operated for removal of the hypo|)hysis. showing the characteristic light color produced by succe.ssful removal, transformed at a body length well below normal. E^ch contained an imperfect hypophysis but fairly well developed thyroid gland.

3. Pituitan-less Rana tadpoles were placed in solutions of Parke Davis' Pituitrin mixed with water in the proportion of 1 to 200, 1 to 1000. 1 to 2()()0.an(l 1 to 4000. In spite of this, the tadpoles showed their characteristic color change at the usual time inter\-al after oiM-ration.

4. The writer, in collaboration with Miss Mary Larson, fetl the anterior lobes of the pituitary glands of cattle to thyroidless tailpoles. The experiment was begun June 22nd and is still being carried on. The.se tadpoles show no greater tendency to metamorphosis or size increa.se than do other thyroidless tadpoles.

.5. The parathyroid glands of thyroidless Rufo ta<lpoles were measuredandfo\md to be much larger than the jwrathyroiils of normal controls, both of corresponding stages and of newly metamorphosed toads. This is still markedly true when allowance is made for differences in body size.

27. Effect of the extirpation of the thyroid gland upon the pituitary gland in bufo. M.\RV Kliz.\hktii L.xkson, I'niversity of Kansas. (Introduced by Helmet .M. .\llenl.

.lames H. Hogers'17 arrived at the conchision that the pituitary gland continues to develop when the thyroid gland is extirpate<l and the anterior lobe reach(>s a larger size actually and relatively than in nonnal specimens. It was felt desirable to test this conclusion in a different amphibian type ami also to make a study of the pars intermedia and the histology of the glanil.

354

AMERICAN SOCIETY OF ZOOLOGISTS

I^ist spring over five hundred thyroiil glands were removed. One hundred and eighty Inaiiis of the thyroidless and control specimens were dissected out and luoasured.

The specimens were ixurod according to body length. They varied from live to twenty-nine millimeters. Not only was it found that the anterior lobe increased in size in the thyroidless specimens but that the pars intermetlia as well grew larger in comparison with their respective controls. The five millimeter tadpoles already showed the effects of the removal of the thyroid gland. Increase in the size of the ghmd increased with the growth in body length. The gra])h will show this fact. In the thyroidless Bufo which measured from five to about twelve millimeters the pars intermedia arched around the anterior lobe while in the control the pars intermedia tended to lie in a straight line. This fact was not nearly so evident in older thyroidless specimens.

The larger thyroidless specimens were paired with still larger controls. The following average measurements for ten pairs of the larger specimens will show distinctly the increase in size of the different lobes:

.•VvernRe total length

.Avorape body length

.\vcragc fore leg length

.Vvcrage hind leg length

Average vertical di.ameter of anterior lobe

Average horizontal diameter of anterior lobe

Average length of pars intermedia

.\verage width of the right part of the pars intermedia. Average width of the left part of the pars intermedia. .

CONTROL

THTROIDLESa

mm.

mm.

22.5

42 4

22.5

18.1

13.0

27.7

4.5

0.3714

0.6822

0.4001

0.6640

0.74r>S

1.0234

0.13(>1

0.3124

0.1386

0.2837

An actual diminution takes place in the size of the pituitary gland at the time of metamorphosis.

There is a distinct difference in the histology of the normal and thyroidless pituitary glands. The nuclei of the thyroidless pituitary are somewhat larger than the nuclei of the control pituitary and in almost everj- ca.se the nuclei of the control arc angular and wedge shaped while in the thj-roitUess they are almost spherical. The control gland presents a very compact appearance while the thyroidless one is quite loose in texture.

Further study will be made upon the histology of the gland.

£8. The solitary and the aggregated generations in salpidae. Maynard

M. Metcalf, Orchard Laboratory.

The comparative study of all species of Salpidae shows a gradual modification in the evolution of the members of the family from the C'yclosalpas to the true Salpas (sensu strictu) along one line, and along another line from the Cyclosidpas to the Oli-gomyari<i. The modification is evidenced very clearly in the condition of the muscles, of the gut, and of the nervous system, especially the eyes.

PROCEEDINGS 355

Arraiinii>»j the several species in order accoriiiiiK to the deRree of diverRence, in these several regards, from the Cyclosalpas and comparing with one another the solitary and aRgreRated forms of each species, one sees that, in the evolution, the aRKrenated Rcneration is the first to respond to the modifying influences, whatever they may be, and the solitary' generation is more conservative. In most species the solitary form shows more archaic character, the aggregated fonn a more divergent structure. However, in the most highly evolved species, namely, the higher (Hujonujaria. even the solitary forms have reached a condition very chvergent from that of the Cyclosalpas. The aggregated form leads in the evolution, but the solitarj- fonn, at the end of the series of species, becomes almost equally modified.

Is not this wholly natural? The Salpa life cycle may be expressed as

. Egg X sjiemi Embryo Solitary forms Stolon with buds Aggregated form with eggs and sperm

The aggregated zooid is therefore the latest form in the ontogeny and might naturally l)e expected to be less conservative than the solitarj' form which is an earlier stage in the ontogeny. This is but another instance of a verj' familiar general principle.

29. Correlation of ferUUtij and fecundity in an inbred stock. RoscoE R. Hydk, Indiana State Normal School, Johns Hopkins University. Over 9o per cent of the eggs isolated from a mating of the wild Dro so|)hila ampelophila gave rise to mature flics. On inbreeding the fertility rapidly declined. The fecumlity of the female was. not affected in this way. The correlation between the number of eggs which a female lays and the percentage which give ri.sc to mature flies is verj' low. This would seem to indicate that the sterilitj' as it affects the female bears no causal relation to reiluccd fertility.

30. The extent of the occurrence of sex intergrades in CIcuiocera. Akthvr M. B.\NT.\, Station for Experimental Evolution.

Sex intergrade strains of Simocephalus vetulus have been reared in the Laboratory for three years (0.5 generations). These all came from the offspring of a single individual. Notwithstanding careful microscojiic examination of thousands of individuals of all the laboratory strains (1.5) of this species, |>articularly during the last 20 months, no other sex intergrades have been found either in the strain which produced them originally or in any of the other strains of Simonephalus.

About 20 months ago sex intergrades were found in one of the strains of Daphnia longispina and from these we have propagated sex intergrade strains for some 3() generations. During the next few months sex intergrades were found (sparingly and only after the microscopic examination of thousands of individuals) in all except one of the six strains of

356 AMERICAN SOCIETY OK ZOOLOGISTS

this si>ecies under ciilliviiliDii. Sex intergradp strains derived from tlirtt' distinct strains of this species are liwing jiropaKated. Two or tliree sex internrades were also seen in a strain of this species in 1'.)15 but no yo\niK were secureil from tlieni.

Long and conliimed seardi of great nuinl)ers of inchviihials of 18 strains of Daplinia pulex, 7 strains of SimocepliaUis serruhitus. and of 11 strains of tiiii'e species of Moina has not revealed a single sex intergrade individual. Hence in these species as well as in Simocephalus vetulus the occurrence of sex intergrades is apparently a rare i)h(>ii<)menon. Sex intergrades are relatively rare in I)ai)hiiia l()iigisi)iiia as well, although iahoriuus searcli has revealetl them, mostly a single individual to a strain, in five of six strains. Once established, however, intergrade strains continue indefinitely the jiroduction of sex intergrades.

In the literature there is, pr«.sumalily, only a single mention of the finding of sex intergrades (K. de La N'aulx). In. view of the large number of workers with Cladoeera and the extensive experimental work on this material the fact that there has been apjjarently only a single occurrence of sex intergrades in other laboratories speaks further for the restrictetl occurrence of these interesting sex forms.

SI. Nuclear reorganization and its relation to con juration and inheritance in Arcella vulgaris. H. M. M.\i('.\rdv, Alma College. The data from pedigreed cultures of Arcella vulgaris maiiilaiiied

from Sept., 1917, to Aug., 1918, have given the following conclusions:

1. A given individual produces a limited number of daughter cells The numljcr varies from none to twenty-seven (the highest found).

2. These daughter cells and in turn their offspring behave in a similar way with the exceptions indicated.

3. After a period of fairl}' regular successive vegetative divisions, a period of 'depression' occurs. Some of the features marking this period are: reduced activity (feeding, locomotion, division), 'Punctate' shells, 'empty' shells, increased mortality. These are incidental, not essential.

4. Individuals pa.ssing successfully through this period may give rise to a new line unlike that from which it came — a marked change in size, for example. This is a 'mutation.' On the other lumd, the new may be like the old line. A new period of vegetative divisions sets in and continues until another period of depression is reached.

5. While some mend)eis of a line are 'de|)ressed,' others conjugate.

(). In pedigreed cultiu'es of exconjugants the two members of the pair tend to produce the same nund)ei>; of daughter cells. This is in agrcH'ment with the fission rate of exconjugants in Paramoecia (Jennings).

7. In Unvs derived from exconjugants, after a period of vegetative divisions, individuals pass again into another period of depression, when the changes noted above luid (or) conjugation may be repeated.

8. Preparations of cells made during 'depression,' and of conjugating cells show remarkably similar conditions of both chromidial net

PROCEEDINGS 3o7

and niiilci. Old nuclei are l)r<ikcii u|) and new niK-lci arc formed. Tliis is the period of Nuclear l{eor(;aiiizalioii. This may occur williin a single individual or through conjunation of two individuals. (In l)o1li iiernianent and temporary mount.s.)

'.). The inheritance of size show.s changes at these periods in individual lines.

10. The followiiiK niodifyiiiK factors .should he mentioned: Cultural conditions influence the procedure -unfavorable conditions appear to hasten "depression' and very favoralile conditions, to delay it. The dilTerent nuclei do not always divide at the same- time or pa.ss through similar stages together. There is also evidence to show that the essential change may occur with no great Iireak in the usual course of events, and the new arise almost or (piite imperee|)til)ly.

(The complete results are being prepared for the printers.)

32. Several u'ays in which (ii/nandromorphis-m in inficds may arise. T. H. MoKG.\N. Columbia rnivei"sity.

Gynandronioiphs have appeared in Diosophila .'? times in lt).()37 flies; 32 times in fJ.lOU; 2 times in 4.<.t7<.t and 3 in 24,()()0; thus in the ratio of 1 to 2200. There is evidence that nearly all of them start as females; 19 were more female than male: 14 were half male half female; and (j were more male than female. Practically all the cases fovmd are ilemonstrably due to elimination of one se.\-chromosome soon after fertilization. A few call for other chromosomal relations. Rarely one may even have begun as a male, but nearly all cases supposed at first to belong to this category have proved to be due to mutation in the sex-chroniosom(\ .\n cases of hybrid Gynandromorphs found in bees can also be explained by the theory of chromosomal elimination. A few cases in Drosophila seem to be explicable oidy on the assumption ofa bi-nudeated egg, and this explanation is the oidy one found so far that will give a consistent explanation of Toyama's two tJynandromorphs in the silkworm moth. Bi-nudeated eggs have been described by Doncastcr in other moths.

33. Duplication. C. B. Bridges, Columbia University.

In Drosophila melanogaster several cases of abnormal inheritance are accounted for by the assumi)tioii that in each case a piece of chromosome has been taken from its normal position and joined to another chromosome.

In the first of these cases a section of the X-chromosome, incluiling the loci for veiinilion antl sable, became detached from its nt)rmal location in the middle of the X-chromosome antl became joim-d on to the 'zero' end (spindle fiber) of its mate. For certain loci this latter chromosome carries two sets of genes — those present in the nonnal location and also the duplicating set. If a male carries the recessive genes for vermilion and for sable in the normal loci and the wild-type allelomorjihs in the dujilicating loci, he is wild-ty|ie in aj^pearance preci.sely as though he were an XX female heterozygous for vermilion

358 AMKKICAN SOCIETY OF ZOOLOGISTS

tiiul sal>l<>. A ft'inulc having one such chioinosoinc iuid a iioriiial chroiiiosoino carryiiiK the vcriiiilioii ami sal)lo pones is triploid for tlicsc loci. It lias thus liocn proved that true recessive genes may dominate .one dominant. .\ female tetrajiloid for the.se loci can he made, and l>v this means it was shown that two recessives an; recessive to two dominants, ("ri.ss-cross inheritance of the Abraxas type can be initiated in Drosopiiila by crcssing one of the above wild-type females to a vermilion sable mate, for the daughters are vermilion sable atid the sons wild-type.

In another case of duplication the duplication piece contains only the locus for sable as far as known. In both of these cases the duplicating piece is joined on at the zero (>nd (spindle-fiber), and experiments can be made in which the linkage of vermilion and sable will indicate a locus at zero instead of at 33 anfl 43, respectively.

A third cas(» is the transposition of a i)iece of the second chromosome to the middle (si)indle fiber) of the third chromosome. The genes of this duplication piece show linkage to both the second and the third chroino.some at the same time. In this third case both the duplicating fragments attached to the III chromosome and the II chromosome that suffered deficiency are on hand. Any gamete that receives this deficient II chromosome dies unless at the same time it receives the third chromosome carrying the missing piece.

The most significant bearing of these cases is upon the idea of evolution of chromosome groups.

THt ANATOMICAL HKCOHD, VOL. 15, NO. 7 rSDHU&HY» 1910

Rcsumido por el autor, Joseph M. Thuringer.

Anatomi ii de un cerdo dic6falo (Monosomus diprosopus) .

La anoinalia (|Vio ;?e describe en el presente trahajo se prct6 en un cerdo a termino que pes6 575 gramos. El cuerpo y cuello aparecen normales : pruebas de una fusi6n del esquelet o comienzan a aparecer en la scjitima \Y'rtebra cervical, haci(ndose mds distintas hacia la cabeza. Esta jiresenta una cara dercha, otra izquierda y una cara compuesta con dos ojos aloeados en una 6rbita comiin. Los dos hocicos estan bien separados, los orificios nasales y las bocas se abren en una naso- y oro-farfnge comunes. L'na lengua rudiment aria situada en el techo de la farfnge oblitera toda la cavidad de esta. Las lenguas derecha e iz(iuierda, las mandibulas y las glandulas salivares de las cara compuesta estan tambien fusionadas. L'na arteria carotida azygos, derivada de las arteria car6tida comiin izquierda, susniinistra sangre a la cara compuesta. Exist en dos ejes cerebrales casi normales que estan unidos entre si en la m^dula oblonga. L'na tlu])licaci6n bilateral completa existe en los nervios craneales, desde el primero al octavo par. El aparato auditivo de la cara compuesta presenta un caso linico de fusi6n.

Traoslalioo by Dr. Jos£ F. Xonidoz Columbia University

,>^

acthor'h AWTHArr ifr Tm« paper ibsubd

BT TUB lllSt.irK)ltAPUIC SEflVICB. lAin'ART 6

THE ANATOMY OF A DICKPIIALIC PIG, MONOSOiMUtf DIPRO.^OPUS

JOSKPH M. THURIXGER Department of Anatomy, School of Medicine, Tulane University

TEN- FICURES

Of the (liccphalio mnnstprs on rorord. tho majority arc oither human or of the calf. Detailed descriptions which wcjukl be of much value in classification or for statistical purposes on the subject of teratology are rather few in number.

Since the publication in The Anatomical Record of The .\natomy of a Double Pig, by Illien Carey, and The .\natomy of a Two Headed Lamb, by .\lbert M. Reese, I came' in possession of a pig belonging to the dicephalie autosites, .subdivision monosotnus dij)rosopus.

A diener in the laboratorj* of the school of medicine of the University of Alabama, who was instructed to empt^ the contents of a museum jar. which was in an advanced" state of decomposition, made the find and brought the specimen to the writer. It presented a number of features which were deemed worthy of recording.

TOPOGR.XPHICAL ANATOMY

The monster was a pig foetus (Fig. 1) apparently full term, cov'ered with black and yellow hair. Its length 24. "> cm. and weighing .")7r) grams.

Tlie body and neck appeared perfectly normal, both ventrally and ilorsally, there being no duplications or deviations in proiiortions between the right and left halves.

The frontal aspect of face (^!g. 2) showed two snouts which, excejjting the mandibles, were nearly normal and synnnetrical in appearance, exhibiting the usual number of teeth found at birth.

350

'M\0 JOSEPH M. TIIURINGER

I lie m;m(lil)l('s, howovor, diverged to such :i degree that proper closure of the mouths was impossible (fig. 3).

The two faces were symmetrical and fused in such a mamier that ill the 'middle"' (the region ix'tween the two heads), the angles of the mouths met. The two 'middle' eyes of the compound face were connected through the ocular conjimctiva; the right eye of the left face, as well as the left eye of the right face, occupying a common orbit, with one fused inferior and two obiifiuely iilaced superior jialpebrae.

Hctween the lower eyelid of the two 'middle' eyes and the 'middle" angles of tiie mouths there was a small orifice concealed in a whorl of hair, which I took to be the everted united ducts of the 'middle' parotid glands, but which subsequently turned out to be the fused external auditory canals. A Imstle introduced into this orifice is shown in figure 2, iliict.

In the occipital aspect, the line of fusion terminated posteriorly in an elevation circular at the base and about 4 mm. high. This was on a line with the two fully developed right and left lateral ears and represented the ' middle' rudimentary ears. Caudad to this point the transverse diameter of the occipital region and the neck diminished very rapidly to normal.

INTERNAL A\ AlOMV

Much difriculty was encountered in the removal of the skin, as the macerated underlying structures showed great tendency to break off with it.

The thyreoid and thymus glands were normal, the former well meriting its name.

The comjiound face presented a large fused and horseshoesliaped parotic! gland, with each of the two vertical or free portions lying in a fos^a formed by the 'middle' .surfaces of the mandil)les and a bony plate jirojecting from the junction of their angles. Ind( pi ndi lit ducts led from the free limbs of the gland to the right and lelt mouths.

' '.Middle,' hcrciifliir ii.scd for siikp of hrevily in .spcilviiig of structures in the rcftion of (he fusion of the two heads.

ANATOMY OF A DUKI'H ALU I'Ki

361

Fits. I. !{iKlil liitcral view nf mlirc spccimpii. Fig. 2 Vontral a.s|>i'ct of Sm-v.

Fig. 3 Siil>n\:ixillary ri'gioii. S.(;.. fiined .suliiiiuvillary gland; (l.H.. geniohyoid muitcleM.

w>

JOSKIMI M. THIRIN'CEH

riic fuM'il cNtrni;!! Miiilitory (■••iiial^ (•(iiitiimctl >lif!;litly to the left UMilrr cover of :i cuimmI Ixhin i)lat(' to disappour hciicatli tlic fused zyjjoiiiatic aicli (('1^. 4i.

The "middle" sul)iua.\illary glands, also fused. ])icsented au irregular, somewhat twisted mass, the greater portion of it lying under eover of the body of the parotid glami and separated from the latter hv the nivelohvoid musrle.

«H«tt C<«'-i>t.A

Oft VfTttttfclA

'jt(c Extft«U CfenfiU

14t C:mj^n CtntiJA ijlfc 54*»«(kv\uvA

ivctu) Atti*iekU» ^twwnkri, A

Fig. 4 i)rii\ cavity. /:..l.('.. u.xiciiial ainlilDry canal: /'.. plate of bomsurroiiniliiigit ; 7.. proximal portiuiiof compound joint ; S. P. .soft palate stretched from palatine process of right to that of left side; A.C., azygos carotid artery, (icvered: H.E., L.E.. riglil and left orbits; /f. 7"., rudimentary tongue; />.D. an<l L.S.. right anil left tongues.

Fig. .") .*<clicni.itic arr.'ingoniciit of circulation.

Tlie suprahyoid giou]) of nniscics was airaiigcd in a jjeculiar way. The "middle" digastric and st>loliyoid nniscics were wanting. Of those attached to the "middle" portions of the mandihles the following was noted: the right nivelohvoid extended from the inyelohyoid line of the right niandilile toward the nndiaii line of the body and was fused with that of the left. The libei-s of ;ill four geniohyoids, exeejiting the most lateral ones, either united or decussated in the median line and were in.serted subsecpiently into the bifid l)ody of the liynjd bone.

ANATOMY OF A DKKPIIAI.K VK.

3()3

III rciii<i\ ill}; tlic >kiti cnxcrinjt tlic nuliiiicntan' cars a ^^mall duct appeared directly IkIuikI tlic line of fusion of the "middle" parietal lioncs (fig. (i).

Oral and pharvnj;e;d cavities (fip. 4). The alveolar processes of the ■ middle' superior maxillae in their |)osterior oneliiilt tinned };radiially toward the incdian line of each oral vault

lu^.!\r Sut'Ur.^

OoTS&tWu:

l"i)i. t) linrics of lu':i(l :iii(l iii'ck. R.I'.'.L.I'.' . rijilit :iiiil left 'niiddli-' parietal iiDtics: rt./'., I.J'.. i-iRlit and left lalcrai i>!irictal.-<; Es..{..\l.. oxtornal aiulitory meatus: L.F., left frontal Imnc.

Fi(j. 7 Dorsal view of cerelird.spinal axis.

Iiroduciii}!; a corre.-pondinfr constriction of the "initMli^' posterior nares.

The posterior nares opened into a large common nasopharynx. A globular mass covered with closely set fungiform papillae and attached to the roof of the pharynx was fouiul to ol>struct jiractically the entire cavity of the iiaso- and oropharynx.

{()4 josKPii M. Tiiriu\«;i
i{

milking n'<pir:iliciii iiiipossil)l(> i KT, lifi.4 i. 'I"liis stnidurc pnivt-d to Ih" a nidimt'iitaiy tunmic

'I'lii' two toiijjups were fused at tluMr l)as(>, their lowot ])oint of junction, coirospondinfj; to the true median line of the l)ody, was the last visible trace of fusion in the respiratory anil digestive systems. The single ejjiglottis was eonneeted to the ra))he of the compound tongue in the identical manner as found when only one tongue is ]iresent, namely, hy a single glossoepiglottic fold presenting nn eitlu'i- .-ide a glo-^(i-e))igliiitic fossa.

(•1H(■^■I.A^<>1!^ .s^stk.m

The heart presented nothing mmsual (Fig. 5). The intraventricular septum was complete. The two atria communicated through the foramen ovale. The ductus arteriosus was also patent.

.\ right and left l)rachiocephalic trunk was given off from the arch of the aorta. A single stem originating fmni the right lirachiocephalic trunk gave rise to the two common carotid arteries. The other branches from this source were the right subclavian and the right vertebral arteries and two minor branr-hes. The left brachiocephalic trunk h.id i)ut three branches, the left subclavian and left vertebral arteries and a small superior cervical ves.-el.

The left connnon carotid artery gave i-ise to a large azygos branch opjjosite the cricoid cartilage. This vessel (Azygos ('arotid. fig. .">) su])|)lied the 'middle' r<'gion of the two faces, taking the place of i)oth internal and external carotids in this region. The right and left lingual and ni;i\illary arteries of the middle region were all derived from this single vessel.

In the 'middle' temju'ral fossa the azygos carotiil divided into two branches, which disa])peared through an opening corresponding to the foramen lacerum, to t.ike part in the formation of the two circles of Willis, as the internal carotids of the right and left sides of the midtlle region.

There was no visible anastomosis between the vertebral .arteries, each one p;i>sing below its re^]iecti\-e jxins as the

ANATKMV OK A DUKI'II AI.IC I'I<; 3(J5

lia.-ilar aittiy to (li\ idi- at a liinlit-r level into the |)i)>terii)r eerobrals (Kin. o).

Only one lar^e vein was jMvsent in the niitldle regimi which (Iraiiicd tlie <iilniia\illary portion of this region ami eMi|)tie(l into till- left external jugular vein just above a level eorri'-|)oniling to the origin of the azygos carotid artery. There were but the two usual external jugular veins.

.SKKLKTo.N

C'audad to tiic first (lor>al vertebra the entire -keletoii was normal, l^ven the cervical vertebrae on superficial examination in the wet specimen presented no great variation from the normal excepting the increased transverse diameter as they apiJioached the skull. Figure six rejiresents their appearance in the dried >peciinen after the cartilaginous portions had shrunk away I'rom the osseous. The duplication of parts, to be seen in the figure, is no doubt due to the fusion of right and left integers.

The frontal. sui)erior maxillary, temjioral jirocesses of the zygomatic, temporal, parietal, great wings of sjjhenoid, occipital bones and manilibles of the right and left heads were fused in the middle region (figs. 3, 4, and 7). Of these, the temporal bones were modified to a gicater d(>gree than any of the others and deserve special mention. No trace was found of anything sinuilating the mastoid portions of the two "middle" temporals. The articular portions, from which normally extend the zygomatic processes of the temporal bones, were united and formed a crescentic articular groove (.1, fig. 4). The zygomatic processes, and perhaps a >mall jiart of the articular jilates as well. I'ormed a curved jilate which covered the fused external auditory canal> (P. fig. 4 and Bony Plate, figs. <t and Kll. This plate formed a compound joint by articulating proxinially with the articular groove just mentioned anil distally with the fused condyl(»id ])rocesses of the 'middl(>' rami of the niamlibli's. The de\'elopnient of the petrous portions was restiicted to the de\elopment of the semicircular canals and the co<'hleae of (Mther side. These wer(> united and arranged in relation to the mid<lle and external ears as indicated in figures '.I and 10.

m\

JOSEPH M. TIHRIN'CJER

llic cr.iiiiiil \.iiilt> wt'ic iiicoiiiplcto alonp the line nt union. Till' riplit aiitl l(>ft <hira fnnninl)ral licMU>pliriv-- in the tcmporo-occipital reijion.

\C AuJ Cuvft^

5: I^r E»r

i"^— Cxt C»r

L Int Ur

Kin. s Vi'iitral view of cerebrospinal axis showing fusion. Fid. !' Right side of fused auditory apparatus. Fig. !(• Left side of fusi-d auditory a!i|i:ir;itus.

CIA ifiAi. .m:i;\<ii > >v.-<ti:m

There were two separate cerehri, two cereljelli and two fu.-ied niethiliae olilongatae. The rigiit and left lateral cerebral henii.•<l>here< and the corresponding lateral halves of the midbrains, pontes, and the fused niedullae oblongatae were normal in size.

WMiiMV nV \ 1)1< Kl'll Al.K' I'lii 'M)7

All aliiiDniuilly l;ir>i;c lateral vciiuiclc was fnuiul in the rinlit latpial f{'rcl)ral licinisjjlu'ro. The rifilit and left lateral rerobellar hcniisplicrcs, also llic median cerelx'llar lobes (veiines). were normal.

riic rinlit and left 'middle' cerebral lieniis])here diminished somewhat in size in the oeeipital region. From tlii- point caiidad a firadual dimimitioii of the "middle" halves of the midbrains, pontes, and mednllae oblonpitae was ob>erved. The middle' cerebellar hemispheres were entirely absent. l"nsir)n of the two cerebrospinal axes was effected in the lower part of the fourth xcntricle .and was limited to the medulla oblontrata. ('omplete bilateral duplication of the cranial ncines. from the first to the eighth pair existed. The 'middle' glossopharyngeal nerves united to foini a single trunk which terminated in and around the rudimentary tongue, ('.•uidal this point no duplicate cranial nerves were found.

.\lDlTOin Al'l'AUATt .s

Most of the uncertainty about the genetic >ignitieance which the various |)arts of the fused auditory apparatus ])resenteil cleared u]) u])on their removal and dissection. Figure •.• is a sketch of the right side of the intact structures. Figure 10 shows the left side of same with the united external ears incised and cvcitcil. The ossicles formed a conglomerate bon\' ma>> in tlic centrally located dilated ])oition corresponding to the middle <ar> which was joined by the fu-ed external .-iiiditory canals, Fu>tachian tubes, and external eai's.

MICKATl IM: CUNSlT/rEO

(."llAl'VKAC llKMi ('(iiii|i:ii-:itivc anatomy of the (loln<■:^tie aniinal.s.

Oitimrcii, (;, !•;. IdlS !)imlil(-lic:iilc(l niciiistrosily. .lour. .\. M. A., vol. .'>I,

l>. io;:{.

I'l.tMKit. .1. ."<.. \M> .SliKKfKii. ('. .1. Kcport on a rase of ('('|>lialo-|litii:u'o|ia|:us inono.synirtros. Hull. no. IV. lutciiialional .\ssociation of .Mcilical .Mu.scuiUM. .\uK.. Itilli.

C.^HKV. ICliKX 1!M7 Tlic anatomy of ji (loul>l(> pijj. .■ivnccplialu.* thoracopagus, witli cspi'cial ronsidrration of tlio pi-nctic siisniticani-r of the circulatory apparalu.s. .Vnat. Hoc. vol. 12. no. I. pp. 177-I!IJ.

Ciii.iK. ICow.Min M.. Jh. I'M" Teratology. Kef. H<ll)k. .\l<-il .•<(•.. new c<l., vol. 7. pp. iLll-l.'JIi.

Kkk.sk, .Vi.HKUT M. HI17 The anat<uny of a t»o-lica>lc<l I:iinl>. Anal. Kcr., vol. I.'t. no. 4. pp I7!» I!I7

Kpsuinitlo pnr ol auti)r, A. (1. I'oliliiwm.

Ureteres dobles en enibrioues huinaiios y en los del cerdo.

Kl ])iosent(> trahajo contiene bcisquejos dc un embri6n humano de 24 iiini. dc lungitud, el cual presenta un doble ureter eom]il('t(t. y del de un eerdo en el cual se presenta la niisina estruetura: taiiibieu contiene una serie de dibujos de un eerdo en el eual un meter aberrante Ionia la posiei6n excepcional de un ureter inferior situado niedialniente resjieeto al ureter superior. Kl autor ])ro])one una posible explieacion de la preseneia de esta anoinalia del ilesarrnllo, tan poeo freeuente.

TraiuIstioD by Dr Jose F. Nonidei Columbia L'oiveraity

AlTllOllfl AmTltACT Or THW PAPKIt UWUED UY THE IIIIILIOUHAPIIK HKHVICH, PKBUUARY 3

1K)T-IU,K TinTKKS IX III .MAN AM) PIC li.Mr.inos

A. ('.. roilLMAN III IKirliHinl I if Anatomy, St. Louis Univemilii

THREE Ftr;rHES

Tlio (louhlc uictiT has a fortain dovoJopinontal importance in that it fiinushcs a clow for the disappearance of the cloacal segment of the Wolflian duct, and its maimer of incorporation into the Ijladder. Two cases of complete double ureter were rejjorted in I'.KIo;' (me is the ^lall embryo, no. 17o (13 mm.) and the other in the Keibel embryo. Piper (24 nvni.). Both of these embr\()s show that the ureter from the lower part of the kichiey lies dorsal to the ureter from the upper pole, and as they swing around to occupy a lateral position on the \\'olHian duct, the dorsal or lower ureter is displaced lateralward, while the ventral or ujiper ureter lies between it and the Wolffian duct. This rotation from a donsal position on the A\'olHian duct to a lateral one is completed at the time when the cloacal segment of the duct has expanded into the lateral funnel-shaped process of lh<> l)ladder proper. The accompanying figures, nos. 27 to 24, inclusive, and 22 and 19, are taken from drawings n>aile of the Keibel embryo, which is at present not available for study. Figure 27, the lowest of the series, shows the convexity of the right ureter A, and 2(1, the section innnediately above it, shows the opening of this ureter into the bladder. The next .section up indicates the convexity of ureter H, indicated in black, and section 2-t its orifice in tlie bladder. In figure 22, ureter H lies slightly dorsolateral to ureter .V, and the convexity of the (■ur\e of the left ureter is shown with its orifice in the blailder

' .Vhiiornmlitics in tlio fi(rm of the kiilnoy and iirotcr ilepciuleiit onthotlcvclopiiioiil of the roiml Inul. .\. (!. I'olilinnn, Johiiit llupkiiis Ilospitul bull., vol. 1(). IVI)., 11105.

.3(i'.»

370 A. a. POHLMAN

three sections higher up (19). This latter figure shows the liouhle urotor on Iho riglit lying ahnost in the sagittal plane, i.e., the ureter H from the lower pole of the kidney practically dorsal to ureter A from the upper pole. Hoth of these ureters have already migrated to a position eephalad to the opening of the Wolfhan duet, and the interval between the orifices is so small that in a gross specimen they might reailil}' be mistaken for a single orifice.

In the Mall embryo the orifices of the two ureters and the \\'olflian duet are, .so far as I was able to determine them, in common. It would seeni, therefore, that where we have wide displacements of the orifices of the two ureters they are to be

^- C^- ^^.- ^^- ..^ /5>

UfDoCDOt*" £<>2'cC3o«»i> '■'^CCXium lxvcCXirwd

°D. fol.. (o)l (a-)) (S)).. \0^

found in displacement downward of the median one, i.e., the one l\ing between the normal ureter orifice and the opening of the Wolffian duet.

I also called attention to the fact that the rotation of the ureter around the Wolffian duct was entirely independent of kidney rotation and position, and that the displacement of the ureter was completed at the time that the cloacal segment of the Wolffian duct was definitely incorporated into the bladder. In the pig the rotation seems to be accomplished before this absdrption is crnnpleted, so that the ui'eter opens laterally into the Wolffian duct at some distance from the orifices of the duct into the urogenital sinus.

DOUBLE URETERS IN HUMAN AND PIG EMBRYOS

371

I h:i\c' fijuncl but two pigs with evident ureteral duplication or divert iculae. Pig 47 has a complete double ureter on the left side. Figure 9-24 shows the emergence of the upper ureter from the pelvis of the kidney. Figure 10-5 shows that of the lower ureter from its pelvis. In 11-1 the ureter from the lower pelvis B is already lateral to ureter A from the upper pelvis, and maintains this position through 11-2, 11-3, and 11-4. In 11-3 the two ureters open into the lateral expansion of the \\'olfhan duct in about a normal position as indicated in the normal ureter on the right ; ureter H lying in front of ureter A.

•"^ e

/

KU '^ AO---iM

The only es^ential difference between the relations as shown in fig. 47 and the human embrj-o is that the twist of the two ureters in the pig appears to nm to a higher level than in the human being, and, second, that a cloacal segment of the Wolffian duct persists in the pig at the time that the rotation has been completed.

Cases have Iieen reported in the human lieing, however, where this general rule, tliat the ureter from the upper pelvis comes to lie medial to th(> ureter from the lower pelvis and in consequence has a lower oritiee in tlie Madder, does not obtain. Weigert.

372 A. C. POULMAN

HjTtl and Kerr^ have reported cases in wliidi tlic iiilc above stated lias not prevailed. KeiT has made a rejjort on a ease of a double ureter in which tiie ureter from the upper jielvis lies lateral to the ureter from the lower pehas as the two curve t<»ward the bladder. In so far as 1 am aware, there is no develojimental exjilanation for this anomaly, however, a sf)mewhat sunilar condition was found in pig 3. Figure 10-S shows the origin of the right ureter from the kidney. Figure 10-15 shows the lower pole of this kidney with th(> two ureters, right and

y ""t sj u

y

s

(0)

bl»p irwAK

(OLv. ... ukdi:;U|t

j^^^^wip -'«-,^^ u,'— N,.;j

'" i»22

.#r '■^'

left, nmning forward. In figure 10-17 shows the right ureter A, and beliind it a blind ending ureteral diverticulum indicated in black and marked RX. Following this down 10-20 shows it somewhat more medial than in 17, and in 10-23 and 10-24 this lower dorsal ureteral diverticulum comes to occupy the position of the normal ureter from the upjier pelvis of the kidney. It appears to end blindly or to fuse with the right ureter in 10-22. I'nfortunately, this pig series was cut at .50;u and the finer details of structure cannot be definitely determined.

' ("omplete double ureter iu man by A. T. Kerr, Anatomical Record, vol. 5, i'Jll, p. 00.

DOUBLE URETERS IN HUMAN AXU I'Ki KMHRYOS 373

It would, however, appear possible that this (iisertieuluin to fonn a seeond ureter might have arisen from the first ureter but close to its orifice in the \\'(jlffian duet, and on its medio-inferior aspeet, and that it was merely draKped out in the upward migration of the kidney. However, had this aberrant ureter connected functionally with the kidney proper, it would have taken exactly the position described in these cases which appear contrary' to the nile, and would also mean that where the ureter from the lower part of the kidney comes to lie medial to the ureter from the upper part of the kidney, that the two orifices into the bhuklcr are close together, in other words, we would not expect a tUsplacement of the medial orifice away from a normal position.

This would, therefore, merely be an exaggeration of an incompletely double ureter, and it would only differ from the latter by reason of the origin of the lower ureter close to the orifice of the upper ureter in the Wolffian duct. It would, of course, be possible to estal)lish this point quite definitely if sufficient number of ureteral duplications could be observed in serial sections. The anomaly is so unusual that the chance of finding intermediate stages, even in the analysis of large numbers of series, is ver>- slight.

I am merely reporting this anomaly because it is so unusual and because it seems to offer some sort of explanation for the few cases of complete ureter duplication which do not follow the rule.

THK AN\TOUICAL IIKCOUO. VOL. 15, NO. 7

Resumido por el autor, A. C Polilinan.

Sobre el empleo clc ua simple m(5todo grafico para anotar las rela ciones de los cortes seriados, particularmente litil

para la ensenanza de la Embriologia.

El presente metodo gnifico para demostrar las relaciones de una serie de cortes de uii enibri6n es simpleiiiente un modo oonvenieiite de anotar los niveles en los cuales pueden encontrarse detemiinadas estructuras. Para conseguir este fin se proyectan los cortes que representan un intervalo de 100 niicras sobre papel rayado con intervalos de 2 mm. ajoidiindose de una guia en forma de escalera con el fin de indicar la secci6n y fila en cada pnrta-objetos. Esta proyecci6n representa el espesor de los cortes seriados aumentado veinte veces, y la posici6n de una estnictura detenninada en un porta-objetos o en un cierto numero de ellos puede indicarse por medio de una linea o marca cuahiuiera. El objeto de este m(?todo grafico es susministrar la infonnaci6n necesaria sobre una serie de cortes de un embri6n de un modo mas accesible al estudiante y hacer tambien mds accesible para el instructor la correcci6n de las observaciones. Este m^todo es tambien aplicable para anotar observaciones en los trabajos de invest igaci6n.

Traodiition by Dr. Jos< F Nonidci Columbia University

ATTHOH fl ABnTHACT OP TBUt PAPER IMUSD

nrTucDiBLinoRApaicacHviccrxaiirABTS

THE Vt^K OF A SLMPLK CiKAPHIC MF:TH0D OF RECORDIXd THE RELATIONS IN SERIAL SECTIONS. PARTICULARLY FOR USE IN TEACHING EMBRYOLOGY.

\. G. POUL.MA.V Department of Anatomy, St. Louis Universitij

FOUR CHARTS

Embrj'ologj' is conpodod to be one of the most difficult courses in the medical curriculum, and this may be true for a number of rea'^on.s: first, because of the position it occupies in the medical schcilulo; secondly, because the cmbrj'ology text-books are descriptive, detailed, and therefore more or less deadly reading; thirdly, because the substance of the course involves a more or less general knowledge of the morphological relations before the student is informed regarding the end-product of the system he is studying; fourthly, because a stud\' of serial sections is imperative and demands the ability of constructing two (liiuciisional pictures into a three-dimensional whole; fifthly, there appears to be a fear in the minds of most teachers that the student will not 'cover the entire subject' in the time allotted to it, and, lastly, many of the teachers of embr>'olog>' are not sufficiently concerned with the pedagogj- involved in so complicated a subject. It may be well to consider some of these topics in the reversed order.

What is true of the pedagogy in embryologj', or the lack of it, may hoUl true of all of the medical subjects and indeed all of the higher branches in learning. The teacher is apt to confuse information with education and, because of his experience in his particular l)raiich and his ac(|uired ability to digest the facts, believe his exposition to be as clear as plate glass. He may come to regard his students as more and more hickorj'-pated because the same fool (]uestions arc asked year after year with

375

37<) A. c. ronLMAN

sickening regularity. The student, however, may not appreeiate and digest the facts so readily because of his inexperience anil because this one i:)articular course is not the only one he is attempting to pigeon-hole in his gray matter. The student may regard these so-called 'self-evident' facts as by no means transparent and may come to look upon his instructor as a sort of human stjuid elected to make inky the otherwise clear and limpid fountain of knowledge. Embrj'ology is not ea.sy to aecjuire and the learning is beset with many pitfalls for the unwary. It may perhaps be a trite suggestion that each man look into his methods of teaching with the same zeal shown in his research and see if it is not possible to make the subject more interesting. Let the psychic juice of interest be wanting, and even the most delectable mental pabulum will neither be digested nor assimilated. The processes in embryology, in so far as we understand them, are mysteriously simple, and do we not make a great mistake in our attempts to make tangible that which is quite beyond our comprehension!

The fifth point mentioned is also one in which embryological teaching is not the only sinner. Each man lielieves his course is important, if not most important, and once given this attitude it is very simple to make the medical student swallow the entire sheaf in order that perchance a few of the grains of truth will stick. The success in imparting the general fundamental principles underlj-ing the development does not include the fatness of the text-book with its dreaded assignments nor the richness in armamentarium in models, series, injected and cleared specimens, and what not. The ver\' accessible facts may be rendered the more inaccessible by placing a halo about them and by saying, In this way only may the relations be understood." A maximum of material must be investigated in the allotted time, but with a minimum of detail so that the facts themselves may not be made too obscure. Do we overload our students with the number of series? .Vre the series cut at a maxiimun rather than at a niininuuu thickness? Do we require our students to spread over too large a territory and obtain as a re.'iult the mere smattering of this, that, and the other system?

METHOD OF RKfORDINT; SEUIAI, SECTrONS 377

Are wo afraid that the practical thing is a thing to be avoided in a sul)ject i>f purely thporctiral value, or do we reniemlier that the practicabilitj- of };he thing is merely a reflection of the theoretical thoroughness? Do we ourselves always know the theoretical and fundamental basis of the things we teach, or do we convenirntly refer to the text and say, "It is so written?" It is true that one can make a student study, but one cannot make him think. Sjjoon-feeding is probably the worst form of instruction, but on the other hand may not the babe, no matter how hungrj', go to sleep over his bottle if the contents is made too inaccessible?

The fourth point made was the inability of the student to resolve two dimensional pictures in the series into the threedimensional whole. This ability seems to be readil}' developed in some, and in others it is well-nigh impossible to acquire. It is necessarj' that the student draw sections in order that his information may be accessible to himself as well as to his instructor. Drawing, however, is a sort of reflex from the eye quite comparable to the stenographer's reflex from the ear and does not necessarily imply information. Drawing in some courses is like the busy work in the kindergarten. It helps to rivet the attention of the student on his work and makes for peace in the class-room. It is a good thing and it may also be overdone. To gain the same end-results as accomplished in the alleged artistic efforts of the students and incidentally to delegate the major part of the work above the region of the cerebellum, the following scheme was tried and is presented without prejudice.

The objection to the usual series issued to the student is that they are of too many forms and of far too many sections. I suggest the study of one form, the pig, and leave it to the instructor to use other fonns where they show particular points of interest. I would also recommend that the details of maturation, fertilization, cleavage, gastrulation, and the varieties of germ-layer formation be ilelegated to the preparatory course in the premedic years, preferably in the department of zoology-. The course of embryologj- in the medical school should be largely one of organogenesis.

•^"S A. G. roilLMAN

Two series of pigs should be selected, one of from 9 to 12 mm. and the other from 14 to IS mm. jir'^atest length. The embryos may be staincul in bulk, embedded in celhudin and out cleared in xylene-cetlar oil or xylene-ca.stor oil after the Fish-(Jage formula. The larger ones may be cut at '>OiJi and the smaller ones at 33;u. A\'hile sections of this thickness arc not to be recommended for research purposes, it is well to employ them for students, because they are too thick for the satisfactory use of an objective of over IG mm. because the sections may be re.scuwl if a slide is cracked or broken, and because the number of sections is reduced materially.

Carmine, paracarmine, or alum cochineal serves the purpose of a bulk stain: the latter perhaps better than the former two. It is well to have a range of embryo sizes varying from 9 to 18 mm. rather than gi\ing out two series of more or less definite developmental stage. The student provides himself with the usual physics laboratory cross-section paper, S x 10',, and ruled 18 cm. by 24 cm. in 2-mm. squares. He takes the two series i.ssued to him and marks the sections at KK) intervals with a dot of ink; that is, every other .section at 50p, and every third one at .33;ti. The shdes are numbered with a Roman numeral, the rows of sections on the slides indicated by a letter A, B, C, D, etc., and the number of dots in each row bv Arabic numerals Thus

Slide I - .\ - 5

B - 4

C -5

D -4 equals 18 dotted sections and the thickness, therefore, of all of these sections, if piled, would e(iual ISOO^, or l.S mm. If each KMV interval is to be plotted on the graph paper, we nmst assume a magnification of twenty times. I suggest the use of the stair-case guide shown in the accompanying charts, niling across for each row lightly, and marking the intervals between the slides with a h('a\y line. When both embryo series have been transferred into terms of thickness of all sections, times twenty, the student is ready to plot the structures to be observed.

MKTHOI) OI HKCORDINfi SERIAI- SECTIONS 379

Tho Kfiicral icloa of this frranhic met hod is vor\' simplo. The length, hrcadtli, and the rchitions of the structures to he shown are entirely eliminated, and all the student does is index the levels at whieh a given structure is to be found by drawing a line which may be bent into any sort of curve, depending on his particular fancy.

We must remember the line only indicates the level and in stnictures which are large, and which twist about, only one wall may be indicated. The lines, therefore, in the charts show, for example, the ventral wall of the neural canal, the ventral wall of the descending aorta, the convexity of the upper branchial arches, the concavity of the pulmonarj' arch, and the point where the lumen of the intestine shows the turning of a corner. If we start with rea-sonably simple things, like the chorda dorsalis and the \entral border of the neural tube, we establish a sort of string tiirough the entire length of the enibrj'o and demon.strate in what way the taU is bent upon the embryo and upon itself. Once the emergences of the spinal ner\-es or the extent of the spinal roots are plotted and the position of the tip of the snout shown, the student usually can orient his schematic diagram to correspond with sagittal sections or the pictures of mid-sagittal sections. The methotl is simple to explain and even nuich easier to do. I can recommend it heartily as a routine laboratory procedure a.s well as a method of establishing the position of various structures in the series of embryos of a large collection so that each obser\-er may indicate where the several things which he has studied are to be found. For example, in pig no. It), 11 nun. the pulmonarj' arterj- arises from the last branchial arch in slide no. 11. row C, .section 4, and the carotiil arch is still complete as shown in row .\, section 2 of the same shde. The hepatic ducts join the cystic in pig 1, slide III, row .\, section 4. This pig is 12 nun. long antl shows no tail gut.

Hy nuiking a master guide for each embryo, any and all sheets of cr()s.s-section paper may be useil in reconling structures, and these observations may be transcribed to the completed graphic representation as indicated in the charts. I have not as yet found two embryos near enough alike to make it po.ssible to confuse one graph with another, and wouKl not hesitate to pick

380

A. C;. POHLMAN

out of eighty or a luindroii unknown graphs the one which would tit the partieuhir embryo slide given nie. Changing the embryo number eaeh year avoids all possibility of eopying. and the student, onee his graphs are made, can readily compare his findings with those of any other student. For example, the carotid arch has disappeared in embryo no. 1, but is to be found in no. Hi. One can readily pick out a pig series and the section in the pig where the dorsal limb of the arch is becoming rudimentary. Instead of studj'ing two series, therefore, the student has two series for each man in his class to draw upon for any one point.

I present the accompanying diagrammatic charts, which include all I require of my men, in the hope that this method may prove satisfactorj- and will make for the elimination of a large amount of the biisj-work wliich detracts from the interest in a course of embryology.

2, optic nip or eyeball

LD, lacrimal duct

J, Jacobson's organ

5, ganglion of trigeminal nerve

7, ganglion of facial nerve

Under Eclo

IE, membranous labyrinth

fl, hypophysis

A', ventral border of neural canal

T, first thoracic spinal ganglion

L', first lumbar spinal ganglion

Under Meso

JC. internal carotid artery

EC, external carotid artery

JIS. right subclavian arterj'

LS. left subclavian artery

//. extent of heart

(>. foramen ovale

1', ventricular defect

P, origin of right and left pulmonary

arte rices SM, superior mesenteric artery

U, umbilical artery

I, II, III, first, second, and third

branchial cartilages SP, spleen .4, adrenal SG, .sex gland M, Muellerian duct ir. Wolffian body and duct K, kidnev and ureter

Under Enlo

L, liver

P, pancreas

UG, urogenital sinus

The Roman numerals indicate the slide number and the .\rabic numrals millilut'ler intervals or finally the length of the pig computed by multiplying the number of sections in the series by the section thickness.

ME. middle-car diverticulum

7U, thymus

TV. thyreoid

T. trachea

(' . chorda dorsalis

METHOD OF RECOKDING SERIAL SECTIONS

381

'1 II '

VI

nW A

I

1 1 ' 1 1 1 ' «

^m ^^M'

o| , '^ iN >^

^4>

1 1

1 \!f^

m

3S2

A. C. I'OMI.MAN

METHOD OK RECORDING SERIAL SECTIONS

38:j

u

384

A. G. POHLMAN

Resuniido por el autor, A. G. Pohlman.

Sobre el enipleo de la cera de bayberry (1) para endurecer los bloques de parafina.

La cera'de bayberry de un punto de fusi6n de 45° a 49°C. se funde y filtra. C'uando so agrega en proporci6n de 10 por ciento a la parafina de un punto de fusion de o2°C. se obtiene un lilocjue C|ue presenta los misnios earacteres favorables para obtener cortes que la parafina dura de 61° a 62°C., con la ventaja de rebajar en 10° el punto de fusi6n de la misnia; un 15 a 20 por ciento de la mencionada cera produce una mezcla de un punto de fusi6n tan bajo como 51° a 52°C., la cual presenta mayor dureza que la parafina mas dura sin la desventaja de la textura cristalina que presenta esta ultima. El presente metodo se propone para cortar secciones de diverso espesor a las teniperaturas extremadamente variables del laboratorio, ajustando el caracter del bloque a dicha temperatura en vez de hacer lo cont ratio.

Translation by Dr. Joai F. Noniiios Columbiu University

(1) Myrica cerifera.

Aornon'n MwrnAcr or rnw p.tpen narKD

BT THE BlUl.lOQHAPUlCaEKVICC. FCBRtAKT 3

THE USE OF RAYI5ERRV WAX IX IIARDEXIXO PARAFFIX RLOC'KS

A. G. POHLMAN Deparlmenl of Anatomy, St. Louis University

ONE CHART

It is well known that paraffin enjoys a limited range of usefulness, particularly whoro temperature fluctuations are marked, especially in extremely warm weather. Soft paraffins 48° to 50° are therefore only available for thick sections at low room temperatures; hard paraffins 60° to 62° develop a crj-stalline texture at the expense of the gummy property, and not only have the undesiraljle feature of re(iuiring a high embedding iieat, but are extremely sensitive to small fluctuations in ordinary room temperatures. We may mix hard and soft paraflins with a resultant of a melting point of about 53° to 55°, gaining good qualities from both, or may procure, already mixed, parawax of about this melting point at low co.st. Parawax has proved a desiT'ablo material for eniliedding, and to this end the writer investigated the possibility of liarilening parawax or soft paraflin without increasing the melting-point. The following experiments were conducted with bayberrj- wax or tallow:

The material is well known and inexpensive. It comes in Uglit brown lumps with a slightly greenish cast (probably chlorophyl) and with a .somewhat floury surface. It is brittle, and may be ground to a powder, if finely flaked, at ordinary room temperatures. Though somewhat variable, its melting-point is about 45° to 50'°C. The wax was heated and filtered. It was found to have a melting-point of 49° C.

This material was added to parawax of determined meltingpoint, 52° to 5.3°(". in projiortions of 10 per cent, 20 per cent and 30 per cent, and blocks constructed of pure parawax (A); 10

3s.-,

8S«i

A. G. POHLMAN

per font l)uyV)on-y (H): 2(1 ])vr cent baybon-y (D); 30 per cent l>!»yl)oiTy (F); aiitl bard paraffin (E), tested niolting-point (il°C. Tbc l)locks, 9 X 22 x 4(1 nun., wore affixed to a slide by a small end so that when the sUde was phiced in a box the long axis of

A-STR.PARAWAX B = 10 X BAYBERRY C=I50=20

f=3o
E=60°-62» PARAFFIN

to 15 to as 90 )9 4C 49 90 99 CO I

GRAPHIC REPRESENTATIOM OF EFFICIENCY IN

each block was horizontal. All of these blocks were introduced into an electric incubator set at 42"'C. and watched for an hour to obser\-e the 'wilting.' The figures at the top of chart 1 represent time; wiiile the figures at the right show the height of the block, i.e., the amount of bending. The parawax began to

BAYBERRY WAX IN HARDENING PARAFFIN BLOCKS 387

bend in ten minutes 10 per cent bayberry at fifteen minutes, hard paraffin at fifteen minutes, and both 20 per cent and 30 per cent bayborry in thirty minutes. At the end of twenty-five minutes the parawax block liad completely turned down and the height of the blocks at the end of the hour is indicated at the right. This showed that while 10 per cent and 20 per cent bayberry had a marked hardening, 30 per cent was beyond the optimum, and the tests were repeated at 38°, using 15 per cent instead of 30 per cent.

Here a wire nail 6 cm. long was melted into the center of each block so that the nail stood horizontal and the heights at the right show the amount of wilting in terms of dl-^placement of the nail head. The blocks were 9 x 9 x 24 mm. Parawax wilted in five minutes, 10 per cent bayberry in fifteen minutes, hard paraffin in seventeen minutes, 15 per cent bayberr\' in thirty minutes, and 20 per cent bayberry not at all.

The same was again repeated at temperature of ^CfC, using blocks 9x9x9 mm. Parawax wilted in seven minutes, hard paraflSn and 10 per cent bayberry at fifteen minutes, 15 per cent bayberry at thirty minutes, and 20 per cent bayberry not at all.

The melting-points of these mixtures, 10 per cent. 15 per cent and 20 per cent, is ^lightlj- lower than the parawax or 50° to 51° — 10 per cent addition created a block which is as hard as hard paraffin Gl° with 10° less melting-point, — while 15 per cent and 20 per cent harden the parawax beyond any hard paraffin obtainable.

The bayberry tallow added in small amounts, say 5 per cent or 10 per cent to hard paraffin, not only hardens it, but eliminates certain undesirable cracking and crj-stalUzation in that material. The wax is about as soluble in .xylene or toluene as paraffin and far more soluble in chloroform. The disadvantages in using the bayberr>' is to be found in lowered transparency and a slight brown color. The material cuts just as well and even better than hard paraffin.

In using bayberry tallow caution must be oi)servctl in letting it iuu-ilen in a pure stat(> in a beaker because it will break the bottom out of it. Again, if thought advisable, the chlorophyl

TIIK ANATOMICAL ■CC^tRD. VOL. 15, NO. 7

HSS A. O. rOHLMAN

may 1)*> di:<solved out by adding 100 grams wax to 250 co. 96 per cent alcohol; bring tho alcohol to a boil and cool off gradually, 'llu' wax will jircciiMtatc out in the form of globules of ilarker brown and a flocculcnt light precipitate. P'iltration and driving off alcohol on a water-bath yields a light brown material with only a trace of green in it. The globular precipitate ha.s a slightly lower melting-point than the flocculent one, but I do not consider the alcohol treatment or the separation of these two elements desiralile or necessary.

I suggest the use of this method for hot-weather work or in extreme hot weather to add 10 per cent, 15 per cent, or 20 per cent to bayberrj- wax hard paraffin to increa-se the efficiency of the iilock and to reduce the melting-point one or two degrees.

Ether embedding technique a.s suggested by Federici for paraffin (Encyclopedia D. Alikro. Technik, 2. Auflage, Bd. 2, S. 3G1) may also be satisfactorily done with bayberry wax.

Resumido por el autor, A. C. Pohlman.

Una modificaci6ii dv la placa de cera y papel empleada en el m^todo de reconstrucci6n de Born.

Esta placa es una laniina de cera obtenida comprimiendo cera caliente por medio de un rodillo, la cual lleva pegado papel de dihiijo a una do sus caras. El oalco del dibujo, obtenido mediant e papel carb6n, se coloca con la superficie que lleva el dibujo sobre la superficie parafinada y comprimiendo por medio de un rodillo se obtiene el dibujo sobre ella; se vierte cera fundida sobre el dibujo hasta la altura que marcan las guias y se nivela con un rodillo caliente. La parafina entre el papel y la superficie de la placa permanece fundida cuando la cera est^ bastante endurecida j^ara retirar la placa o])tenida. Esta m^^todo combina ciertas ventajas de la placa de Born y del de la placa obteuitia vertiendo cera y remedia algunas de las desventajas de ambos.

Translation by Dr. Joe^ F. Nonidez Columbia University

ACTROH II AfWTItACT OP TnOI rAPKR OMrKO AT TMi: UIOLIfXlHAPHICItEKVICE. PCBHt'AKT 3

A MODIFICATION OF THE BORN PAPER-WAX RECONSTRUCTION PLATE

A. G. POULMAN

Department of Anatomy, St. Louis University

The Born mothod of applying the third dimension to serial drawings for purposes of rt-coiist ruction has nianj' advantages over the blotting-paper and 'poured' wax plate. All three methods work well in the hands of experienced men. The great disadvantage of blotting-paper is that it cannot be used for thick plates, i.e., over 1 mm., while under 1 nun. thickne-sses are readily handled, especially if impregnated with hard paraffin, when they may be cut finite readily with a sharp knife on a gla.ss plate. The i)oured plate of 1 mm. in thickness is too friable and is likely to undergo distortion in the cutting and piling. Hence it is used practically only in 2 mm. thicknesses. The Born method of applying the wax to the carbon copy of the serial drawings has many advantages, especially if made on specially devised machines, such as that of Huber at Ann Arbor or my own at .Johns Hopkins. The latter is calibrated to make accurate plates at twentieths of millimeters, and has the great advantage of making possible resulting models of uniform size no matter what the section thickness and magnification. The Born plate is briefly made as follows: The carbon copy of the serial drawing is squeegeed to the surface of the stone or metal slab with turpentine. Melted wax, preferably wax paraffin resin, is poured over the drawing to a thickness slighly exceeding that of the strips or guides and leveled with the hot roller. .\s soon as the surface of the wax is firm, it is brushed lightly with turpentine and a sheet of tissue-paper applietl and again rolled. The plate is trimmed and placed between blotters to dry. The great disailvantage in this method is the use of turpentine which is very disagreeable, particularly when hot, and

3S9

390 A. (i. poll I.MAN

which makes the wax sticky. Further, it tends to dissolve off tiie carbon copy, takes a long time to dry, and, finally, does not incorporate either the drawing or the tis.suc-papcr into the wax, so that they tend to peel off in the cutting. The tissuepaper backing also makes it more difficult to fuse the plates successfully in building the model.

The modification suggested is the following: Place the carbon copy of the drawing face down on the stone slab. Pour a few teaspoonfuls of melted paraffin on the paper and squeegee the paper to the .slab with the hot roller, passing it at right angles to the guides. Scrape the upper surface of the paper with a broad painter's knife, to get rid of the excess of paraffin and avoid blisters. Pour on the wax and level with the hot roller as before. As soon as the plate is firm, trim to tlie edges of the paper and lift off. The pure paraffin between the paper and the slab is still melted when the wax is quite firm. Place the plate on some smooth surface, cover the wax surface with paper and put a board on it to overcome the curling. The plate is read}' for use as soon as it is cold.

The advantages of this method are as follows: First, the turpentine is entirely eliminated and, second, the plate is not backed with tissue-paper. It compromises the poured and the rolled plate with all of the advantages of both and with none of the disadvantages of either. The carbon copy is fixed with the paraffin and the paper is so thoroughly incorporated into the wax that it may only be torn off bit by bit. The plate is cut, paper side up, and gives a sharp edge without the thickening found in the poured plate.

Resuniido por el autor, Harvpy Ernest Jordan.

I^ histogenesis de las plaquetas sangufneas en el saco vitelino del embri6n del cerdo.

Mediante el empleo de la t^cnica de Wright se puede demostrar la prcspnria de ])laciuctas sangin'neas ti'picas en los sinusoides del saco vitelino del enibri6n de cerdo de 12 nun. Esta.s plaquetas se fomnan principalmente a expensas de c^lulas gigantes y tanibi(?n, en cierto grado, a expensas de los linfocitos priniitivos o '"henioblastos" y a veces se derivan de las c(lulas endoteliales. Esta.s ultimas deben interpretarse conio hemoblastos que se estdn diferenoiando del endotelio. Las c^lulas gigantes son, en esenoia, hemoblastos hipertrofiados. Tanto los hemoblastos oomo las celulas gigantes que de cllos derivan se caracterizan por la pre.sencia de griinulos metacromdticos en su citoplasma. Las plaquetas se originan de dos maneras diferentes: por segmentaci6n de pseud6podos y por fragmentaci6n de areas oitophismicas de mayor tamano. L'na de estas maneras esta a.sociada con una funci6n aparentemente normal, la otra con procesos degenerativos. como indica la condici6n anormal del nucleo. Tanto los hemoblastos como las celulas gigantes pueden diferenciarse en eritrocitos. Las cdlulas gigantes hemogenicas del saco vitelino estdn representadas en la m^dula roja de los huesos por elementos hom6logos. Los osteoclastos no contienen griinulos metacroniaticos; su ausencia susministra un criterio exacto para diferenciar las celulas gigantes hemogenicas de las osteoliticas, en la m^dula 6sea. La formari6n de las plaquetas es un proceso id^ntico en el saco vitelino y en la medula roja de los huesos. Las plaquetas aparecen como un product o accesorio de la actividad Aormai de los leucocit'os con grdnulos metacromdticos, la cual se manifiesta por la formaci6n de pseud6podos, y tambi^n como resultado de procesos degenerativos, que se manitiestan por cambios nucleares acompanados de una fragment aci6n del citoplasma granular metacromatico.

TtmraUtion by Dr. loti K. Nonidd Columbia Univeniity

At'THOK » ABHTWAfT Of THW FAPEK UMt'BD ■ r TIIK BIBLIOOnAPIlIC SKRVfCK. rEBRL'ABT 3

THE HISTOGENESIS OF BLOOD-PLATELETS IN THE YOLK-SAC OF THE PIG EMBRYO

H. E. JORDAN Laboratory of Histology and Embryology, University of Virginia

INTROOrCTIOX

In his article on the origin of blood-platelets from megakaryocytes in tlie bone-marrow of certain mammals, ^^'rigllt"' describes and figures also certain megakaryocyte 'forerunners' in the blood of young guinea-pig embryos. In this paper also he offers the hypothesis tliat the amphibian homologue of the manunalian megakaryocyte is the spindle cell. The fundamental question here involved concerns the significance of the bl()()(l-i)lat(>l('ts. The present investigation aims to further elucidate this jjroblem through an approach by way of the giantcells of the yolk-sac of the pig embr>'o.

As regards the embryonic 'forerunners' of the megakaryocytes, ^^'right states that blood-platelets are present only after these cells have made their ajipearance, in guinea-pig embryos of about 4.5 mm. length. After this stage of development the 'forerunners' of the megakaryocytes occur free in the bloodvessels; they then have a size about that of the erythrocytes, and contain the characteristic metachromatic (red to purple) granulation of the larger megakaryocytes. Both the smaller 'forerunners' and the transition forms are said to break up in the l)lood-vessels into typical blood-|)latelets just as do the fully developed megakarjocytes. Certain of these 'forerunners' are described as originating from the endothelium of the bloodvessels, and one such progenitor is figured still in connect it)n with the endothelium, but containing the cytoplasmic granules characteristic of megakaryocytes. These 'forerunner' cells

i«»2 H. E. JORDAN

would soom to call for further study. A more complete interpretation is made possible on the basis of the data given below jis derived from a study of the yolk-sac of the pig.

It seems desirable at this point to recall the scojie of Wright's work, and to emphasize the cogency of his arguments, ba.sed upon data which amount to a practically comi)lete demonstration, that hlood-jilatelets arise by a process of segmentation of the pseudopods of megakaryocytes at certain stages of their development. Wright studied the red bone-marrow and spleen of the cat, kitten, man, mouse, dog, rabbit, guinea-pig, white rat, and opossum. The results of the study of tliis variety of material consistently support the same conclusion. This conclusion was confirmed by the work of Bunting' and that of Downey* for the rabbit. Ogata" also has confirmed Wright's conclusion in ever>' respect (cited from Downey). A careful study of the red bone-marrow of the rabbit and of the guineapig, treated according to Wright's technic, has convinced me also of the accuracy of Wright's conclusion regarding the giantcell origin of the blood-platelets.

Wright's hypothesis of the homology between the thrombocytes of ichthj-opsid and sauropsid bloods and the hemogenic giantcells of mammalian hemopoietic organs is based chiefly on his observation with regard to the spindle cells of the blood of Hatrachoceps attenuatus, where also the cytoplasm contains metachromatic granules and where portions are regularly pinched otT to form corpuscles structurally and tinctoriallj' very like mammalian blood-platelets. Downey' describes similar 'azurophil' granules in the spindle cells of Amblystoma, but fails to find cytoplasmic constrictions; he nevertheless believes that Wright's conclusion 'that the spiiidle cells correspond to circulatory megakaryocytes is justified' (p. 313). I have seen this same j)h(>nomenon of pseudopod fragmentation also in the case of the thrombocytes of the blood of the frog, Rana pipiens. An attempt will be made to formulate inclusive and consistent reinterpretations of this body of data in the light of evidence derived from the study of the yolk-sac of the pig, combined with certain observations regarding the primary lymjihocytcs (hemo

BLOOD-PLATELETS IN YOLK-SAC OF I'lO EMBRYO 393

blasts) of tlu' marrow of the frog. The latter will be diseusscd more in detail in a separate paper.

Attention should here be directed also to the difTerences in details, as rovoalod partieularly by the illustrations, in the process of platelet origin from megakarj-ocytes as describeil in the papers of Wright'* and of Downey.* Wright views and illustrates the process chiefly in terms of a segmentation of pseudopods of apparently health}' cells at a certain stage of their development (Wright's fig. 14); Downey, on the contrary, figures the process as one of disintegrating cells, as indicated by their complexly lobulated, wrinkled, non-graindar nuclei (Downey's fig. 15). This dilTerence in detail is actuall}' of much importance and demands an explanation. In my study of the red marrows of the guinea-pig and the rabl)it I find that both processes (segmentation and fragmentation) occur abundantly. They are quite difTerent in nature, but lead to practically identical results. The matter will be further discussed below.

Still other essential points in this connection concern: 1) The genetic, morphologic, and tinctorial dissimilarity between the osteolytic and hemogenic giant-cells of hemopoietic foci; that is, the osteoclasts and the giant hemoblasts, respectively, the essential phagocytic nature of the former, and the en,-throblastic significance of the latter (Jordan'"). Contrarj- to the conclusion of Dickson,* who identifies all types of giant-cells and ascribes to them in common a phagocytic function, the evidence indicates that the so-called megakar>'ocytes are not primarily and generally phagocytic. 2) The demonstration that the genetic history of the giant-cells of the yolk-sac traces back to hemoblasts, which may in some cases be traced to the endotheUum, and the further demonstration that certain mononucleated giant-cells (genuine megakarj-ocytes) or large hemoblasts become polymorphonucleated and subsequently multinucleated, in which phase they may under certain conditions become transformed into erytlirocytes (Jordan'").

394 H. E. JORDAN

MATERIAI, AM) MKTHODS

Portions from the proximal pole of the yolk-sac of the pig embrj'o of about 12 mm. length constitute the chief body of material for this investigation. The tissue was fixed for twentyfour hours in a mixture of 10 parts of formalin to 100 parts of a saturated normal-salt solution of HgCl«, as recommended by Downey. It was then passed through several changes of 70 per cent alcohol, treated with tincture of iodine, and embedded in paraflin. Sections cut at 5m were stained on the slide according to the technic employed by Wright. Similar tissue fixed in Helly's fluid and stained with eo.sin-azure, or hematoxylin and eosin. was used for comjiarison. The several marrows (femurs of frog, guinea-i>ig, and rabbit ) employed for comi)aris()n with yolk-sac, certain observations from which enter into the present discussion and contributed to the interpretations here arrived at, were also preserved and stained according to Wright's technic.

DESCRIPTION

We are interested at this time only in the hemopoietic tissue of this yolk-sac, namely, the middle mesenchymal layer Cangioblast') with its network of blood-vessels containing cells at all stages of differentiation from original hemoblasts or even endothelial cells to erythrocytes (erythroblasts and normoblasts). Hemopoiesis is still very active at this stage of development in the proximal portion of the yolk-sac. Giant-cells and platelets are very abundant. The metachromatic (red to purple) granules of these and other cells stand forth with remarkable clearness in this tissue treated with Wrights technic. Comparative studies of the various types of cells in yolk-sacs of the same age prepared by the Helly and Wright technics, respectively, were very helpful in interpreting especially the giant-cells.

In my previous studies of the yolk-sacs of pig" and mongoo.se' embryos, I arrived at the conclusions: 1) that certain hemoblasts differentiate directly from endothelium; 2) that the giantcells are enlarged hemoblasts; 3) that the iiolynudeated giantcells are derived from the mononucleated forms, chiefly by

BLOOD-PLATELETS IN YOLK-SAC OK PIG EMBRYO 395

nuclear aniitosis leading throup;h polyniorphonueleated forms, and 4) that the polynucleated types may produce en,'throcytes by a process of intracellular differentiation. This endogenous er>"throcytogenesis is practically limited to binucleated forms of giant-cells, though it may occasionally be seen in cells with four nuclei. Such binucleated types occasionally differentiate into structures corresponding to an endothelial cell enclosing an erythroblast. The polynucleated giant-cells are accordingly multiple hemoblasts comparable to blood-islands, an interpretation con.sistent with the facts of their origin and their function. In a subsecjuent work (Jordan'") it was shown that the hemogenic giant-cells of red marrow, as distinct from the invariably multinucleated osteolytic giant-cells or osteoclasts, have a like origin from hemoblasts, and may under certain conditions apparently function as sources of erythrocyte formation. Only the polynucleated forms apparently differentiate into er>-throcytes; transition stages between the hemoblasts and the multinucleated giant-cells are polyniorphonueleated forms like the so-called megakaryocytes, and are apparently identical with the latter. Mononudeated, polyniorphonueleated and the polynucleated types of hemogenic giant-cells have in common a fundamentally homogeneous and basophilic cytoplasm and contain tine spheroidal metachromatic granules. The three types may give rise to blood-platelets by segmentation of p.seudopods or by fragmentation of larger peripheral portions of their cytoplasm.

In the yolk-sac of the pig embryo several forms of giant-cells occur, which after staining according to Wright's technic show structural and tinctorial features identical with those of the coiTesponding cells of the marrow. These cells likewise produce platelets of varying sizes both by a segmentation of pseudopods and by a fragmentation of their cytoplasm. The former cells are characterized by more normal nuclear features than the latter. There are in addition to these cells still others with a similar cytopla.sm and metachromatic granulation: 1) Certain endothelial cells difTerentiating into hemoiilasts and just separating from the endothelial wall, and 2) hemoblasts.

396 H. E. JORDAN

Then* an- apparently cprtain exceptions to the more typical granulated types among the free young hemoblasts. Such have a deeply blue staining homogeneous cytoplasm. The micleus is of the same \esicular type, with delicate reticulum, a plasmosome and several net-knots, as that of the hemoblasts with metachromatic granules. Careful examination will in many cases rewal a few metachromatic granules in the cytoplasm of such apparently non-granular hemoblasts. ^Moreover, the granules first appear about the attraction sphere, thus in a restricted region, and sections may obviously pass through a plane of the hemoblast at right angles to the plane passing through this initial ma.ss of granules. The granulation may make its appearance at variable stages of development, sometimes earlier, even while the diflferentiating hemoblast is still in connection with the endothelium, .sometimes relatively late in the hemoblast stage. In the case of the marrow of the guinea-pig and the rabbit, the Ix'giiinings of the gramdatioii can be traced likewise, but the granules apparently first ajipear in relatively later stages of hemoblast develo})ment. As the metachromatic granules increase in amount they scatter through wider areas of the cytoplasm, and the orignally deeply blue-staining cytoplasm changes to a pall' blue color. .V non-granular hyaline border of variable V idth can almost invariably be distinguished peripherally in ttiese granular hemoblasts and giant-cells of the yolk-sac. The coincident decrease in the basophily of the cytoplasmic substratum with the appearance and increase of the azurophil granulation suggests a genetic relationship, but the actual steps in the origin of the granules from out of the cytoplasm cannot be discerned.

As the hemoblasts differentiate into erj-throblasts, the metachromatic granules disappear. Several granules may occasionally still be ."^een in the later phases of erythroblast differentiation, scattered in the hemoglobin-containing cytoplasm. In the Helly-fixed tissue the young erythroblast ('megaloblast/ Maxiinow) has a gi'anular cytoplasm; the hemoglobin seems to originate as initial granules, for this granulation does not occur in the hemoblast forerunner nor in the erj'throblast derivative.

BLOOD-PLATELETS IN YOLK-SAC OF PIG EMBRYO 397

Since such granulation is absent from the cytoplasm of the henioblasts in Helly-fixed tissues, it cannot be identical with the metachromatic granules of the hemoljlasts in tissues treated according to Wright's method. This fact contravenes any suggestion that the hemoglobin of the erythroblasts has its direct (jrigiii in the metachromatic granules of the hemoblasts. However, tlie ability of cytoplasm to produce metachromatic granules and hemoglobin undoubtedly resides in the same cell and to some extent at least coincidentaly. Binudeated giantcells in process of endogenous erj'throcyte formation undergo similar perinuclear cytoplasmic alterations in the elaboration of hemoglol)in, with a coincident development of a limiting membrane.

The process of platelet formation from the giant-cells and hemoblasts of the yolk-sac presents nothing essentialh* new. It is exactly like that in the red bone-marrow as regards the megakaryocytes. The process is twofold, that is, either by segmentation of pseuodpods or by disintegration of large peripheral masses of cytoplasm. The resulting platelets are identical and vary much in size. The nuclear characteristics associated with this twofold process are relativeh' specific. Cytoplasmic disintegration is as.sociated with a peculiar type of nucleus. This nucleus is very extensively lobulated; the lobules arc small with a wrinkled contour; certain lobules are verj' pale, while others are pycnotic; the lobules have a non-granular vesicular character and present a sort of cloudy appearance; net-knots are practically lacking and the nuclear network is either very faint or entirely lost. On the contrary, the nuclei of the giant-cells producing platelets by segmentation of pseudopods consist of relatively fewer and much larger lobules. The relatively robust nuclear wall has a sharp contour. The clear vehicular lobules have a distinct chromatic network with many larger and smaller karyo.somes and an occa.-*ional pUtsmosome. The nucleus as a whole has a distinctly healthy appearance in comparison with that of the giant-cells contributing platelets by cytoplasmic fragmentation.

398 U. K. JORDAN

The endothelial cells with metachromatic granules apparrently produce platelets only by terminal constriction of short pseudopods. None were seen with long pseudopods, nor were any endothelial cells seen in process of fragmentation. This cell is identical with that described by Wright'* for the bloodvessels of the guinea-pig embryo.

Certain hemoblasts also are covered with psuedopods. These can be seen to segment off typical blood-platelets. Other hemoblasts suffer cytoplasmic disintegration, producing thus groups of typical platelets and leaving naked nuclei. These cells are identical with those described by Wright for the guinea-pig as 'forerunners' of megakaryocytes. Hemoblasts in all respects like these platelet ancestors differentiate into erythrocytes.

The giant-cells of the yolk-sac are essentially enlarged hemoblasts. As such they may contain a single large spheroidal or reniform nucleus, a bilobed or polylobular nucleus suggestive of the 'megakaryocyte' nucleus of red marrow, or they may be multinucleated. The cytoplasm is identical with that of the giant-cells of the marrow, consisting of a light-blue staining substratum with metachromatic lilac-colored granules. Like the conesponding cells of the marrow, the yolk-sac giant-cells produce blood-platelets by segmentation of pseudopods and by fragmentation of larger cytoplasmic areas. The later steps of this latter process leave a naked nucleus.

This material shows also occasional giant-cells among the en todermal cells hning the yolk-sac. They can be readily identified by reason of the metachromatic granules of the cytoplasm, which causes them to contrast sharply with the entodermal cells with their proximal content of basophilous substance and ergastoplasmic filaments. Spee" reported similar polynuclear giantcells among the entodermal cells of the human yolk-sac and describeil them as arising from the entoderm. He, moreover, interpreted them as progenitors of ret! cells. Saxer'= likewise interpreted similar giant-cells in the yolk-sacs of pig; sheep, and cat embrjos as ancestors of normoblasts. In my studies of the yolk-sacs of a 9-mm. and a 13-mm. human embryo' I failed to find giant-cells among the entodermal cells; only a few were

BLOOD-PLATELETS IN YOLK-SAC OF PIG EMBKYO 399

seen pxtruvascularly within tho inosonchyintil layer. Possibly the staining ti'chnic cniployi-d tlid not dearly reveal such cells that might have been present among the entodemial cells in these sections. Howcxer, the evidence is coinj)Iete that these giant-cells do not originate from entodermal cells as claimed by Spee, but that they may occasionally wander into this layer from the underlying mesenchyma. Hut it is of much interest that both Spee and Saxer also interpreted these cells as ancestors of erythrocytes.

Conditions identical with those of the yolk-sac appear also in the- liver sinusoids of this stage of development. Certain endothelial cells elaborate metachromatic granules, round up into typical hemoblasts, and separate from the endothelial wall as free cells. Such may produce platelets either during their origin from endothelium or subse(|uently. Free hemolilasts and giant-cells of the liver likewise jjmduce platelets abundantly.

The liver contains also the peculiar elements, previously described for the yolk-sacs and the intra-embryonic blood-ve.s.sels of certain mammals, namely, structures that appear like a crosssection of a capillary containing an erythrocyte, the wall of the capillary being formed by a single endothelial cell with its nucleus at thel('\('l of the section. Such structures are interpreted as originally binudeated hemoblasts in which one nucleus with its enveloping cytoplasm has differentiated into an erythroblast, the other into an endothelial cell. The presence of such cells in both the yolk-sac and liver ve.ssels of this stage gi\-es additional support to this interpretation.

The pig embryo of this stage shows also numerous large celldusters along the ventral jxirtion of the abdominal aorta. These ha\e been iireviously interpreted as clusters of hemoblasts differentiating from endothelium which has been invaginated locally into the lumen of the vessels, in some cases at least in consecjuence of a shrinkage of underlying mes(>nchyma, due to atrojjliy of a ventral segmental ramus." The \\ right's staining technic reveals metachromatic granules in the cytoplasm of these cells. Thus, on the basis of still another feature is indicated the classification of the constituent cells of the.se dusters as differentiating erythrobhusts.

IIIK ANATOMICAL RECORD, VOL. 15, NO. 7

400 H. E. JORDAN

DISCUSSION AND CONCLUSIONS

The foregoing doscription of conditions in the yolk-sac of the pig embryo indicates the complete correspondence of the cells with metachromatic granules (endothelial cell derivatives, primitive free lyniphocyfes or liemoblasts. and hemogenic giantcells) with similar cells in the blood-vessels of guinea-pig embryos and the red bone-marrow of adult mammals as first described by Wright.* It becomes evident also that similar cells occur in the li\er sinusoids, and that the aortic cell clusters consist of elements corresponding with the hemoblasts of the yolk-sac. The exidence shows also that typical blood-platelets may originate from any cell with metachromatic granules. It shows, further, that all types of cells with these granules are deri.vatives of a common lymphocyte-like cell or 'hemoblast.' At least a certain number of the latter differentiate from endothelium. Bloodplatelets occur abundantly in the blood-vessels of the yolk-sac of the 12-mm. pig embryo. Since they arise from hemoblasts at all stages (except possibly the very earliest), in some cases before the cell has separated from the contributing endothelium, the conclusion seems justified that a certain few are present almost coincidental!)- with the appearance of the first bloodcell progenitors (primitive lymphocj'tes or hemoblasts). It does not seem probalile, therefore, that platelets first arise at al)out the 4.5-mm. stage in the guinea-pig embryo as stated by ^^'right. An examination of the yolk-sacs of earlier stages might reveal platelets in abundance.

In previous studies of the yolk-sac of the 10-mm. pig embryo' and ot the mongoose embryo' I have shown that the giant-cells of the yolk-sac arise from hemoblasts and may function as erythroblasts; that is, they may differentiate erythrocytes intracellularly. The present stud)- shows that all of these cells involved in the hemoblast and giant-cell history contain metachromatic granules and that such cells may produce tj-pical blood-platelets. Wright's work reveals identical conditions in the body blood-ves.sels of the guinea-pig embrj-o and in adult red bone-marrow. I can abundantly confirm Wright's conclu

BLOOD-PLATELETS IN YOLK-SAC OK HKi EMBKYO 401

sion rf'nariiiiiK these cells as progenitcirs of platelets in the rase of the inarrcnv of the femurs of the guinea-pig and the rabbit. The more complete evidence now permits the conclusion that the giant-cells are derived from hemoblasts, that they produce platelets wherever found, in yolk-sac, liver, and ret! marrow, and that they may at the same time function as multiple en.'throblasts; that is, they are in fact hemogenic giant-cells in contrast with the osteoclastic giant-cells.

The foregoing leads to a closer analysis of the phenomenon of platelet ft)rmation by cells with metachromatic granules. The question resolves itself e.ssentially into one regarding the significance of platelet formation by g)"rit-cclls Cmegakarj'ocytes' and 'polykarjdcytes'). .\n extensive microscopic study of the red bone-marrow of the femur of the frog — the details of which will be published elsewhere — reveals the following e.-sential facts: All types of primitive lymphocyte derivatives (lymphocytes, thrombocytes, and granulocytes) may form pseudopods, which may constrict or segment to form platelet-like bodies. Thus the young hemoblasts and lymphocytes may produce hyaline bodies; the polymorphonucleattxl special (neutrophilic) granulocytes produce corpuscles with neutrophilic granules that is, elements suggestive of platelets; the thrombocytes, especially in the circulation, produce similar bodies; the basophilic granulocytes or mast-cells produce platelet-like bodies with coarse basophilic granules; the eosinophilic granulocytes produce platelet like liodics which may occasionally contain eosinophilic granules, but more generally lack granules ( hyaline bodies) ; and certain lymphocytes of the circulation which contain metachromatic granules may also produce by a similar method typical platelets.

The evidence from the study of tije frog's marrow indicates that pseudopod formation and constriction is a common characteristic of lymphocytes and their leucocyte derivatives. Blood-platelets are accordingly a by-product of this phenomenon, and genetically belong in the same cla-ss with hyaline bodies and the cytoplasmic fragments of neutrophilic, basophilic, and eosinophilic granulocytes.

402 H. E. JORDAN

The suggestion presents itself that pseudopod segmentation and localized cytoplasmic fragmentation may to some extent be related to the nuclear amitosis also characteristic of these cells. It seems a reasonable assumption that the fundamental factors which cause a relative increase of nuclear substance by nuclear fission operate also to the same end by a decrease in the amount of cytoplasm by pseudopod constriction. The metabolic re(luirements as expres.sed in the nudeo-cjtojjlasmic relationship could conceivably be met either by increase of nuclear surface or by decrease of cytoplasmic volume, or still more effectively by a combination of both proces.ses.

In the frog's marrow no naked nuclei seem to occur. In the circulation, however, naked nuclei of thrombocytes occur. The latter phenomenon alone seems to place these cells closer to the megakaryocytes of mammalian marrow than are the ami)iiibian neutrophilic granulocytes. However, no strict homology obtains between the s))indle cells or between the neutrophilic leucocytes and the megakarj'ocytes. In the frog's marrow occasional mononucleated giant-cells occur; thej' result from hypertrophy of certain primitive lymphocytes. These are the true homologues of the heniogenic giant-cells (mononucleated, polymorphonudeated, and polynucleated) of manuiialian red marrow. These few giant-cells of the frog's marrow also contain metachromatic granules during later stages and may produce platelet-like bodies by pseudopod constriction.

As above described, platelets arise also by another method from the hemogenic giant-cells of mammalian red marrow, namely, by a process of fragmentation of larger areas of peripheral cytoplasm. This mode of origin was recognized also originally by Wright and subsequently by Downey. But neither seems to have gra-sped thg full implication of the phenomenon. .\ similar double mode of origin of platelets is exemplified also in the ca.se of the hemoblast and the giant-cells of the yolk-sac. In his report on the origin of platelets from megakaryocytes Wright paid special attention to the segmenting pseudopods; Downey, as judged by his illustrations, saw principally the other mode, namely, origin by cytopla.smic fragmentation. A

BLOOU-PI.ATKLKT.S IN YOLK-SAC OF PIG E.MURYO 403

comparison of their illustrations (fig. 14, Wright :'^ figs. 15, DownoyM shows that the first mode is associated with healthy inulear condition, the latter with a degenerating nucleus. The platelet prf)genitor of the yolk-sac of the 12-mni. pig embrj'o shows the same nuclear conditions associated with these two modes of platelet formation. It is obvious that either mode leads to practically the same morphologic result, namely, small globules of slightly bjisophihc cytoplasm containing metachromatic granules.

In the light of these obaen'ations, platelet formation is apparently simply a by-product of cytoplasmic fragmentation of certain cells with metachromatic granules. A careful study of the marrow of the femur of the rabbit and of the guinea-pig has convinced me of the accuracy of this interpretation in part as applied also to these marrows. The evidence, then, from a comparative study of the marrows of guinea-pig, rabbit, and frog, and of the j'olk-sac of the pig embryo, consistently points to the same conclusion, namely, that a giant-cell is a hypertrophied hemobla.st, that it produces platelets as a by-product of apparently normal pseudopod formation and constriction -a process perhaps related to metabolic conditions as expressed in the nucleo-cytoplasmic relationship — and of cytoplasmic fragmentation, and that under certain conditions in a multinucleated form the giant-cell may function as a multiple erythroblast.

That the multinucleated giant-cell arises from the polymorphonucleated inegakarj-ocyte by a process of separation of the lobules of the basket' nucleus can be readily demonstrated in the marrow of the rabbit and of the guinea-pig. Moreover, in the red marrow of the guinea-pig the polynucleated types predominate, while in the marrow of the rabbit the polymorphonucleated are by far the most common forms of giant-cells.

It seems desirable, finally, to attempt to bring the foregoing morphologic data into relation with the mechanism of coagulation. According to Howell,* clotting of blood phu^ma and of lymph involves the cooperation of four elements: 1. fibrinogen and 2, antithrombin, both present in both lymph and plasma;

404 H. E. JORDAN

i, im)thr()mhin. liberated by blood-platelets and by lymphocytes

and 4, thromboplastin, elaborated by platelets, lymphocytes and tissue cells generally, and operating to neutralize antithroinbin. Though the lymph of the thoracic duct lacks platelets ( Howell;' Jordan'^) it nevertheless clots like blood plasma under similarly favorable conditions, only somewhat more slowly. The blood of birds, reptiles, and ampliibia likewise clots in the absence of circulatory platelets; in these forms occur additional blood-cells, the thrombocytes or spindle cells, which appear to be analogues of the mammahan platelet. Spindle cells and mammalian platelets contain apparently identical metachromatic granules. Lymphocytes likewise contain a certain amount of similar granules. The source of the prothrombin of lymph would seem to be restricted, at least largely, to the preponderant lymphocytes. In non-manunaUan bloods, as for example that of frog, the prothrombin could apparently be liberated by the spindle cell or by the lymphocyte or by both. The evidence suggests that the specific source of the prothrombin is the metachromatic granule. The combined physiologic and morphologic data seem to indicate that the metachromatic granules of hemoblast, hemogenic giant-cell, Ijnnphocyte, spindle cell, and free platelets, whether of giant-cell or spindle-cell origin, are functionally similar. This suggestion seems the more plausible in view of the fact that the lymphocyte, hemogenic giant-cell, and spindle cell are all direct and but relatively slightly differentiated derivatives of the hemoblast. The precise relationship of the cytoplasmic granules of the polymorphonucleated neutrophilic granulocytes of certain mammals and amphibia to the closely similar metachromatic ('azurophil') granules of the abovespecified group of cells remains undetermined.

SUMMARY

1. In the blood spaces of the yolk-sac and of the liver of the 12-mm. pig embryo typical blood-platelets occur in large numbers. They are produced l)y the primitive lymphocytes or hemoblasts and their giant-cell derivatives, occasionally also by

BLOOD-PLATELETS IN YOLK-SAC OF PIG EMBRYO 405

endothelial cells in process of differentiation into hemoblasts and separation from the vessel wall. The mode of platelet formation is twofold: a, by segmentation of pseudopods, and h, by fragmentation of larger portions of cytoplasm. .\11 of these cells contain a homogeneous, slightly basophilic substratum filled with fine spheroidal metachromatic granules. The smaller mononudoatcd cells correspond with those described by \\'right as megakaryocj'te 'forerunners' in the guinea-pig embrj'o and in the red bone-marrow of certain mammals. Cytopla.smic fragmentation (disintegration) is associated with abnormal (or senile) nuclear conditions and leads to naked nuclei. Pseudopod segmentation is apparently a common phenomenon of normal lymphocytes and their leucocyte derivatives, and may be a method of maintaining the nucleo-cytoplasmic relationship at an optinmm, an end aided also by the nuclear amitosis characteristic of these cells.

2. The giant-cells are essentially hypertrophied hemoblasts and in the yolk-sac may function as multiple erj'throblasts.

3. Blood-platelet formation appears to be a by-product both of the normal activity and of the disintegration of potentially erj'throcytogenic giant-cells.

4. The true amphiliian homologue of the mammaUan "megakarj-ocyte' is not the thrombocyte, but a mononucleated giantcell derived by hypertrophy from a primitive lymphocyte or hemoblast. Spin<ll(> cells of ichthyopsid and sauropsid i)loods, and platelets of mammalian bloods apparently have a similar function in relation to thrombus formation; therefore they maj' be considered analogous elements, but no strict homology' obtains between them.

5. Blood-platelets occur in the pig embryo coincidentally with the appearance of primitive lymphocytes, or yolk-sac hemoblasts, with metachromatic granules.

0. The evidence suggests that the seat of the prothrombin is the metachromatic granule, whether of hemoblast, megakarj-ocyte, spindle cell, lymphocyte, or platelet origin.

4()() H. E. .lonnAN

LITERATURE (1 TKD

1 BrNTiNO, C. H. 1U09 Hlood platelet ami megakaryocyte reactions in the

rabbit. Jour. Exp. Med., vol. 11, p. 541. '2 l)i«K8o.\, \V. E. ('. UtOS Tlie bone marrow. Thesis. Longmans, Green

& Co., London. 11. (Jiant colls, pp. 64-72.

3 DowNKV, H.^L 191.3 a The granules of the polymorphonuclear leucocyte

of Amblystonia, with a few notes on the spindle cells and erythrocytes of this animal. Anat. .\nz.. Hd. 44, .S. 309.

4 1913 b The origin of blood platelets. Folia Hacmatologica, Bd. 15, S. 25.

5 Howell, W. H. 1914 The coagulation of lymph. .\m. Jour. Physiol.,

vol. 35, p. 483.

6 Jordan, H. E. 1910 A microscopic study of the umbilical vesicle of a

13-mm. human embryo, with special reference to the entodermal tubules and the blood-islands. Anat. Anz.. Bd. 37, S. 12.

7 1916 The microscopic structure of the yolk-sac of the pig embryo, with special reference to the origin of the erythrocytes. Am. Jour. Anat., vol. 19, p. 277.

8 1917 Hemopoiesis in the mongoose embryo, with special reference to the activity of the endotheliimi, including that of the yolk-sac. Pub. no. 251, Carnegie Institution of Wash., p. 291.

9 1918 The histology of lymph, with special reference to platelets. Anat. Rec, vol. 15, p. 37.

10 1918 A contribution to the problems concerning the origin, structure, genetic relationship, and function of the giant-cells of hemopoietic and osteolytic foci. .\m. Jour. .\nat., vol. 24. p. 225.

11 Ogata 1912 Untersuchungen iiber <lie Herkunft der HUitpliittchen. Zieg ler's Bietr. z. pathol. .\nat. u. z. allgem. Pathologic, Bd. ,52, heft 1 (cited from Downey).

12 Saxer, F. 1896 Tber die Entwicklung und den Bau der normalen Lymph driisen und die Entstehung der roten und weisen Blutkorpcrchen.

Anat. Hefte. Bd. 6. S. 349. 1.3 Spee, Grak V. 1896 Zur Demonstration iiber die Entwicklungen der

Drtlsen des menschlichen Dottersackes. Anat. Anz., Bd. 12. p. 76. 14 Wbioht, J. H. 1910 The histogenesis of the blood platelets. Jour. Morph.,

vol 21, p. 263.