American Journal of Anatomy 1 (1901-02)

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Amer. J Anat.: 1 - 1901-02 | 2 - 1902-03 | 3 - 1904 | 4 - 1905 | 5 - 1906 | 6 - 1906-07 | 7 - 1907-08 | 8 - 1908 | 9 - 1909 | 10 - 1910 | 11 - 1910-11 | 12 - 1911-12 | 13 - 1912 | 14 - 1912-13 | 15 - 1913-14 | 16 - 1914 | 17 - 1914-15 | 18 - 1915 | 19 - 1916 | 20 - 1916 | 21 - 1917 | 22 - 1917 | 23 - 1918 | 25 - 1919 | 26 - 1919-20 | 27 - 1920 | 28 - 1920-21 | 29 - 1921 | 30 - 1922
Historic Journals: Amer. J Anat. | Am J Pathol. | Anat. Rec. | J Morphol. | J Anat. | J Comp. Neurol. | Johns Hopkins Med. J | Ref. Handb. Med. Sci. | J Exp. Zool. | Yale J Biol. Med.
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PEOPLE Full text of "The American journal of anatomy" See other formats MARINE BIOLOGICAL LABORATORY.

Received SL^^^Jl^

Accession l^o.....3.. .■^...^...JL

Given hy .}^I:.^(:.dZt2^..J)..<:y^L<2^^

Place, ^^.^.i^.[.Jj24^:,.J^...}7:^^ -7.i^-^yi^


^* No book or pamphlet is to be removed from the Lab oratory without the permission of the Trustees


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University of Chicago. Columbia Univernity


Harvard University. Johns Hopkins University.


Cornell University. Harvard University.


University of Michigan. University of l^eniisyivaflia.

HENRY Mc E. KNOWER, Secketaky, Johns Hopkins University.







No. 1. November 7, 1901.

' I. Charles Eussell Bardeen and Warren Harmon Lewis. The Development of the Limbs, Body-Wall and Back 1

With 9 Plates and 27 text figures.

II. Preston" Kyes. The Inti-alobular Framework of the

Human Spleen 37

With one text figure.

III. G. Carl Huber. Studies on the Neuroglia .... 45

IV. Alfred Scott WarThin. The Normal Histology of

the Human Hemolymph Glands 63

V. Charles Sedgwick Minot. On the Morphology of the Pineal Region, based upon its Development in

Acanthias 81

With 14 text figures.

No. 2. February 28, 1902.

VI. B. F. Kingsbury. The Spermatogenesis of Desmog nathus Fusca 99

With 4 Plates.

VII. John Lewis Bremer. On the Origin of the Pulmonary

Arteries in Mammals 137

With 9 text figures.

VIII. Warren Harmon Lewis. The Development of the

Arm in Man 145

With 2 Plates and 14 text figures.

IX. Arthur B. Lamb. The Development of the Eye Muscles in Acanthias 185

With 9 text figures.

X. Charles Eussell Bardeen. A Statistical Study of

the Abdominal and Border Nerves in Man .... 203 With 8 figures and 14 tables.

iv Contents

No. 3. May 26, 1902.

XL Fkederic T. Lewis. The Development of the Vena

Cava Inferior 229

With 11 text figures and 2 double colored Plates.

XII. John Bruce ]\L\cCallum. Notes on the Wolffian Body

of Higher Mammals 245 With 17 text figures.

XIII. Franklin Dexter. On the Vitelline Vein of the Cat, 261

With 8 text figures.

XIV. Joseph Marshall Flint. The Ducts of the Human

Submaxillary Gland 269

W'ith 9 text figures.

XV. S. W. Williston. On the Skeleton of Nyetodactylus,

with Kestoration 297

With 1 text figure.

XVI. Frederick Adams Woods. Origin and ]\Iigration of

the Germ-Cells in Acanthias 307

With 14 text figures.

/ XVII. Katherine Foot and Ella Church Strobell. The

Spermatozoa of Allolobophora Foetida 321

AVith 1 Plate.

XVIII. Franklin P. Mall. On the Development of the Connective Tissues from the Connective-Tissue Syncytium 329 With 18 text figures.

XIX. Florence E. Sarin. On the Origin of the Lymphatic System from the Veins and the Development of the Lymph Hearts and Thoracic Duct in the Pig . . . 367 With 12 text figures.

No. 4. September 15, 1902.

XX. ]\Iervin T. Sudler. The Development of the Nose,

and of the Pharynx and its Derivatives in Man . . 391 With 1 3 text figures.

XXI. Alice Hamilton. A Case of Heterotopia of the White

Matter in the Medulla Oblongata 417

With 4 text figures.

XXII. Harris Hawthorne Wilder. Palms and Soles . . 423 With 21 text figures.

Contents v

XXIII. Daniel G. Eevell. The Pancreatic Ducts in the Dog, 443 With 14 text figures.

XXIY. AViLLiAM A. Hilton. The Morphology and Development of Intestinal Folds and Villi in Vertebrates . 459 With 3 tables and 7 Plates (87 figures).

XXV. Proceedings of the Association of American Anatomists, 507


The Ameeicax Journal of Anatomy has been founded to collect into one place, and present in a worthy manner, the many researches from our investigators, now scattered through many publications at home and abroad.

Human Anatomy in America needs as high a standard of reference as it has in other countries. Without such a standard it fails to make for itself any proper, satisfactory or stimulating impression. The best interests of modern scientific medicine will be greatly advanced by the upholding of such a standard in this fundamental subject through a journal of high character.

Many asi^ects of Comparative xVnatomy, Embryology, Histology and Cytology are so intimately bo and wp with the problems of Human Anatomy that these subjects will be included within the scope of the new journal. It will be the aim of The Ameeican Journal of Anatomy to recognize this close natural relationship between the various branches of the science, and to publish results of the best original work of American students of anatomy.

The most cordial assurance of support has been given by the collaborators, and this we believe is sufficient indication of the results to be expected.

A number of generous persons, whose names will appear later, have given some financial support to help us in gaining a foothold in a suitable manner. The journal must however look to those who are to be benefited by its publication for its real and permanent support; and a good list of regular subscribers is expected and required to maintain it.

It is hoped that those interested in promoting a worthy development of research in America, in the subjects included within the scope of this journal, will energetically assist us.

'/{r^i.r. ^?^



CHARLES RUSSELL BARDEEN, M. D. AND WARREN HARMON LEWIS, M. D. From tlm Aiiatomical Laboratory of the Johns Hopkins University, Baltimore, 31(1.

With 9 Plates and 27 Text Figukes.

The purpose of the following paper is a description of various typical stages in the development of the back, the limbs, and the body-wall in man. The work is based primarily upon reconstructions, according to the method of Born,' of parts of five human embryos; it has been extended and controlled by a study of the external form and of serial sections of several other human embryos. Dr. Lewis has devoted special study to the formation of the arm. Dr. Bardeen to that of the leg, the body-wall and the back.

In the accompanying table a list is given of the embryos utilized. Those marked with an asterisk have been reconstructed.

We shall consider the early stages in the development of the limbs, the body-wall and the back, first, from the point of view of the external form and, secondly, from that of internal structural differentiation.

I. External Form.

The external form of the embryos we have used has been compared with that of embryos of a corresponding stage of development pictured ^-- XI, „ TT-:^ A+inc.2 T^io-o ^-^^ on naffes 3 to 9 represent a series of

ERRATUM FOR Vol. I, No. 1.

Vol. I, No. 1, page 83, Fig. 2, of this Journal should read:

Fig. 2. Embryo of 13.0 mm. Sagittal series, No. 224, section 6.5. x 30 diams.




From the. Anatomical Laboratory of the Johns Hopkins University , Baltimore^ Md.

With 9 Plates and 27 Text Figures.

The purpose of the following paper is a description of various typical stages in the development of the back, the limbs, and the body-wall in man. The work is based primarily upon reconstructions, according to the method of Born,' of parts of five human embryos; it has been extended and controlled by a study of the external form and of serial sections of several other human embryos. Dr. Lewis has devoted special study to the formation of the arm, Dr. Bardeen to that of the leg, the body-wall and the back.

In the accompanying table a list is given of the embryos utilized. Those marked with an asterisk have been reconstructed.

We shall consider the early stages in the development of the limbs, the body-wall and the back, first, from the point of view of the external form and, secondly, from that of internal structural ditferentiation.

I. External Form.

The external form of the embryos we have used has been compared with that of embryos of a corresponding stage of development pictured in the His Atlas.^ Figs. 1-15, on pages 3 to 9 represent a series of embryos belonging some to the Mall collection and some to the His collection. The general relation of the limbs and body-wall in embryos between two and seven weeks of age,"* and between 2.1 and 20 mm. in length, are here represented by simple outline diagrams, based in part upon published drawings and in part upon photographs and upon

1 See Bardeen : Wax plate reconstruction according to tlie method of Born as utilized in the Anatomical Laboratory of the Johns Hopkins University. The Johns Hopkins Bulletin, April-May-June, 1901.

-Anatomic menschlicher Embryonen, Leipzig, 188.5.

3 The ages given are for the most part only roughly approximate. 1

2 Development of the Limbs, Body-wall and Back in Man








Length in mm.

Probable ag-e in weeks.







NB 5


VB 4.5

NB 7


VB 6

NB 9 VB 9




VB 11

NB 12 VB 14


NB 13



NB 15.5 VB 17


NB 14.5



NB 14

VB 16

NB 18


VB 20

Size of Ovum in mm.

Thickness of section

in micro mm

























Number of Myotomes.


14 8c.


So. 5s.

35 8c. 2- 3c. or 12t.

36 51.

2o. 2s. 8c. 27 lot. 51.

cannot be counted with certainty.

3o. 5s. 8c. 5-6c. 38 12t. 51.

8c. 5s. 34 12t. 4c. 51.




Myotomes opposite


arm leg



7-11 .5c- It





21-2^5 or 26 11-ls


11-1 or 2s

No. of Myotomes between tlie arm and leg regions





The Roman numerals refer to embryos in the collection of human embryos belonging to Prof. Mall, in the Anatomical Laboratory of the Johns Hopkins University. To Dr. Mall we are gi eatly indebted for the use of these embryos.

Reference to the embryos given in Table I will be found in the following articles by Dr. Franklin P. Mall. No. II, A Human Embryo Twentj^-six Days Old, Jour, of Morph., Vol. V; Nos. II, XII, XXII and XLIII, Development of the Human Coelom, Jour, of Morph., Vol. XII; Nos. II, XII and XXII, Ueber die Bntwickelung des Menschlichen Darmes, Arch, fiir Anat. und Phys., Special Bd., 1897; Nos. II, XLIII and LXXVI, Development of the Internal Mammary and Deep Epigastric Arteries in Man, Johns Hopkins Hospital Bulletin, 1898; Nos. II, XH, XXII, XLIII, LXXVI and CIX, Development of the Ventral Abdominal Walls in Man, Jour, of Morph., Vol. XIV, 1898; Nos. II, XII, XXII. LXXX CVI. CIX and CXLVIII, A Contribution to the Study of Pathology of Early Human Embryos, Johns Hopkins Hospital Reports, Vol. IX, 1901.

Charles Eussell Bardeen and Warren Harmon Lewis 3

sketches of the embryos indicated. On Plate I the photographs utilized are reproduced. On Plates II to IX are represented several typical stages in the general development of the body-wall and limbs. Figs. A and B, Plate II, are drawn from wax-plate reconstrnctions. Figs. C to E, Plates III to Y, are based, in the main, npon a reconstrnction of the regions of the arm, abdomen and leg of embryos CLXIII and CIX, and npon excellent photographs. Figs. F to I, Plates VI to IX, are based npon wax-plate reconstrnctions of Embryo XXII.

Fig. 1. X about 10 d.

Fisr. 2.

Fig. 3. Fig. 4.

X about .5 d.

Fig. .5.

i:mbryo XII.

The development of the neuro-muscular apparatus begins in the human embryo in the cervical region. In Fig. 1 is represented Embryo XII, 2.1 mm. in length and about two weeks of age. The axis of the embryo is curved in a semicircle about the heart and the umbilical vesicle. The axis contains neural tube, notochord, myotomes, dorsal-aortas, and mesenchyme (see Fig. 16). There are fourteen myotomes on each side. Mall considers three of them occipital, eight cervical, and three thoracic. The first cervical and the first thoracic myotomes are numbered " 1 " in Fig. 1. Caudal to the fourteenth myotome, an unsegmented band of tissue extends along each side of the spinal cord. The neural tube is open dorsally anterior to the fourth and posterior to the fourteenth myotome. Opposite the twelfth myotome a solid band of cells, the "' neurenteric canal," unites spinal-cord and entoderm. The notochord extends from a point opposite the cephalic margin of the heart to the region of the neurenteric canal. The dorsal aortae run a course parallel with the notochord, but extend further than the myotomes caudally. A considerable amount of mesenchyme is formed at the cephalic extremity of the axis of the body, in the region of the heart, but toward the caudal extremity little exists. The heart and the pericardial and pleural cavities are developed in the cephalic regi'on of the wall of the umbilical vesicle. Between the region of the heart and the neural

4 Development of the Limbs, Body-wall aud Back in Man

tube the pharynx extends forwards. Trom it project the hrst and second branchial pockets, Seessel's pocket, and the thyroid diverticulum. Into the caudal end of the embryo the hind-gut extends. The umbilical vesicle projects forwards from the region opposite the 1-6 cervical myotomes (see Fig. 1). At this period the amnion arises on each side along the length of the axis of the embryo as far forward as the region of the heart (Fig. 1). Externally and internally the amnion is covered by a layer of epithelial cells. From epithelium lining the coelom, several layers of cells have arisen (Fig. 16). There is, however, as yet, no true body-wall caudal to the region of the heart. There are no external visible signs of limb buds.

Embryo Lr.

In Fig. 2 is represented the His embryo Lr; length, neck-breach, 4.2 mm.; age, about three weeks. The back of this embryo presents a slight concavity opposite the ninth (first thoracic) myotome. It is probable that this is an artifact, due to the removal of the embryo from the ovum, and that in the natural condition the back curved about the viscera as it does in the embryos represented in Figs. 1, 3, 4 and 5." "Lr'^ shows externally thirty-one myotomes (8c, 12t, 51, 5s, Ic). The ninth (first thoracic) and twenty-first (first lumbar) myotomes are designated by the numeral " 1. Lateral to the region of the myotomes lies the Wolffian ridge, a band of tissue which represents the anlage of the limbs and body-wall. The arm is represented by a slight swelling opposite the 5th to 8th cervical and 1st thoracic myotomes. The leg is represented by a slight swelling opposite the 1st to 5th lumbar and 1st sacral myotomes. The amnion was probably attached, in this embryo, to the umbilical cord. Between the Wolffian ridge and the umbilical cord the menibrana reuniens extends, at this period, so as to cover over the thoracic and abdominal viscera. It is represented as torn along the heavy irregular line.


In Fig. 3 is represented Embryo CXLVIII; length, neck-breach, 4.3 mm.; age, about three weeks. A photograph of the embryo is given on Plate I. Though more advanced in development than Lr, but twenty-seven myotomes are present (2o, 8c, lOt, 51, 2s). This has been determined by careful counting of the myotomes in serial sections of the embryo. The base of the arm-bud appears to lie opposite the sev 4 See Mall, Human Coelom, op. cit., p. 421.

Charles Eussell Bardeen and Warren Harmon Lewis 5

entli to the eleventh myotomes. It is, therefore, probable that two occipital myotomes are present. But nine myotomes lie in the area between the arm-bud and the leg-bud. The base of the latter lies opposite 31st to the 25th or 86th myotomes. If two myotomes be considered occipital myotomes, the leg, in this instance, lies two segments nearer the head than usual. It is therefore probable that this embryo has an unusually short body-wall.

Embryo LXXVI (length, 4.5 mm.; age, about three weeks) is of essentially the same stage of development as CXLVTII. It has thirtyfive myotomes (3o, 8c, 13t, 51, 5s, 2c). The base of the arm lies opposite the eighth (fifth cervical) to the twelfth (first thoracic) myotomes. The base of the leg lies opposite the twenty-fourth (first lumbar) to the twenty-ninth (first sacral) myotomes. Eleven myotomes lie between the regions of the arm and leg-buds. In CXLVIII the limbbuds protrude more than in LXXVI and the body-wall extends further ventrally.

Embryo LXXX (length, 5 mm.; age, about three weeks), a photograph of which is given in Plate I, is similar, though slightly more advanced in development than Embryo CXLVIII.


In Fig. 4 is represented the His embryo a ; length, neck-breach, 4 mm.; age, about 23 days. The back of this embryo is very greatly flexed. Thirty-five myotomes are present (8c, 12t, 51, 5s, 5c). The arpabud lies opposite the 5th to 8th cervical and 1st thoracic myotomes; the leg-bud opposite the 1st to 5th lumbar and 1st sacral myotomes. Both protrude further than in CXLVIII. The arm-bud projects in a caudal direction. The leg is represented, not as in the original His drawing, but instead in the more normal position shown in the His drawing of the right side of the same embryo.

Embryo R.

In Fig. 5 is represented the His embryo E; length, 5 mm.; age, about 3^ weeks. Thirty-five myotomes are pictured (8c, 12t, 51, 5s, 5c). The arm-bud lies opposite the 5th to 8th cervical and 1st thoracic myotomes; the leg-bud opposite the 1st to 5th lumbar and 1st sacral. Both point somewhat caudally.

Embryo II.

In Fig. G is represented Embryo II; length, neck-breach, 7 mm.; vertex-breach, 6 mm.; age, about 4 weeks. Thirty-eight myotomes are

6 Development of the Limbs, Body-wall and Back in Man

present (3o, 8c, 12t, 51, 5s, 5-6c). The extensions of the myotomes within the body Avail are pictured. The base of the arm-bud lies opposite the 5th to 8th cervical and 1st thoracic myotomes; that of the legbud opposite the 1st to 5th lumbar and 1st sacral myotomes. The armbud projects caudally, the leg-bud outwards and slightly caudally.

Embryo A.

In Fig. 7 is represented the His embryo A; length, 7.5 mm.; age, about 4 weeks. Thirty-five myotomes are pictured (8c, 12t, 51, 5s, 5c). The arm-bud lies opposite the 5th to 8th cervical and 1st thoracic; the leg-bud lies opposite the 1st to 5th lumbar and 1st sacral myotomes.^ Both project caudally. Both show a slight division into segments. This, however, is much more marked in the following embryo.

Fig. 7. X about 5 d.

Embryo CLXIII.

In Fig. 8 is represented Embryo CLXIII; length, 9 mm.; age, about 4|- weeks. Two photographs of this embryo are shown on Plate I. Thirty-three myotomes are present (8c, 12t, 51, 5s, 3c). The base of the arm lies opposite the 4th to 8th cervical and 1st thoracic, and that of the leg opposite the 1st to 5th lumbar and 1st to 2nd sacral myotomes. The arm projects nearly caudally. A constriction on the cephalic and caudal borders separates the rounded upper arm from the flattened lower arm and hand. The constriction on the caudal border is close to where the arm joins the body-wall, while that on the cephalic border is at a point some distance from the body-wall. This difference on the two borders is to be correlated with the caudal projection of the arm.

5 This statement is based on the drawing given in Fig. 3, Plate I* of the Atlas.

Charles Eussell Bardeen and Warren Harmon Lewis 7

The medio-lateral flattening of the distal portion of the arm-bud is especially well marked. Proximal to this flattened portion swellings on both medial and lateral surfaces indicate -where pre-muscle tissue is developed. A constriction may likewise be seen dividing the leg-bud into two distinct divisions. Owing to a slight torsion the lower portion o± the leg-bud presents to view the anterior margin instead of the flattened lateral surface.

Fig. 9.

X about 5 d.

Fig. 10.

Embryo Bi\. In Fig. 9 is represented the His embryo Br,; length, 11 mm.; age, about 4^ weeks. Thirty-five myotomes are pictured (8c, 12t, 51, 5s' 5c).' The base of the arm lies opposite the 4th to 8th cervical and' 1st thoracic spinal ganglia. The division of the arm into its main segments is advanced beyond that pictured in Embryo CLXIII. The upper arm still projects caudally. The lower arm, owing to flexion at the elbow projects caudo-ventrally. The hand is flattened and can be distinguished from the forearm. Swellings of the digits are visible. The flrst indications of the shoulder are present. The posterior limb shows a differentiation of foot, leg and thigh regions.

Embnjo CIX. Fig. 10 represents Embryo CIX; length, 11 mm.; age, about 5 weeks Two photographs of this embryo are reproduced in Plate I. The base

"Externally visible segmentation at this stage is due to the spinal ganglia not to myotomes. The latter have lost their identity.

8 Development of the Limbs, Body-wall and Back in Man

of the arm lies opposite the 3d to 8th cervical and 1st thoracic spinal ganglia. The upper arm still projects caudally. The forearm is more flexed and projects ventrally; it is now quite well marked off from the upper arm and hand. The digital swellings have increased and are now visible on the margin as well as on the flattened surface of the hand. The shoulder is more marked. The base of the arm is larger and extends higher in the cervical region than in the younger stages. The posterior limb shows a distinct differentiation of the foot. The kneebend may be distinguished. The hip region is not clearly marked externally.

Fiff. 11.

Fig. 12. X about 2^ d.

Fig. 13.

Embryo CLXXV. Fig. 11 represents Embryo CLXXV; length, 13 mm.; age, about 5-| weeks. Two photographs of this embryo are given in Plate I. The various regions of the arm and the swellings of the digits are well marked. The forearm has a more caudal projection than in Fig. 10, In the posterior limb the various regions are more or less distinctly indicated. In the foot digitation has begun. The body wall has advanced half-way across the surface of the liver.

Enibryo CVI.

Fig. 12 represents Embryo CVI; length, vertex-breach, 17 mm., neckbreach, 15.5 mm.; age, about 5^ weeks. A photograph is reproduced on Plate I. The limbs and body-wall are similar in development to those of Embryo CLXXV, but the flexion at the elbow and knee is more marked, and the body-wall lias advanced further across the abdomen.

Embryo CLXVII. Fig. 13 represents Embryo CLXVII; length vertex-breach 14.5 mm., neck-breach 13.5 mm.; age, 5-|- weeks. A photograph is shown on

Charles Eussell Bardeen and Warren Harmon Lewis

Plate I. While the embr3'0 is similar in general differentiation to Embryos CLXXV (Fig. 11) and CVI (Fig. 13), the development of the digits of the hands and feet is further advanced. The flexion of the forearm is also more marked.

Fie:. 14.

X about 3^ d.

Fig. 15.


Emhryo XLIII.

14 represents Embryo XLIII; length, vertex-breach 16 mm., neck-breach 14 mm.; age, about 6 weeks. The forearm and leg have grown considerably beyond the stage shown in Figs. 11-13, but without marked alteration in external form.

Emhryo XXII. Fig. 15 represents Embryo XXII; length, vertex-breach 20 mm., neck-breach 18 mm.; age, about 7 weeks. A photograph is reproduced on Plate I. The limbs have begun to resemble those of the adult. In the hand the clefts between the digits are well marked. The forearm and hand are somewhat pronated. The tips of the digits now reach nearly to the ventral mid-line. In the hind-limb the foot still lies in the same plane with the leg. The twist at the ankle which brings the foot into adult position has not begun. The toes are fairly distinct.'

' A number of embryos have been pictured wliich correspond essentially in external form to those above described. The following list gives reference to the articles in which several of these have been described and pictured. To the His embryo R. Fig. 5, corresponds :

Foil's 5.6 mm. embryo, Fig. 217, p. 386, Minot's Embryology. To No. II, Fig. 6, corresponds :

Keibel's embryo H. S., length 8 ram., Arch, fiir Anat. und Phys., 1891, Taf. TvIX, Fig. 2.

10 Development of the LimbS;, Body-wall and Back in Man


In noting the main features of development in early human embryos, the most convenient landmarks for describing relative positions of structures are the myotomes. By the end of the second week of embryonic development about fourteen myotomes have been distinctly differentiated in the human embryo (see Fig. 1). The formation of myotomes continues until by the end of the fourth week about thirtyeight have been differentiated (see Embryo II, Fig. 6). Anterior to the eight cervical myotomes, three occipital myotomes seem usually to be developed; posterior to the cervical region, twelve thoracic, five lumbar, five sacral, and five to six coccygeal myotomes are formed. The division of the myotomes into occipital, cervical, thoracic, luinbar, sacral and coccygeal groups depends upon the nerves and skeletal structures related to the body segments in which the myotomes lie.

The occipital myotomes are transient structures developed in conjunction with roots of the Twelfth Cranial Nerve. Before the spinal nerves have appeared they cannot with certainty be distinguished. No account of occipital myotomes is given in the description of the embryos pictured in the His Atlas. In the Mall embryos, LXXVI. CXLVIII

To the His embryo A, Fig. 7, correspond :

His's embryo Pr, length 10 mm., Atlas, Taf. XIII, Fig. 4, and Taf. X, Fig. 10.

His's embryo B, length 7 mm.. Atlas, Taf. I, Fig. 1. To No. CLXIII, Fig. 8, correspond:

Kollmann's 10.3 mm. embryo, Arch, of Anat. and Phys., 1891, Taf. Ill, Fig. 1.

Ruge's 9.1 mm. embryo, His's Atlas, Taf. XIII, Fig. 5, and Taf. X, Fig. 12. To the His embryo Br, Fig. 9, corresponds :

Keibel's embryo, H. S. Bui., length 11. .5 mm.. Arch, fiir Anat. und Phys., 1891, Taf. XIX, Fig. 13a. To No. CIX, Fig. 10, corresponds : ' His's embryo S, length 13..5 mm.. Atlas, Taf. XIII, Fig. 7, and Taf. X, Fig. 16. To No. CLXXV, Fig. 11, corresponds:

His's embryo^Schj, length 13.8 mm., Atlas, Taf. XIV, Fig. 3, and Taf. X, Fig. 18. To No. CVI, Fig. 13, correspond :

His's embryo Br^, length 13.3 mm.. Atlas, Taf. XIV, Fig. 1.

Ruge's 13.6 mm., embryo, His Atlas, Taf. XIV, Fig. 4, and Taf. X, Fig. 19. To No. CLXVII, Fig. 13, corresponds :

His's embryo Pr, length 14..5 mm.. Atlas, Taf. XIV, Fig. .5, and Taf. X, Fig. 30. To No. XLIII, Fig. 14 corresponds :

His's embyro 8^, length 1.5..5 mm., Atlas, Taf. X, Fig 31. To No. XXII, Fig. 1.5 correspond :

His's embryo XCI, length 16 mm., Atlas, Taf. X, Fig. 33.

His's embryo Ltj, length 17..5 mm., Atlas, Taf. X, Fig. 33.

His's embryo Z. W., Atlas, Taf. X, Fig. 34.

Charles Eussell Bardeen and Warren Harmon Lewis 11

and II, the occipital myotomes have been determined by a careful study of serial sections.

The cervical myotomes are those developed in conjimction with the cervical spinal nerves. In the vast majority of instances these are eight in number. The occasional absence in the adult of a cervical vertebra indicates that in the embryo less than the normal number of cervical spinal segments sometimes develop. In all of the embryos we have studied, the arm-bud begins its development opposite the fifth to the eighth cervical segments and opposite the following spinal segment. The most caudal myotome lying opposite the arm-bud may, therefore, be taken to represent the first thoracic segment.

Between the base of the arm-bud and that of the leg-bud, as a rule, eleven myotomes intervene. A marked exception to this is found in Embryo CXLVIII, where but nine myotomes seem to lie in this region. In other instances, twelve myotomes have been pictured. Such is apparently the case in the His embryo B (Atlas, Taf. I, Fig. 1) and in the His embryo Pr (Atlas, Taf. XIII, Fig. 4). It is difficult to determine this with certainty from the figures. Variation in the number of myotomes intervening between, the regions of the arm and leg corresponds with well-known variations in the length of the spinal axis in the adult.' ,

Bardeen, Costo-vertebral Variation in Man, Anat. Anz., 1900. In the His wax-models of young human embryos, Embryo Lr (No. 6) shows nine myotomes between the regions of the swellings which indicate the arm- and leg-buds. Embryo A shows fifteen myotomes between the arm and leg areas. The number of the myotomes in the thoraco-abdominal region in each of the models is probably incorrect. Fig. .5, Plate XX, of the His Atlas seems to show the usual number of thoraco-abdominal myotomes in Lr. Fig. 2, Plate I*, shows twelve instead of fifteen thoraco-abdominal myotomes in A. The model of A presents, in case of the thoracoabdominal myotomes, conditions characteristic of the pig. Out of twelve young pig embryos of various sizes, in eleven instances we have found sixteen myotomes intervening on each side between the arm and leg regions, and in one instance fifteen. In the pig, therefore, five more body segments than in man lie between the arm and leg areas. This is also to be seen in the adult. In the pig there are two more thoracic segments than in man, and, as indicated by the nerve distribution to the abdominal wall, three more abdominal segments. The third lumbar nerve in the pig corresponds in distribution somewhat to the first lumbar in man, but it has a more extensive abdominal distribution. While therefore in man the first lumbar segment is counted as belonging to the leg area, in the pig the third lumbar segment may be considered to belong to the thoraco-abdominal region. There are six lumbar vertebrie in the pig. The furcal nerves are the fifth and sixth lumbar usually, but sometimes the fifth, probably sometimes also the sixth, may be the sole furcal nerve.

In the Keibel " Normentafeln " of the pig the number of myotomes pictured between the arm and leg areas varies from fifteen to eighteen in different embryos. It

12 Development of the Limbs, Body-wall and Back in Man

The leg-bud arises opposite six myotomes. These usually are the twenty-fourth to the twenty-ninth, corresponding to the five lumbar and 1st sacral myotomes. Caudal to this region arise the remaining sacral and the coccygeal myotomes.

The myotomes give rise to the dorsal musculature, to the thoracoabdominal musculature, and to the musculature of the neck, tongue (?) and caudal region. When differentiation of body musculature takes place the distinction between the myotomes becomes lost, and they can no longer be used as landmarks. This occurs by the time the embryo has reached a length of 11 mm. and an age of five weeks. Hereafter segmental skeletal structures and spinal ganglia may be used as landmarks. The former present the more stable relative conditions.




Development of the spinal axis begins in the cervical region. At the end of the second week fourteen myotomes are present (3o, 8c, 3t); at the end of the fourth week, thirty-eight (3o, 8c, 12t, 51, 5s and 5c). During the fifth week the myotomes, owing to fusion of their dorsal surfaces, cease to be externally visible. The spinal ganglia, however, give rise to a segmentation externally visible for a somewhat longer period.

The limbs and body-wall arise in the region where the amnion joins the axis of the embryo. By the end of the second week, at the stage represented in Embryo XIT, the amnion is attached directly to the axis along a line extending from a region anterior to the heart to the caudal extremity (Fig. 1). The amniotic cavity rapidly enlarges. That part of the amnion near the axis of the embryo is carried ventrally so that it closes in the viscera which have previously protruded free into the general coelomic cavity. Finally the amnion reaches the allantoic stalk (or umbilical cord) and becomes attached to this. That part of the amnion extending from the umbilical cord to the axis of the embryo is now known as the menibrana reuniens. It forms the chief covering of the pericardial, pleural and peritoneal cavities. Fig. 2 probably, and Fig. 3 certainly, represent stages in which the viscera are completely inclosed by the menibrana reuniens.

is possible tliat no great care was taken in determining accurately the number of myotomes in tbis region. In the " Normentafeln " of the chick the number of myotomes pictured in this area varies from seven to ten. Apparently seven in the normal number.

Charles Eussell Bardeen and Warren Harmon Lewis 13

During the second half of the third week the membrana reuniens becomes markedly thickened along its line of attachment between, usually, the fourth and twenty-sixth spinal segments. This thickening is known as the WoMan ridge. In Figs. 2-7 the ventral margin of this ridge is emphasized by a heavy line. Opposite the fifth to the ninth and the twenty-first to the twenty-sixth spinal myotomes this thickening is especially marked. The two latter areas represent the inception of the limb-buds; the intervening area represents the rudiment of the lateral body-wall. These three areas first become well marked toward the end of the third week (Figs. 2-3).

The limb-buds increase very rapidly in size. At first the limb-buds extend directly laterally from the Wolffian ridge (Figs. 2 and 3), but as development proceeds they project more and more in a caudal direction (Figs. 4, 5 and 6). Meanwhile two distinct divisions appear in each limbbud. The part which arises directly from the Wolffian ridge we may call the basal division. This portion continues to grow in the ventrolateral direction taken by the limb-bud originally. Beyond this basal part the limb-bud bends in a ventro-median direction. The part of the limb-bud beyond the bend we may call the " distal part " of the limbbud. For some time the basal portion of the limb-bud continues to grow in a ventro-lateral direction, while the distal part grows in a ventro-median direction. The limb as a whole meanwhile points distinctly in a caudal direction.

Dorsally the base of the limb becomes continuous with the dorsal margin of the Wolffian ridge, ventrally with its ventral margin. The dorso-lateral surface of the base of the limb-bud is therefore extensive; the ventro-median surface is small in area. Owing to the caudal direction assumed by the limb, the anterior (cephalic) surface of the base is extensive, while the posterior (caudal) surface is limited in extent.

The distal part of each limb-bud is flattened so that it presents median and lateral surfaces and anterior (cephalic), ventral and posterior (caudal) margins. A constriction may be seen immediately beyond the region where the distal flattened portion of the limb joins the thicker rounded base. In Fig. 8 the constriction is emphasized by heavy lines. In the arm it is seen as one looks at the limb directly from the side. In the leg, which is here slightly twisted, one may see the constriction as it appears when the limb is viewed on the anterior margin. The constriction is just beginning to appear in the leg-bud shown in Fig. B, Plate II, and in Fig. C, Plate III.

From the basal ^portion of each limb-bud are developed the limbgirdle (shoulder and pelvic-girdles), and the upper limb (upper arm and

14 Development of the Limbs, Body-wall and Back in Man

thigh), from the distal portion are developed forelimb (forearm and leg) and extremity (hand and foot). The place where the distal part of the limb joins the base represents the future elbow or knee. These two joints are formed in a flexed position (see especially Figs. F, G, H and I, Plates VI-IX).

The constriction mentioned above serves to designate the differentiation of the distal part of the limb-bud into the forelimb and extremity. Opposite the constriction the forelimb is formed; immediately distal to it the extremity (Figs. 8, 9, 10, 11, 12, 13, 14, 15).

The hand and foot are at first flattened, disk-shaped bodies. Digitation is first marked on the lateral surface (see the hand in Fig. 9), and then in the free margin also (Fig. 10).

Differentiation of the base of the limb-bud is somewhat less easy to follow than that of the distal part. This is due mainly to the fact that many distinguishing structures are at first deep-seated. As the musculature, however, becomes distinctly differentiated, the various main groups of muscles give rise to distinctive external characteristics.

The limbs at the stage shown in Fig. 15, p. 9, and in Figs. F, Gr, H and I, Plates VI-IX, exhibit rudiments of most of the structures characteristic of the adult limbs. Much growth and shifting of parts, however, is necessary before adult conditions are reached.

The great curvature of the axis of the body opposite the limbs during their formation has been noticed by His."

The arm-bud at first lies opposite the 5th to 8th cervical and 1st thoracic myotomes. As it grows in size the area of attachment to the body enlarges and extends on its cephalic side to the level of the 3rd cervical neural process (see Embryo CIX, Fig. 10). We find also that the shoulder comes to lie closer and closer to the precervical sinus until the embryo reaches a length of about 20 mm. As development still further proceeds the shoulder gradually migrates caudally and the distance between the shoulder and precervical sinus increases imtil the adult position is attained. Simultaneous with this caudal migration of the arm, the lower portion of the ventral wall of the neck appears.

The posterior limb arises slightly later than the anterior. Throughout its development the leg lingers somewhat behind the arm. The base of the leg-bud is at first opposite the 1st to 5th lumbar and the 1st sacral myotomes. Gradually the base extends so as to include the region opposite the second and third sacral myotomes and the upper

9 Zur Geschichte des Gehirns etc., Bd. XIV, der Abhandl. der Mathematisch-pbysiclien Classe der Konigl. Sachs. Gesellschaft der Wissenschaften, p. 381.

Charles Eiissell Barcleen and Warren Harmon Lewis


extremity of the base ceases to extend over the region of the first lumbar segment. As adult condition is approached, the leg assumes a much more caudal position.

During the growth of the limbs the body-wall grows forwards at the expense of the membrana reuniens. In the- various figures the ventral line of the body-wall is indicated. The abdominal wall has closed in against the umbilical cord by the time the embryo has reached a length of 6 cm. and an age of about 3 months.

Having thus considered the more general external features presented in the early development of the limbs and body-wall, let us turn to a consideration of the formation of the main structures within these areas.

Fig. 16. Cross section

through the fourth cervical

segment of Embryo XII. X 55 d.

II. Internal Structiteal Differentiation. Second Week.

•-»;4V,.';jv^ ;>■,.-;% «?,*.' -jTi

Fig. 17. Cross section through the fifth thoracic segment of embryo LXXVI. x 55 d. The right side of the section passes through the middle, the left side through the posterior third of the segment.

In an embryo of two weeks, No. XII, Fig. 1, the viscera are attached to the ventral surface of the spinal axis of the embryo, and the amnion is attached to its lateral margin. Fig. 16 shows the conditions which are seen in a transverse section through the fourth cervical segment. The spinal cord is, in this region, a closed tube, the lateral walls of which are composed of five or six layers of epithelial cells. On each side lie the myotomes. These are oval in cross-section, square with rounded corners when viewed from the lateral surface. Each has a well-marked myocoel, surrounded on all sides by four or five layers of epithelial cells. On the left in Fig. 16 the section passes through the centre, at the right it passes through the anterior margin of a myotome. Ventral to the

16 Development of the Limbs, Body-wall and Back in Man

spinal-cord lies the notochord; on each side of this a dorsal aorta. Between notochord, aortse, spinal-cord and myotomes lies a certain amount of mesenchyme, which arises apparently from the ventro-median surface of the myotomes.

In the region between the dorsal margin of the myotomes, the spinalcord and the ectoderm lie spinal ganglia cells.

Ventral to the aorta on each side the splanchnopleure is attached to the axis of the embryo; ventral to the myotomes on each side, the somatopleure. The latter is continuous with the amnion. The coelom is surrounded by epithelium, and this is surmounted by several layers of mesenchyme cells which apparently have arisen from the epithelium lining the ccelom.

Third WeeTc.

During the third week of embryonic development marked changes take place in the spinal axis of the embryo and in the adjoining somatopleure. A section throvigh the fifth thoracic segment of an embryo of the third week. No. LXXVI, is shown in Fig. 17. The spinal ganglia are definite, well-developed groups of cells on each side of the spinal-cord. In the spinal-cord the ventral-root zone is well marked, and the first ventral-root fibres have appeared. The myotomes have become flattened and elongated. The median surface of each of these has become converted into muscle cells, shown in cross-section in the figure. There is a single dorsal aorta, on each side of which a Wolfl&an body has developed. Dorsal to the Wolffian body lies the cardinal vein. The axial mesenchyme, which arises, at first at least, apparently from the myotomes, and the mesenchyme which springs from the coelomic wall have increased very greatly in amount, and have fused so as to form a common mass of tissue which surrounds- the other structures. None, however, intervenes between the dorso-lateral surface of the myotomes and the ectoderm.

A vascular plexus is developing in the mesenchyme. The axial mesenchyme of the caudal third of each segment has become dense. This is shown at the left in Fig. 17. It represents the anlage of the intervertebral disc and of the vertebral arch and costal processes. The somatopleure is considerably thicker than at the stage shown in Fig. 16. This increased thickness is due to an increase in the amount of the mesenchyme. This mesenchyme extends for a short distance dorsally between the ventral tip of the myotome and the ectoderm. The myotomes have not yet entered the body-wall.

In the region of the posterior limb the increase of mesenchyme between coelom and ectoderm precedes the formation of muscle on the

Charles Eussell Bardeen and Warren Harmon Lewis


median layer of the myotomes, the formation of the spinal ganglia, and the appearance of the ventral nerve roots. This is shown in Fig. 18, taken throngh the leg region of Emhryo LXXVI.

Fig. 18. Cross section tbrougli tlie lumbar region of Embryo LXXVI. x 55 d.

Fig. 19, taken through the leg region of Embryo CXLVIII, shows an older stage in which the limb-bud is considerably more advanced in development. The spinal ganglia are distinct. Formation of muscle cells has begun on the median surface of the myotomes.

Fig. 19. Cross section tbrough the lumbar region of Embryo CXLVIII. x 55 d.

The Wolffian ridge and limb-buds, which appear during the third week, as shown in Figs. 2, 3 and 4, consist, therefore, at the end of this period, merely of a mass of mesenchyme which intervenes between the coelom and the ectoderm lateral to the axis of the body. This mesenchyme contains a vascular plexus. Along the free edge of the limbbuds the ectoderm consists of several layers of epithelial cells (Fig. 19). 2

18 Development of the I-iimbs, Body-wall and Back in Man

Fourth Weelc.

During the fourth week certain structures extend from the spinal axis into the Wolffian ridge and the limb-buds. Let us consider first the relation of these axial structures to the body-wall and then their relation to the limb-buds.

Fig. 20. Cross section through the fifth thoracic segment of Embryo II. x .55 d. The right half of the section passes through the middle, the left half through the posterior third of the segment.

coelom sympathetic branch spinal i

Fig. 21. Diagrammatic cross section through the mid-thoracic region of Embryo II. About 2.5 d. In looking at the section the si)ectator is supposed to be facing towards the head of the embryo. In the background one sees the fourth thoracic scleromere with arch and costal process, and at the right the intersegmental artery and the distal edge of the fourth myotome. In the foreground the spinal cord and spinal ganglia in section, and the spinal nerves of the fifth thoracic segment are shown. At the left the fifth thoracic myotome is shown in section.

Fig. 20 shows the general appearance of a typical thoracic segment (the fifth) in an embryo at the end of the fourth week. In Fig. 21 similar structures are shown somewhat more diagrammatically. Fig. A, Plate II, shows a reconstruction of the axis of the body and a part of the right lateral wall and the leg in the same embryo.

The skeletal portion of each axial segment consists of a condensation of the mesenchyme at the caudal third of the segment as shown at the left in Fig. 20. To this skeletal tissue the term scleromere may be applied. It represents the intervertebral disc, the arch or neural process and the costal process of a segment of the future spinal column. The general form of the scleromeres at this stage may be readily seen in Fig. A. The scleromeres do not as yet extend into the body-wall.

The general form of the myotomes may be seen in Fig. A. At the

Charles Eussell Bardeen and Warren Harmon Lewis 19

right the 11th and 13th thoracic and the 1st and 3d lumbar myotomes are viewed from the side and slightly in front. At the left a portion of the median surface of several myotomes may be seen. In Fig. 20 two myotomes are shown in cross-section. The myocoel has disappeared. The median surface of each myotome has been entirely converted into musculature. The lateral surface and ventral and dorsal tips are covered by epithelium. A certain portion of the dorsal surface of the thoracic myotomes is, however, converted into musculature. This is shown in the myotomes at the right in Fig. A. The thoracic myotomes extend for a short distance into the body-wall. In Fig. 20 this is shown in a cross-section. In Fig. A the projecting tips of the myotomes may be seen through tlie membrane lining the coelom.

The thoracic spinal nerves project for a shorter distance into the bodywall than do the myotomes. Each is divided at the dorsal margin of the coelom into two portions, a median which becomes the sympathetic ramus, and a lateral which extends outwards between the median surface of the myotome and the lining membrane of the coelom and becomes the ventral trunk of the spinal nerve (see Figs. A, 20 and 21).

From the aorta intersegmental arteries arise and send branches dorsally toward the spinal ganglia, laterally between the myotomes and ventrally into the body-wall (see Fig. 21). A considerable vascular plexus is developed in the mesenchyme. The general relations of the latter are shown in Fig. 20. The venous blood is collected in the cardinal veins and in branches of the umbilical vein.

The general relation of the formed structures of the axis of the body to the leg-bud at the end of the fourth week are shown in Fig. A, Plate II. The limb-bud lies opposite the five lumbar and the first sacral segments. The coelom extends to a point opposite the first sacral segment, but in the region of the limb it does not extend as far dorsally as in the thoracic region. From the model represented in Fig. A, several of the myotomes of the left side, the axial mesenchyme, the aorta, the left cardinal vein, the intestines and urino-genital organs have been removed. A ^^ortion of the right cardinal vein and a portion of the right umbilical artery are shown, reduced in size for the sake of clearness. The umbilical artery curves about the distal extremity of the coelom. From the umbilical artery a branch passes into the limb-bud. Veins pass from the limb-bud into the cardinal vein. The blood-vessels of the limb exist at this time in the form of an irregular sinusoidal plexus.

The second, third and fourth lumbar nerves may be seen sending spreading bundles of nerve fibres into the dense tissue of the limb, dorsal


Development of the Limbs, Body-wall and Back in Man

to the cardinal vein. They extend, however, for no considerable distance into the limb-bud. The myotomes do not extend into the limbbud.

By following the successive spinal segments from the fourth sacral to the first lumbar in Fig. A, an idea may be gained of the development of segmental structures in the axial region. The scleromeres are represented at the caudal third of each segment. On the left side of the body the myotomes are omitted from the 3d sacral to the 3d lumbar segments. The spinal nerves first appear in the first and second sacral segments.

Fig. 33. Tangential section tlirougli the leg region of Embryo II. 35 d.

Fig. 22 shows the general conditions existing in the limb region of Embryo II when seen in section. At the left the leg-bud is shown cut through an area near the distal extremity of the coelom. At the right the cut is more dorsal and extends through the tips of the lumbar spinal nerves.

In the arm region of Embryo II, the cervico-brachial plexus is formed and limb nerves extend into the limb-bud. The conditions are essentially similar to the conditions found in the leg region of Embryo CLXIII and described below.

Charles Eiissell Barcleen and Warren Harmon Lewis


Fifth Week.

During the fifth week of development a considerable amount of organization occurs in the spinal axis, the body-wall and the limbs. The nature of the processes taking place are indicated in Embryo CLXIII (length, 9 mm.; probable age, 4^ weeks).

The structure of the back, the limbs and body-wall in this embryo is shown in Fig. B, Plate II, and Fig. C, Plate III. The areas mentioned are drawn from reconstructions. The remaining parts of Pig. C are drawn from an excellent photograph.

Fig. 28. Diagrammatic cross section tlirough the 5th-Gth thoracic segments of Embryo CLXIII. x 25 d. The general arrangement of the structures represented is like that described for Fig. 21.

Fig. 23 shows diagrammatically the general nature of the structures in a typical thoracic segment (the 6th) of Embryo CLXIII. The changes taking place in the thoracic region during the first half of the 5th week may be readily followed by comparing Fig. A with Fig. B, and Fig. 21 with Fig. 23.

The skeletal portion of the segment consists in Embryo CLXIII, as in Embryo II, of a condensed mesenchyme at the distal third of the segment, but the scleromere is far more definitely outlined. Xeural and costal processes are well developed. The latter present something of the general form of ribs in the thoracic region (see Fig. 23 and Fig. B). A sheet of condensed mesenchyme connects the neural and costal process of the scleromeres of neighboring segments. That connecting the

22 Development of the Limbs, Body-wall and Back in Man

neural processes is shown in Fig. B at the right. That portion of each scleromere representing an intervertebral disc is likewise united ventrally and dorsally with its neighbors by a dense sheet of tissue at the periphery of the disc. The ventral portions of these sheets of tissue are represented in Fig. B. Within the space lying between the scleromeres is developed the chondrogenous tissue which gives rise to the vertebral bodies. Marked alterations have taken place in the myotomes. In the thoracic region the two layers of the myotome have, with the exception of the dorsal and ventral tips in the more distal segments, become converted into musculature (see Fig. 23 and Fig. B). As shown at the left in Fig. 23, the myogenous tissue which has arisen from the myotomes is being divided into three portions — a dorsal, a lateral and a ventral — by the ingrowth of a vascular mesenchyme. The finer changes taking place during this period are essentially similar to those previously described as taking place in the body-wall of the pig.'°

The tissue of the superficial lateral layers of the myotomes has formed into a continuous layer (see Fig. C, Plate III), Segmentation, however, is still visible.

Both the costal processes of the scleromeres and the myotomes have extended well into the body-wall (Fig. 23).

The thoracic spinal nerves likewise have kept pace in growth with the myotomes. The ventral extremities of the spinal nerves are caught between the tips of the myotomes (see Fig. B, Plate II). From the spinal nerves, dorsal and lateral branches have arisen as well as the sympathetic. A sympathetic cord has arisen from the extremities of the sympathetic rami.

The segmental arteries (see Fig. 23) are similar in nature to those of the stage shown in Fig. 21, but the branching is more extensive. The mesenchyme is much more developed and now surrounds all formed structures in each spinal segment. It has begun to invade the myotomes.

In the region of the posterior limb bundles of fibres from the five lumbar and first two sacral nerves have become anastomosed into a plexus, from which in turn four nerves have sprung. These represent the femoral, obturator, tibial and peroneal nerves (Fig. B). Within the legbud the central mesenchyme, near the axis of the embryo, has become condensed. This condensed mesenchyme represents the femur and hip bone of the adult limb. In the drawing the outline of this sclerogenous mass is made dia grammatically sharp. The femoral portion of the

'"Bardeen, Development of the musculature of the body wall in the pig, including its histogenesis, and its relations to the myotomes and to the skeletal, and nervous apparatus. Vol. IX, Johns Hopkins Hospital Reports, 1900.

Charles Eussell Bardeen and Warren Harmon Lewis 23

skeletal mass fades gradually into the undifferentiated mesenchyme of the distal portion of the limb. It. is this skeletal mass which seems to divide the bundles of nerve fibres of the plexus into the four main divisions which form the origin of the four chief nerves of the limb. The main artery and vein of the limb are represented, but smaller in proportion to the other structures than in nature. The border vein at this period is well developed. The conditions just described are well shown in I'ig. C, Plate III, which represents the conditions seen in the limb from the lateral side after removing the ectoderm and the undifferentiated mesenchyme. It is to be noted that the myotomes do not extend into the limb-bud.

The arm is somewhat more advanced in development than the leg. The following description applies to the conditions represented in Fig. C, Plate III. A detailed account of the structure of the arm at this stage is reserved for a later paper.

Lateral to the myotome system in the arm region is an ill-defined mass of mesenchyme extending from the upper cervical to the 7th thoracic myotomes. At the level of the 1st and 2d ribs it divides into several masses. The first passes ventral to the arm and brachial plexus. The main portion of it joins the arm pre-muscle sheath. From this mass the pectoral muscles develop, hence we may designate it the pectoral pre-muscle mass. It is continuous ventrally with an in-egular mass of condensed tissue, the ventral neck pre-muscle mass. The second division of the lateral pre-muscle mass passes dorsal to the arm and brachial plexus and joins the arm pre-muscle sheath. It constitutes the latissimus dorsi pre-muscle mass. The third division, parallel to the ventral portion of the cervical myotome column, represents the levator scapulge and serratus anterior pre-muscle mass. Lateral to it is an ill-defined mass of pre-muscle tissue. The fourth and most dorsal division is thinner and less clearly marked than the others. We may call it the rhomboid pre-muscle mass. The caudal limit of the trapezius pre-muscle mass appears at the upper end of the arm region. It is at the level of the 4th cervical neural processes, and from here the muscle mass extends to the occipital region. Most of the arm premuscle sheath which surrounds the skeletal core has been dissected away. Toward the distal end of the arm the skeletal and pre-muscle tissues blend (Fig. C). Beyond this point differentiation is less advanced. The proximal portion of the arm sheath is continuous with that around the scapula.

Part of the skeletal core is seen projecting caudally beyond the cut edge of the pre-muscle sheath. The lowej end of the humerus, ulna

24 Development of the Limbs, Body-wall and Back in Man

and radius are represented. The ulna and radius are continuous with the hand plate, which is composed of less differentiated tissue. There is a slight bend at the elbow. The upper end of the humerus is continuous with the scapula. The scapula is a flattened oval mass embedded in the scapular pre-muscle tissue. No coracoid or arromiom processes are present. The clavicula is not present. The skeletal core is composed of dense mesenchyme. It shades oft", however, into the surrounding pre-muscle sheath.

The brachial plexus is formed by the 5th to 8th cervical and 1st thoracic nerves. The spinal nerves, as well as the plexus they form, have scarcely any caudal inclination, but pass ventrolaterally into the arm. The plexus is fairly well formed. The main nerves arising from it are present. The presence of the condensed skeletal core separates these into two groups. The musculo-spiral and circumflex on the dorsal and the ulnar, median and musculo-cutaneous on the ventral side. In Fig. C only the tips of the musculo-spiral and median nerves are shown. At this stage they have scarcely grown to the elbow.

Sixth Week.

During the sixth week the limbs and the body-wall and the back begin to approach the structural conditions characteristic of the adult.

The conditions present at the end of the fifth week are shown in Embryo CIX; length, 11 mm.; age, about 5 weeks. This embryo is pictured in Figs. D and E, Plates IV and V. These figures, with the exception of the head in Fig. D, are drawn from wax-plate reconstructions. Fig. 24 represents a cross-section through the 6th to 7th thoracic segment of a slightly older embryo, CVI (length, 15.5 mm.; age, about 5-| weeks).

'The typical thoracic segment during the first half of the sixth week exhibits the following conditions:

The skeletal structures have begun to assume adult form. Between the intervertebral discs the bodies of the vertebrae are now formed of chondrogenous tissue. This may be seen in the dotted area of the scleromere in Fig. 24 and in the darker areas of the spinal column in Fig. E. The chondrogenous tissue extends into the neural and transverse processes of the scleromere (see the left side of Fig. 24). Each costal process extends considerably further ventrally than at the stage shown in Fig. 23. Within the dense tissue of the costal process chondrogenous tissue is formed similar to, but without direct connection with, that of the vertebral body and transverse process. A lateral view of the

Charles Eussell Bardeen and Warreu Harmon Lewis


neural and transverse processes of the vertebrae is shown in the cervical region in Fig. D. The extent of development of the ribs is shown in Fis. E.

Fi^. 24. Diagrammatic cross section through the 6th-7th thoracic segments of Embryo CVI. x 35 d. This figure is made to correspond with Figs. 31 and 23. See legend for Fig. 31.

The musculature has undergone most marked changes. The dorsal musculature is slightly separated from the ventro-lateral by vascular mesenchyme. The dorsal musculature is further subdivided into three muscle columns. These correspond to the ileo-costal, longissimus dorsi and spinalis groups of dorsal muscles in the adult. The ventral musculature is likewise subdivided into two main portions, the rectus muscle and the lateral musculature. The last is further subdivided into external oblique, the intercostals, the internal oblique, and the transversalis muscles. In the intercostal muscles alone is the segmentation characteristic of the mvotomes fullv maintained.

26 Development of the Limbs^, Body-wall and Back in Man

In Fig. D the separation of dorsal from ventro-lateral musculature is clearly shown. The dorsal musculature is shown divided into three columns in the upper thoracic region; caudally differentiation is not so extensive. The musculature of the external oblique may be seen, in Fig. D, covering the external intercostal muscle, the ribs and in part the rectus musculature. In Fig. E the rectus musculature, internal intercostal and internal oblique may be seen. Differentiation, however, is not so far advanced at the stage shown in Figs. D and E as it is at the stage shown in Fig. 34. The ventro-lateral abdominal musculature of Embryo CTX is connected by an irregular dense band of tissue Avith the pubic process of the anlage of the pelvic bone. This band of tissue is represented with diagrammatic distinctness in the draAvings.

The neural apparatus has undergone rapid development. Fig. 21 shows clearly the main branches arising from the typal thoracic nerve. Muscle twigs are arising. The general appearance of the spinal nerves at this stage is shown in Fig. E.

The mesenchyme is extensive in amount. The blood-vessels are similar in general distribution to those described in Embryo CLXIII, but the vascular plexus is more extensively developed.

In the posterior limb the central skeletal mass has assumed somewhat definite outlines. Fairly good views of it are presented in Figs. D and E. The pelvic portion of the skeleton of the limb consists of a central region continuous with the head of the femur. From this central acetabular portion spring iliac, ischial and pubic processes. The femur is short and thick. It is indistinctly shown in Fig. D. The tibia and fibula are of fairly definite form (Figs. D and E). The skeleton of the foot has the form shown in Figs. D and E. It is composed of condensed mesenchyme. No accurate division into parts can be distinguished.

The main nerve trunks have continued their growth into the limb. From them many of the principal muscular and cutaneous branches have sprung. The general distribution of the lateral nerves of the limb, the femoral and peroneal, and their branches, may be seen in Fig. D. That of the median nerves of the limb, the obturator and tibial, in Fig. E, In both figures the anterior border nerves (the ilio-hypogastric and genito-crural) and the posterior border nerves (the pudic and posteriorcutaneous) are shown.

About the main branches of the nerves of the limb a differentiation of musculature has begun. This is indicated in Figs. D and E. In Fig. 25 are shown the appearances in cross-section presented by the early developing musculature of the leg. The blood-vessels of the limb are shown in Figs. D and E. The sciatic artery is still the chief source of

Charles Eussell Bardeen and Warren Harmon Lewis


supply, but the femoral and obturator arteries also have appeared. The blood is carried into the cardinal (iliac) vein partly by the femoral vein and partly by the sciatic. The formed structures of the limb are surrounded by a vascular mesenchyme.

spiDal ganglion

sciatic nerve, Atid niusculatiir


femoral nerve, and musculature

/ '-.Os'^.


abdominal musculature

Fig-. 25. Section through Embryo CIX. x 25 d.

The conditions existing in the arm are considerably in advance of those in the leg. The following description of the conditions in the arm region can be followed from Figs. D and E, Plates IV and V.

The lateral pre-muscle mass has become completely divided into several groups. The tissue of these groups is now fibrillated. The first division, the one which passes ventral to the brachial plexus, is seen in Plate V. It constitutes the pectoral muscle mass, representing

38 Development of the Limbs, Body-wall and Back in Man

both pectoralis major and minor muscles. This mass extends from the level of the third rib to the humerus and clavicle. There is no attachment to the ribs. The intercostal muscles have been dissected away in Plate V to show its costal end. The second division of the lateral pre-muscle mass has developed into the latissimus dorsi and teres major muscle mass. It extends, from the level of the 4th rib to the humerus, where it blends with the scapulo-humeral mass. Its development and differentiation have not proceeded so far as the pectoral mass. The third division of the lateral pre-muscle mass has developed into a long muscle extending from the 1st cervical vertebra to the 9th rib, Digitations extend to the transverse processes of the cervical vertebra and to the cephalic 9 ribs. The muscle lies in a more median plane than the scapula and has as yet no attachment to it. It represents the levator scapula? and serratus anterior muscles. The trapezius mass has extended to a lower level than found in CLXIII. There is no scapular attachment. Only the ventral half of the mass pictured in Plate IV consists of muscle fibres, the remaining connective tissue portion connects with the dorsal ends of the neural processes.

Considerable differentiation in the pre-muscle sheath has taken place. The scapulo-humeral mass is with difficulty separated into the various muscles. These are more blended into a single mass than would appear from Plates IV and V. Here portions of the deltoid and trapezius have been dissected away.

The extensor mass of the forearm can be separated into three groups which are more or less blended however. The larger superficial mass has been partially dissected away. It represents the extensor digitorum communis, extensor carpi ulnaris, and extensor digiti quinti proprius muscles. The second group arises beneath the first group, taking a course at nearly right angles to it. It represents the deep extensor muscle of the forearm. These two groups fuse distally with the general condensed mesenchyme of the digits, where all traces between premuscle and pre-cartilage are lost. The third group represents the brachio-radialis and the extensor carpi radiates longus et brevis. A • portion of the brachialis can be seen in Plate IV. The flexor surface of the arm is shown in Fig. E, Plate V. The biceps and coracobrachialis are obscured by the pectoral mass. The flexor mass of the forearm has split into two layers. It shows less differentiation than the extensor mass.

The arm skeleton shows considerable advance. The shape of the scapula has changed, both coracoid process and acromion are present and of large size. The clavicle has begun to develop and consists of

Charles Eussell Bardeen and Warren Harmon Lewis 29

an ill-defined mass of condensed tissue projecting about one-half the distance from the acromion to the tips of the first rib. The humerus is fairly well defined and is continuous with the scapula as well as the ulna and radius. The elbow bend is well marked. The ulna and radius are farther advanced than in Embryo CLXIII. They end distally in the carpal plate. In the carpal plate indications of formation of the carpal elements are seen. The digits consist of undifferentiated tissue in which are blended skeletal muscle and tendinous elements. The humerus, ulna and radius have a core of hyaline cartilage. The rest of the skeletal tissue consists of condensed mesenchyme and pre-cartilage.

In Plate IV are shown the circumflex, radial and musculo-cutaneous nerves. The brachial plexus with portions of the nerves arising from it are seen in Plate V. The plexus is well formed. It has only a slight caudal inclination. The spinal accessory nerve is seen on the median surface of the trapezius. Branches from the 3d and 4th cervical nerves join it.

Seventh WeeJc.

By the end of the seventh week most of the structures characteristic of the adult back, body-wall and limbs have appeared. Subsequent development depends in the main upon growth and upon relative shifting of parts.

The structures of the abdominal wall in a seven-week embryo (XXII, length 20 mm.) are shown in the Figs. F, G, H and I, Plates VI, VII, VIII and IX. The vertebra are composed of embryonic hyaline cartilage. Each presents a neural and a transverse process on each side. The cartilaginous portions of the vertebrae are shown in the upper thoracic region of Fig. H. The. ribs are likewise composed of embryonic cartilage, shown in the same portion of the figure. The cartilage of the ribs is at no time continuous with that of the vertebrae. The ribs and vertebras are surrounded by a dense mesenchyme. This is continuous from the ribs to the transverse process of the vertebra. Between the bodies of the vertebrae it forms the intervertebral discs (Fig. I). It is continuous over the surface of the spinal column. From it are developed the ligaments characteristic of the thorax and spinal column together with the perichondrium and periosteum. No thirteenth rib is present in this embryo or in any of the other young embryos we have studied. Fig. 26 shows at the right the portion of the skeleton composed of embryonic cartilage, at the left the covering of dense mesenchyme.

30 Development of the Limbs, Body-wall and Back in Man

The mnscnlatnre of the back and abdominal walls has a general resemblance to that of the adult. Fig. I shows the musculature of the abdomen and thorax as seen from within, Figs. F and G that of the more superficial layers of the abdomen and thorax, and Fig. H that of the deeper layers of the abdomen and thorax. The dorsal muscles are not clearly shown in any of the figures, but they are divisible into the three distinct groups, the ileo-costal, longissimus dorsi, and spinalis muscles, characteristic of the adult.

Fig. 26. Skeleton of distal half of Embryo XXII. At the left side the covering of dense embryonic connective tissue is shown, at the right the parts composed of embryonic cartilage, x 10 d.

The nerves, like the muscles, have a distribution essentially similar to that found in the adult. The figures indicate with sufficient clearness the distribution of the thoraco-abdominal and border nerves.

The main blood-vessels are those characteristic of the adult. ■ In the posterior limb the skeletal tissue has undergone extensive differentiation. The rudiments of all of the bones of the leg may be seen in the form of cartilage except that the terminal phalanges of the

Charles Russell Bardeen and Warren Harmon Lewis 31

three onter toes have not yet appeared (see Fig. 26, right side). The cartilaginous skeleton of the limb, like that of the spine, is covered by a dense mesenchyme. Torsion has not yet begun at the ankle-joint.

The musculature of the posterior limb is so far differentiated that all of the individual muscles characteristic of the adult may be distinguished except the lumbricales. The muscles lie in distinct groups, as is shown in the various figures.

The femoral or extensor group of muscles is shown in Figs. F and H. The groups of muscles belonging to the peroneal nerve and its branches, the gluteal, peroneal, and pedal extensor muscles may be seen in Figs. F and H. The adductor or obturator group of muscles is best seen in Fig. G. The groups of muscles belonging to the tibial nerve may be seen in Fig. I. A detailed account of these muscles is reserved for a subsec{uent article.

The nerves of the posterior limb, like the muscles, are so well developed that most of them may be readily compared with those of the adult. To reach the adult position a considerable amount of shifting, however, must take place.

At the period under consideration the blood-vessels of the limb are those characteristic of the adult. The main artery and the chief vein are the femoral.

An idea of the general condition of the tissues of the limb at this stage may be obtained from Fig. 27, which represents a photograph taken through a section passing through the limb region.

The anterior limb presents similar conditions of structure.

The muscular system shows well-marked fibrillation. All of the muscles of the adult arm are present and in about the relative adult positions.

The bones of the arm are represented by hyaline cartilage except the distal row of phalanges of the 2d to 5th digits. This row is represented by masses of undifferentiated condensed tissue, into which the long extensor and flexor tendons merge.

The clavicle is well developed and extends to the 1st rib, where it comes in contact with the sternal anlage. The sternal anlage is composed of condensed mesenchyme. The ribs, vertebrse and their neural processes are composed of cartilage. All of the cartilages of the arm are surrounded by a condensed mesenchymal sheath, the perichondrium.

The nervous system presents nearly the adult conditions. It has not been possible, however, to resolve the brachial plexus into its usual distinct cords. They appear from study of sections and the reconstruction to be fused into one mass.


Development of the Limbs, Body-wall and Back in Man



In an embryo of two weeks, XII, Figs. 1 and IG, fourteen myotomes are present, three occipital and eleven spinal (8c, 3t). In an embryo of

Fig. 37. Photograph of a section through the leg region of Embryo XXIL

four weeks, II, Figs. 6, 20, 21 and A, thirty-eight myotomes have been counted, three occipital and thirty-five spinal (8c, 12t, 51, 5s and 5c). At this stage the formation of myotomes ceases. Soon hereafter the

Charles Eussell Bardeen and Warren Harmon Lewis 33

occipital myotomes disappear. From the thoracic myotomes processes enter the hody wall. During the fifth week the myotonies give rise to a dorso-ventral muscle-mass in which the segmentation characteristic of the myotomes mainly disappears. This muscle-mass becomes divided longitudinally into two great divisions — a dorsal and a ventro-lateral. Into the composition of the dorsal division all of the spinal myotomes enter. From it is derived the dorsal musculature of the adult. The ventro-lateral muscle-mass is formed from the processes which extend from the thoracic myotomes into the body wall. From it are derived the intrinsic muscles of the thorax and abdomen. The differentiation of these muscles takes place during the fifth, sixth and seventh weeks.

From the median surface of the myotomes near the ventral margin mesenchyme springs to surround the intrinsic structures of the spinal axis (Fig. 16). This mesenchyme is at first non-segmental in distribution. Gradually, however, it becomes denser at the posterior third of each spinal segment. This condensed tissue forms the scleromeres. From the scleromeres are developed the intervertebral discs, the arches and transverse processes of the vertebrae, and the ribs. Between the scleromeres the bodies of the vertebras are formed. The vertebral column at first surrounds only the ventral half of the spinal-cord. It is at a comparatively late period that the vertebral arches from each side meet dorsally to form the vertebral spines.

Owing to the accurate studies by His, the main stages in the development of the spinal-cord and early formation of the spinal nerves are too well known to demand further description. We find, however, that the dorsal divisions of the spinal nerves are given off after the division of the spinal nerve into somatic and sympathetic branches, a period later than that described by His. When the dorsal musculature begins to be formed from the tissues derived from the myotomes, the dorsal divisions appear and pass into the differentiating musculature, where they give rise to the characteristic median and lateral trunks from which muscular and cutaneous branches spring. The ventral trunks of the spinal nerves in the cervical and lumbo-sacral regions unite to form plexuses from which in turn the nerves of the neck and limbs arise. In the thoracic region the ventral trunks pass inio the body wall as intercostal nerves. Sympathetic branches are given off at the end of the fourth week, at the period when the thoracic nerves reach the dorsal margin of the coelom. The lateral and ventral cutaneous branches are given off during the fifth, the main muscular branches during the sixth week.


34 Development of the Limbs, Body-wall and Back in Man

Soon after the two dorsal aortas fnse into a single dorsal aorta intersegmental arteries are given off. From these, main branches pass between the myotomes, between the spinal ganglia and to the ventral surface of the spinal-cord. Longitudinal anastomosing branches are formed between these vessels and an extensive vascular plexus arises. The blood is collected again in the cardinal veins and into the abdominal branches of the umbilical vein.

The limbs and body-wall are developed in the Wolffian ridge. This first appears as a thickening of the membrana reuniens along its attachment to the axis of the body between the 4th and 26th spinal segments. The limbs are developed from special bud-like projections from the Wolffian ridge, the anterior extremity appearing opposite the 5th to 9th spinal segments, the posterior extremity opposite the 21st to 26th. At the end of the third week the Wolffian ridge and limb-buds are well marked, but are without special internal differentiation.

The body-wall is developed by an ingrowth into the Wolffian ridge of processes from the myotomes, scleromeres, nerves and blood-vessels belonging to the twelve thoracic spinal segments, and a gradual differentiation of adult structures from embryonic. Ingrowth begins during the fourth week, structures essentially similar to those characteristic of the adult are differentiated by the end of the sixth week. The body-wall does not complete its growth to the midline, however, until toward the end of the third month.

Into the limb-buds blood-vessels and nerves extend from the axis of the embryo, but neither myotomes nor scleromeres send processes into the limbs. The skeletal and muscular structures of the limb are differentiated from the mesenchyme of the limb-bud. Ingrowth of bloodvessels precedes ingrowth of nerves. The brachial plexus is formed and nerves grow into the anterior limb during the latter half of the fourth week. The lumbo-sacral plexus is formed, and nerves grow into the posterior limb during the first half of the fifth week. Skeletal differentiation begins in the region of the shoulder or hip, and extends distally and proximally. This differentiation immediately precedes ingrowth of the nerves of the limbs. The skeletal structures serve in part to guide the nerves in their distribution. Muscle differentiation immediately follows the entrance of a motor nerve into a given region. From this, however, it must not be concluded that a causal connection exists between the nerves and differentiation of muscles. "

"See: Leonowa. Ein Fall von Anencephalle combinirt mit totaler Amyelie Neurol. Centralbl. Leipsic. Bd. XII (1893), s. 218, 363.

Charles Eussell Bardeen and Warreu Harmon Lewis 35

The structures of the upper arm and thigh are differentiated before those of the forearm and leg, and the latter before the hand and foot. Differentiation in the anterior limb precedes that in the posterior limb. Most of the main structures of the anterior limb may be distinguished at the end of the sixth week; most of those of the posterior limb at the end of the seventh week.

During the first two months of embryonic life, therefore, are developed the rudiments of the muscles, nerves, blood-vessels, and skeletal structures characteristic of the back, the body-wall and the limbs. Adult conditions are reached by an increase in size and complexity of the various organs and by a relative shifting of parts.


A series of photographs of human embryos in the collection belonging to Dr. Mall m the Anatomical Laboratory of the Johns Hopkins University The Roman numerals refer to the numbers by which these embryos are designated. The Arabic numbers indicate the ratio between the size of the photog-raphic image and the size of the embryo.




CXLVIIl— 3-1



CLXMI— 2-1

CLXVII— 24-13


CVI — 55-37


PLATE II. Figure A. Magnification aboiit 20 d.

Drawing from a reconstruction of the region of the posterior extremity in Embryo II; length, 7 mm.; age, 26 days. The xvii (9th thoracic) to the xxix (4th sacral) spinal segments, and, at the left, the right leg and a portion of the body-wall are represented. From the region ventral to the spinal-cord unformed mesenchyme, the aorta and left cardinal vein, and the smaller blood-vessels, the intestines and urino-g'enital organs have been removed. From the spinal segments the myotomes of the left side have been removed with the exception of the last two thoracic, the first two lumbar and the fourth sacral. Half encircling the spinal-cord the scleromeres may be seen at the distal third of each spinal segment. The chorda dorsalis runs ventral to the midline of the spinal-cord. The 9th to the 12th right thoracic mj^otomes and nerves of the right side may be seen extending for a short distance behind the lateral surface of the coelom. The thoracic nerves give off sympathetic branches at the dorsal margin of the ccelom. The first four lumbar nerves give off spreading branches towards the limbbud. The 5th lumbar and 1st sacral nerves are but slightly developed. The umbilical artery curves about the posterior tip of the coelom and sends an arterial branch into the leg-bud. Below this the cardinal vein sends a branch into the limb.

Figure B. Magnification about 20 d.

Drawing from a reconstruction of the region of the posterior extremity in Embryo CLXIII; length, 9 mm.; age, about 4% weeks. The xvi (8th thoracic) to the xxx (fifth sacral) spinal segments are represented. The right leg and a portion of the right body-wall are shown. From the region ventral to the spinal-cord unformed mesenchyme, the blood-vessels, the lining membrane of the coelom, the intestine, and the urino-genital organs have been removed. The 11th and 12th thoracic myotomes only are represented on the left side. The 9th to the 12th myotomes, costal processes and spinal nerves extend ventrally in the right body-wall. Lateral and dorsal branches have arisen from the spinal nerves. The sympathetic cord receives branches from the thoracic and the first two lumbar nerves. The five lumbar and the first two sacral nerves combine to form a plexus. From this the four main nerve trunks of the posterior limb are beginning to spring. The sciatic artery and vein are represented entering the limb. At the centre of the base of the limb-bud the femur and hip-bone are beginning to be differentiated by the formation of a dense mass in the mesenchyme. The mesenchyme lying median to the limb-bud has been removed so as to expose this skeletal mass and the lumbo-sacral plexus.





PLATE III. Figure C. Magnification about 15 d.

Lateral view of Embryo CLXIll; length, 9 mm.; age, 41/0 weeks. The areas from which the skin has been removed are drawn from reconstructions, the remaining portions are drawn from excellent photographs. The myotomes hide from view most of the deeper structures of the back and body-wall. The superficial tissue of the myotomes has to a certain extent fused, so that segmentation is becoming indistinct. In the region of the arm, certain dense masses of tissue are represented in which later the musculature of the arm is differentiated (see p. 23). In the region of the forearm and hand this " premuscle " tissue has been removed so as to disclose the dense mass of mesenchyme which at the centre of the limb-bud represents the forerunner of the skeleton. The skeletal tissue is represented with sharper outlines than in nature. Ulna, radius and hand plate are shown. The musculo-spiral and median nerves may be seen reaching about to the elbow.

In the region of the leg the superficial tissue has been moved so as to disclose the border vein, the sciatic artery, the skeletal rudiment of the femur and hip-bone and the lumbo-sacral plexus. Into the formation of the latter enter the five lumbar nerves and the first two sacral.






Figure D. Magnification about 12 d.

Lateral view of Embryo CIX; length, 11 mm.; age, about 5 weeks. The areas from which the skin has been removed are drawn from wax-plate reconstructions, the remaining portions are drawn partly from photographs, partly from an embryo of corresponding age. The arches and transverse processes of the 4th to 8th cervical vertebrae have been exposed by the removal of the dorsal musculature in that region. The embryonic cartilage of which these structures are formed has been exposed by the removal of the perichondrial mesenchyme. The dorsal musculature has likewdse been removed from the 5th lumbar and first three sacral segments. In this region, however, is shown the dense mesenchyme which incloses the cartilaginous portions of the spinal column.

The heads of the first three ribs may be seen median to the transverse processes of the first three thoracic vertebrae. The third to the eleventh ribs may be seen through the lateral musculature of the body-wall.

The dorsal musculature is distinctly separated from the ventro-lateral. In the thoracic region little evidence remains of segmentation in the dorsal musculature. In the lumbar, sacral and coccygeal regions myomeric structure is still visible. The ventro-lateral musculature, which has developed from processes from the twelve thoracic myotomes, is beginning to assume a differentiation into the muscles characteristic of the thorax and abdomen.

The dorsal divisions of the spinal nerves are shown in the regions where the vertebrae are exposed. The lateral branches of the ventral divisions are shown in the thoracic region.

In the region of the anterior limb superficial tissues have been removed so as to expose the main structural features. Near the spinal column the trapezius and serratus anticus muscles are shown, the former being represented as semi-transparent. From the shoulder the greater portion of the deltoid muscle has been removed, from the upper arm the greater portion of the triceps, and from the forearm the greater portion of the extensor digitorum communis.

In the region of the leg the more superficial tissue has been removed so as to expose the skeletal, muscular, nervous and vascular apparatus.

The skeleton consists of hip-bone, femur, tibia and fibula, which are composed of embryonic pre-cartilage covered by a dense mesenchyme, and of a dense mass of tissue which represents the anlage of the ankle and foot.

The five lumbar and the first three sacral nerves enter into the formation of the lumbo-sacral plexus. From this arise the femoral nerve, which enters a mass of tissue that represents the extensor muscles of the thigh; the peroneal nerve, which gives off gluteal branches to the gluteal muscle mass, a posterior cutaneous branch, branches to the extensor musculature of the foot, and a peroneal branch, which extends a short distance along the fibula. The peroneal musculature has not yet become differentiated from the mesenchyme. At the posterior extremity of the plexus the pudic nerve may be seen.

The border vein empties into the femoral and sciatic veins. The sciatic artery is shown terminating in the extensor musculature of the foot.






Figure E. Magnification about 15 d.

Drawing from a reconstruction of the regions of the arm, leg and bodywall of Embryo CIX; length, 11 mm.; age, about 5 weeks. In the arm region the cervico-brachial plexus, and the nerves and muscles of the arm and hand are exposed (see text, page 27); in the region of the body-wall the ribs, the spinal nerves, and the thoracic musculature (see text, page 26); ' and in the region of the leg the lumbo-sacral plexus and the main nerves of the limb, the anlage of the limb musculature, the skeletal rudiment and the blood-vessels.

The five lumbar and the first three sacral nerves enter into the formation of the lumbo-sacral plexus. The twelfth thoracic nerve, however, sends a communicating branch to the ileo-hypogastric. From the first lumbar nerve arise the ileo-hypogastric nerve and a communicating branch to the lumbar plexus. From the lumbar plexus the femoral nerve may be seen passing behind the pubic process of the hip-bone into the extensor muscle mass. Just above the femoral nerve the genito-crural nerve arises from the plexus. Between the pubic and ischial processes of the hip-bone the obturator nerve passes forward into a mass of tissue which represents the adductor musculature. Below the ischial process the sciatic nerve passes into the limb, and from this the tibial nerve extends distally on the median surface of the skeleton of the leg and terminates in the flexor musculature of the foot. Along the course of the tibial nerve several muscle masses may be distinguished. These represent the perineal, obturator internus and quadratus femoris, ham-string and soleus-gastroenemus muscle masses. Posterior to the tibial nerve the pudic nerve arises. The sciatic artery passes in company with the tibial nerve, the obturator artery with the obturator nerve, and the femoral with the femoral nerve. The border, sciatic and femoral veins may be distinguished.

lA portion of the interosseal musculature has been removed near the tips of the first three ribs.





PLATE VI. Figure F. Mag-nification about 10 d.

Drawing- from a reconstruction of Embryo XXII; length, 20 mm.; age, about 7 weeks (see also Figs. G, H and I). From the arm, leg, body-wall, and the adjacent dorsal region the ectoderm and the superficial tissues have been removed. The muscles and nerves of the body-wall may be recognized readily from their likeness to adult structures. The shoulder muscles and the brachialis and triceps muscles of the upper arm are likewise plain. In the forearm the following muscles may be distinguished from above downwards: brachio-radialis, extensor carpi radialis longus et brevis, abductor pollicis longus, extensor pollicis brevis, extenso;r pollicis longus and extensor indicis proprius, extensor digitorura communis, extensor carpi ulnaris, and flexor carpi ulnaris; and in the hand the abductor minimi digiti. Branches from the circumflex and radial nerves may be seen.

In the posterior limb the sartorius and the extensor muscles (the vastus internus, rectus and vastus externus) may be seen above the femur. Between the femur and ilium the tensor vaginae femoris and the gluteus minimus, medius and maximus muscles may be seen. The biceps curves below the knee-joint. In the leg the tibialis anticus, extensor hallucis longus, extensor digitorum communis, and peroneus tertius muscles may be distinguished, and below the last the peroneal muscles. The middle and lateral cutaneous nerves lie over the thigh; the long saphenus, musculocutaneous and lateral saphenus lie exposed in the region of the leg and foot.

The perichondrium has been dissected away from the phalanges of the hand, leaving the cartilaginous cores visible. In the leg and foot the condensed mesenchyme or perichondrium surrounding the cartilages has been left intact.





PLATE VII. Figure G. Magnification about 10 d.

Drawing- from a reconstruction of Embryo XXII; length, 20 mm.; age, about 7 weeks (see also Figs. F, H and I). The left arm was not reconstructed, but has been drawn in part from the reconstruction of the right arm, in part from a photograph. In the region of the right arm the pectoralis major, biceps, coraco-brachialis, brachialis, brachio-radialis, extensor carpi radialis longus et brevis, the extensor communis digitorum and extensor carpi ulnaris; and the interossenis muscles may be seen. In the abdominal region the external oblique and rectus muscles are exposed. In the region of the posterior limb the adductor and ham-string muscles, and the tibialis anticus, the extensor pollicis longus and extensor digitorum communis muscles may be distinguished. The ventral tips of the thoracic nerves and the long saphenus nerve, and the tip of the anterior tibial nerve are shown.

The dense mesenchyme covering the cartilaginous parts of the skeleton is pictured intact.






Figure H. Magnification aboiit 10 d.

Drawing from a reconstruction of Embryo XXII; length, 20 mm.; age, about 7 weeks (see also Figs. F, G and I). The dorsal musculature has been removed. From the thoracic region all intrinsic muscles lateral to the internal intercostal muscles have been removed down to the 8th rib. The serratus anterior, however, has been left in position. The attachments of the external oblique musculature are shown. The rest of the external oblique muscle and a considerable portion of the internal oblique muscles have been removed. The thoraco-abdominal nerves are exposed. The most anterior nerve shown is the 7th cervical. The 7th cervical vertebra bears a rib-like process. The cartilaginous portions of the vertebrae and ribs of the first eight thoracic segments are represented. The ribs and vertebrae distal to this point are shown covered with a dense embryonic connective tissue.

The skeletal portions of the arm are drawn without the condensed mesenchyme or perichondrium which surrounds all the cartilages. Portions of the cartilages of the scapula, clavicle, humerus, ulnacarpus, metacarpus and phalanges are shown.

Most of the superficial muscles of the arm have been partially dissected away. Most of the deltoid, the infraspinatus and teres minor, the teres major and latissimus dorsi, the triceps and anconeus, the extensor digitorum communis and the extensor carpi ulnaris muscles have been dissected away except for their attached ends which can be readily recognized. The senatus anterior, supraspinatus brachialis, brachioradialis, extensor carpi radialis longus et brevis, supinator brevis, abductor pollicis longus, extensor pollicis brevis, extensor pollicis longus and extensor indicis muscles are left intact. The intrinsic muscles of the hand as well as the insertions there of the muscles of the forearm have been entirely cut away.

The suprascapular, circumflex and radial nerves with their main branches are shown.

In the posterior limb portions of the rectus and sartorius muscles have been removed so as to expose the chief branches of the femoral nerve; and portions of the tensor vaginae femoris, gluteus medius and gluteus maximus muscles, so as to expose the chief branches of the gluteal nerves. In the leg a portion of the extensor digitorum communis muscle has been removed so as to expose the extensor hallucis longus and the extensor digitorum brevis muscles, and the distribution of the anterior tibial nerve. Portions of the peroneal muscles have been removed so as to expose the distribution of nerves to these muscles.

In the anterior limb the cartilaginous portions of the skeleton are represented except at the joints, where some condensed tissue is pictured. In the posterior limb is shown the condensed tissue which incloses the cartilaginous portions of the skeleton.





PLATE IX. Figure I. Magnification about 12 d.

Drawing from a reconstruction of Embryo XXII; length, SO mm.; age, about 7 weeks (see also Figs. F, G and H). The thoracic, abdominal and pelvic viscera have been removed. The attachment of the diaphragm to the body-wall is shown. The intrinsic muscles and nerves of the thorax, abdomen and pelvis are shown intact, and may be readily distinguished by their relative positions.

The ventral ends of the upper four ribs and the median end of the clavicle are not shown.

In the region of the shoulder and upper arm the deltoid, biceps, brachialis, coracobrachialis and subscapular muscles are shown intact. The attached ends of the pectoral muscles may be seen. In the forearm the following muscles may be distinguished from above downwards: brachio-radialis, pronator teres, flexor carpi radialis, palmaris longus, flexor digitorum sublimis and flexor carpi ulnaris. The brachial plexus is shown together with the main nerves of the arm. Some of the interossei muscles are shown in the hand.

The brachial plexus arising- from the 5th to 8th cervical and 1st thoracic nerves is seen. The relatively large size of the nerves and plexus is at once noticed. The plexus itself forms a closely packed mass of fibers in which it is just possible to distinguish the position of the three main cords from which the principal nerves of the arm arise. The posterior cord is not visible in this figure. The suprascapular, small nerve to the subclavious, branch to the pectoral muscles, musculocutaneous, median, ulnar, and internal cutaneous nerves are seen arising from the plexus.

In the region of the posterior limb the psoas muscle is shown cut away over the lumbar plexus. The sartorius and vastus medius muscles may be seen above the femur. In the region of distribution of the obturator nerve the belly of the gracilis muscle has been removed so as to expose the adductor muscles. The bellies of the semi-membranous and semi-tendinosus muscles have been removed so as to expose the sciatic nerve, below which The long head of the biceps may be seen. In the leg the gastroenemeus, soleus, popliteus muscles, and the flexors of the toes may be distinguished. The lumbo-sacral plexus arises from the 12th thoracic to the 3d sacral spinal nerves. The inguinal nerve arises from in front of the first lumbar nerve, the genito-crural from in front of the second, and the lateral cutaneous from a point just above the region where the obturator and femoral nerves are given off. From the lumbar plexus a large nerve bundle passes into the psoas muscle. After passing Pouparts ligament the main trunk of the femoral nerve may be seen below the sartorius muscle. The long saphenus nerve may be traced to the ankle. The main branches of the obturator nerve are shown. The pudic nerve may be seen passing out between the great sacro-iliac ligament and the levator ani muscle, the posterior cutaneous nerve is given ofE on the lateral side of sacro-iliac ligament. The main branches of the tibial nerve in the leg and foot may be readily followed.








{Associate in Anatomy, the University of Chicago.)

With one Text Figure.

The coarser framework of the human spleen divides the organ into small masses of parenchyma — the lobules, each having an afferent artery near its center and the larger efferent veins at its periphery.

The finer fraraev/ork of the spleen subdivides each lobule and supports the individual spleen cells and smaller blood-vessels contained therein. The finer framework is an extension within the lobule of the coarser framework of the trabeculse and is here indicated as the intralobular framework.


Oesterlen/. writing in 18-13, mentioned, without description, a substance between the individual cells of the spleen pulp.

Atto Tigri," iji 1853, described as a constant element of structure an intercellular network giving support to the spleen cells.

Billroth/ in 1857, coufirmed the presence of this intercellular framework, and described its minute structure more in detail. From a comparative study of the vertebrata, he concluded that the framework was formed by stellate cells M'hose anastomosing processes appeared as fibers connecting the cell bodies proper with one another. Thus, according to Billroth, the cell bodies appeared as local expansions of the fibrils, each containing a definite nucleus. From the spleens of amphibia he isolated stellate cells that he asserted were the branched cells forming the intralobular framework. From the human spleen, however, he could not isolate the corresponding cells, and stated that their demonstration in situ was more difficult than in lower forms because of their relative infrequency.

'.Oesterlen, F. : Beitrage zur Physiologic des gesunclen unci kranken Orgaiiismus, Jena, 1843.

2 Tigri, A. : Schiarimenti sulla struttiira e sulla funzione clella milza, Gazz. tried, ital. feder. tosc, Firenze, 2 s., T. Ill, 1853, pp. 25-27.

3 Billroth, T. : Beitrage z%ir vergleichenden Histologic der MHz, Arch. f. Anat., Physiol., u. wissensch. Med., Berl., 1857, S. 88-108.

38 The Intralobular Framework of the Human Spleen

Henle/ in 1859, questioned the findings of Billroth and denied the existence of nuclei within expansions of the fibers. He described the intralobular framework as composed, not of anastomosing cells, but of fibrils similar to those of tendon.. These fibrils crossing one another in all directions and at varying angles were, according to Henle, direct continuations of the white fibrous tissue of the trabeculas. He further described certain circular and spiral fibrils anastomosing with one another to encircle the capillary veins.

W. Mliller/ in 1865, corroborated the findings of Henle as to the fibrillary character of the intercellular framework. He further described a finely granular intercellular ground substance within which he considered the individual fibrils to be imbedded.

Oppel," in 1891, employing precipitation methods, was able to determine with some accuracy the general distribution of very delicate intercellular fibrils within the pulp cords and Malpighian follicles of the human spleen. Such fibers he termed " Gitterfasern," but he gave no data as to their intimate structure.

Mall,' in a series of contributions, commencing in 1888, established a lobule as the unit of structure of the dog's spleen and determined that its framework is composed largely of connective tissue differing from both the white fibrous and yellow elastic forms. The component fibrils of this tissue he found to be devoid of nuclei and everywhere anastomosing with similar fibrils to form a delicate but resistant network. Unlike elastic tissue, these fibrils resisted pancreatin digestion and differed from white fibrous tissue in their extensive branching and anastomosis. To this tissue he applied the specific term " reticulum." The most delicate framework within the pulp cords he described as a distinct "variety of reticulum not resisting pancreatin digestion. The reaction of the reticulum within the Malpighian follicle he did not determine.


In studying the framework of fifteen human spleens, I have employed the so-called "destructive" methods, the removal of the spleen pulp by maceration and digestion permitting inspection of the isolated frame 4 Henle: Zeitschr. f. rationelle IfecUzin, III Reihe, Bd. VIII, S. 201-230.

5- MiiUer, W. : Veber der feineren Bau der MHz, Leipzig, 186.5.

"Oppel, A.: Ueber Gitterfasern der menscldichen Leber mid Milz, Anat. Anz., Jena, Bd. VI, 1891, S. 16.5-173.

'Mall: Anat. Anz., 1888. AbJiandl. d. K. S. Oes. d. Wiss., Bd. XVII, 1891. Johns Hopkins Hospital Reports, Vol. I. Johns Hopkins Hospital Bidletin, Nos. 90-91, 1898. ZeitscMft f. MorpJtolofjie u. Anthropologie, Bd. II, 1900.

Preston Kyes 39

work. Pancreatin digestion was used as suggested by Mall and Spalteholz. According to Mall's method, sections of fresh tissue are digested in the following solution :

Pancreatin, 5 gms.; bicarbonate of soda, 10 gms.; water, 100 ccm. The resulting specimen is washed by shaking in a volume of water and then allowed to dry on the slide. To stain the section, a few drops of the following solution are allowed to dry on the specimen:

Picric acid, 10 gms.; absolute alcohol, 33 ccm.; water, 300 ccm. The specimen is then immersed for one-half an hour in the following solution :

Acid fuchsin, 10 gms.; absolute alcohol, 33 ccm.; water, 66 ccm. Upon removal, the tissue is washed with the picric acid solution, dehydrated, cleared in xylol and mounted in balsam.

The method of digestion suggested by Spalteholz has been of especial service because of the support given to the delicate framework by the glass slide and because of the accuracy of the control. According to this method the tissue to be digested is fixed in a one per cent solution of mercuric chlorid in thirty-three per cent alcohol for twenty-four hours. Each succeeding twenty-four hours the tissue is transferred to fresh alcohol increased ten per cent in strength, until absolute alcohol is reached. The tissue is imbedded in paraffin and cut in serial sections from 6f to 20 microns thick. The sections to be digested are fixed to the slide by the water method and the process continued as follows:

Eemove the paraffin with xylol; wash in absolute alcohol; immerse in benzine at 38° C. for 24-36 hours; wash in absolute alcohol followed by 95 per cent alcohol; wash in water five minutes; digest in a solution of pancreatin at 38° C. for 12-48 hours; wash in water 10 minutes; stain with iron hsematoxyliu; mount in balsam. By substituting acetic alcohol ' for the graded alcohols, the time required for fixing may be reduced from several days to a few hours. The pancreatin solution was employed as follows:

Pancreatin ferment (Griibler), 1 part; sodium bicarbonate,

20 parts; thymol, 10 parts; distilled water, 10,000 parts.

I^he digestion of hardened tissue in bulk was employed as follows:

Small pieces of tissue were hardened 4-12 hours in acetic alcohol and

dehydrated in absolute alcohol. They Avere then extracted with ether

for 10-14 days. The tissue was then digested in an aqueous solution

sCarnoy: La Cellule, T. Ill, 1886.

40 The Intralobular Framework of the Human Spleen

of pancreatin and washed in water. The resulting specimens were in some cases stained, imbedded and sectioned; in others immersed in glycerin and studied with a low-power stereoscopic microscope. The thick sections thus obtained afforded most instructive pictures in three dimensions.

Controls of the digested specimens were stained with orcein, Weigert's elastic tissue stain, hasmatoxylin and eosin, and the connective tissue stains of Mallory and Van Gieson. In searching for elastic fibers within the lobule the so-called differential stains of Weigert, Mallory and Unna-Tanzer were employed.

Digested specimens were stained by immersing them in a five per cent solution of the ammonio-sulphate of iron for 12-18 hours, and subsequently removing them to a five per cent aqueous solution of hsematoxylin for four hours. By difi!erentiating such specimens in a one per cent solution of acetic acid a sharp and intense black stain was obtained. Weigert's elastic tissue stain was also used to stain digested specimens.

Dilute aqueous solutions of potassium hydrate, acetic and hydrochloric acids were employed in testing and differentiating the connective tissues of the framework.


By macerating and washing fresh human spleen tissue it is possible to remove the parenchyma cells and expose a framework of bloodvessels and anastomosing fibers that outlines the general form of the specimen in all dimensions. This is the coarser framework of the spleen formed by the trabeculse and is of imiform density throughout the organ. Its meshes, incompletely marked off by the surrounding fibers, average 20 microns in diameter and are the compartments occupied by lobules. In a specimen thus prepared each compartment appears as a vacuole, its outline alone being indicated.

If, however, spleen tissue be hardened in alcohol, thoroughly extracted with ether, and the parenchyma entirely removed by pancreatin digestion without mechanical injury, there is preserved in addition to the coarser framework a delicate network of fibrils within each individual compartment — the intralobular framework. (Fig. 1, C.)

The delicate fibrils composing the intralobular network vary from 1 to 5 microns in diameter. They branch and anastomose in all directions to form a network with meshes from 16 to 40 microns in diameter. The fibrils are directly continuous with those of the coarser interlobular framework at the periphery of the lobule

Preston Kyes


and with the sheaths of the- blood-vessels within the lobule. The network is devoid of nuclei and the picture is that of a continuous system of branching and anastomosing threads of fairly uniform caliber. The extent of an individual fibril canuc^t be determined.

Fig. 1.— Congested human spleen ; digested with pancreatin ; stained with iron h?ematoxylin ; Zeiss, Obj. AA, Oc. 2.

A. Capsule.

B. Trabeculae of coarser framework.

C. Intralobular framework.

D. Intralobular capillary veins [Billroth].

E. Malpighian follicle with afferent artery.

The network extends throughout the entire lobule between the capillary veins and in a modified form within the Malpighian follicles. Thus the arrangement is such as to outline in the digested specimens all the structures appreciable in the undigested specimens. The general distribution of the network throughout the lobule is interrupted by irregularly, circular channels averaging 0.18 mm. in diameter. These channels freely communicate with one another, and in thin specimens appear as circular and oval lacunae surrounded by the framework of the pulp cords. They represent the lumina of the intralobular capillary veins first described by Billroth. (Fig. 1, D.)

The arrangement of the fibrils immediately surrounding the capillary veins is so modified as to form a definite framework limiting the venous spaces. Continuing from the general network of the pulp cords

42 The Intralobular Framework of the Human Spleen

the fibrils encircle the venules and anastomose with one another to form a series of circular and spiral rings completely surrounding the Imnen. The}' are an integral part of the framework of the pulp cords, modified only as to their distribution. Encircling and limiting the venous spaces the spirals are not compactly apposed to one another, but separated by intervening spaces of varying width. They are identical with the fibrils first described by Henle as white fibrous tissue and by more recent writers as elastic fibers. The nature of the fibril per se will be discussed below.

In fortunate preparations the intralobular framework may be seen to extend throughout the Malpigliian follicle and become continuous with the coats of the afferent artery. (Fig. 1, E.) The fibrils are much more delicate and fewer in number than those of the pulp cords. The meshes are larger and the network is uninterrupted by the spaces representing the capillary veins. There is no membrane or condensation of the framework at the periphery of the follicle to mark it off from the surrounding portion of the lobule, the only transition being from the coarser to the more delicate network.

In all their reactions the component fibrils correspond to reticulum as described by Mall. They differ from elastic fibrils in that they are not digested with pancreatin ferment and are resistant to weak acids and alkalies. Unlike white fibrils, they continually branch and anastomose. Their reaction with pancreatin is constant, and in specimens subjected to the enzyme for forty-eight hours the framework remains intact where sufficiently protected from mechanical injury. The fibrils within the Malpighian follicles, because of their extreme delicacy, are easily broken away in the process of washing, and in a majority of specimens this portion of the framework is wanting. In fortunate preparations, however, this framework is present, intact, after the continued action of the enzyme.

The staining reaction of these fibrils is intermediate between that of elastic and white fibrous tissue. By prolonging the time of staining or by heating, reticulum fibrils may be tinted and in cases somewhat deeply stained by the so-called specific elastic tissue stains of Weigert, Mallory and Unna-Tanzer. Sections of human spleen from which all elastic tissue has been removed by digesting forty-eight hours in pancreatin, may be tinted a deep purple by staining two hours in Weigert's elastic tissue stain. By continuing the staining for three hours at 38° C. the intensity of the color is much increased. The reaction is not the same as that with elastic fibrils, the intense black color never being attained with reticulum as with elastic tissue. Likewise with the orcein

Preston Kyes 43

method, the fibrils may be stained brown and with Mallory's stain a blue-black. I have not been able to obtain similar reactions with the white fibers of tendon.

Such staining reactions would seem to be responsible for the continued descriptions of elastic fibrils within the pulp cords, the walls of the capillary veins and the j\Ialpighian follicles. Fibers within all of these structures may be brought into view by overstaining with the socalled elastic tissue stains. The staining reaction of such fibers, however, is not that typical of the elastic fibers seen within the trabecule and the arterial walls, and unlike elastic fibers, they resist pancreatin digestion. I quite agree with Hohl' that the fibrils within the walls of the capillary veins of the human spleen described by v. Ebner "" as elastic tissue cannot be such, since they resist pancreatin digestion. In addition their staining reactions are those of reticulum found elsewhere.


1. Within the lobule of the human spleen there is a delicate network of fibrils continuous throughout the pulp cords and the Malpighian follicles.

2. The fibrils of this entire network are reticulum in the sense of Mall.

3. The fibrils encircling the capillary veins are an integral part of this reticulum network and are not elastic tissue.

4. The so-called specific elastic tissue stains yield a positive reaction with reticulum as well as with elastic fibers.

SHohl, E. : A7iat. Am., Bd. XVII. i»v. Ebner: Anat. Anz., Bd. XV.




From the Anatomical Laboratory of the University of Jlichigan.

Our present conception of the structure of neurogiiar tissue is based in the main on results obtained by the employment of two fundamentally different methods, each of which is regarded by its supporters as a differential stain for neuroglia. We refer here to the chromesilver method of Golgi, for many years the only method at our disposal by means of which the structural elements of this tissue could be brought to light. To this method we are indebted for the results obtained by Golgi, v. Lenhossek, v. Kolliker, Ramon y Cajal, Retzius, Sala y Pons, Van Gehuchten, Eurich, Reinke and others in their investigations of the structure and histogenesis of the neuroglia. The other method was the one fully described by Weigert in 1895 in his large monograph on the structure of neurogiiar tissue, giving the results of seven years' labor in this field. Almost simultaneously appeared Mallory's publication giving an analogous method. In the Weigert and Mallory methods, the staining is the result of a chemical differentiation of the neuroglia fibers. Their results have been corroborated by Pollack, Ivrause and Aguerre working with normal tissues and by Taylor, Storch and Bonome in their study of pathological tissue and new growths. Quite recently Benda has published a differential stain for neuroglia, which seems destined to become very useful.

All recent writers on neurogiiar tissue have called attention to the apparently contradictory results obtained by those observers working with the chrome-silver method as compared with the recorded observations of investigators who have used the more modern differential stains, and a glance at the more recent literature is sufficient to convince one that a classification of the current views of the structure of neurogiiar tissue may with propriety be based on the methods used in the investigation of the tissue. It is, however, not our purpose at the present time to extend this controversy, and a repetition of what has been previously stated in several of the discussions of neuroglia literature seems uncalled for; it Avill therefore be entered upon only to the extent necessary to present our own observations as clearly and succinctly as possible.

46 Studies on the Neuroglia

As is now very generally known, the chrome-silver method shows the neurogliar tissue to consist of cellular elements possessing a varying number of tibrillar processes. These structural elements have been designated by v. Lenhossek by the very appropriate name of astrocytes, and are subdivided into (a) astrocytes with long processes, found in the white and grey matter, and (b) astrocytes with short processes, found only in the grey matter. In these few words we have endeavored to reflect, as well as possible in so short a space, the views of the majority of the early observers who have used the chrome-silver method in the study of neuroglia. V. Kolliker, while agreeing in the main with the above statement of the structure of the neuroglia, describes the fullydeveloped astrocyte, which he terms a Golgi-cell, as consisting of two portions — a cell-body containing the nucleus which is intimately associated with a cell-plate, from which the cell processes arise. He suggests the hypothesis that " a Golgi-cell with a portion of its protoplasm develops a cell-plate from which the cell processes arise; this plate originally and as long as the processes possess the power of growth is intimately connected with the nucleated portion of the Golgi-cell; in many instances, however, the cell-plate attains a different consistency and perhaps also a different constitution and, under certain conditions, may sever its connection with the nucleated portion of the Golgi-cell." Andriezen, who has used the chrome-silver method in studying the neurogliar tissue of the brain cortex, recognizes two varieties of neuroglia cells. One of these, which he has designated as the neuroglia fiber cells, corresponds in the main with the astrocytes with long processes described by other writers. It should, however, be stated that this observer was able to make out in certain of his preparations that not all neuroglia fibers are processes of cells, but that many of them "pass right through the cell body." The other variety of neuroglia cell is 'described by him under the name of protoplasmic glia cell, and has stout, coarse and very shaggy processes, which vary very greatly in size. These latter cells are said to be of mesoblastic origin. Eeinke, who, in his study of neuroglia tissue, has made use of a modified chrome-silver method (after obtaining a chrome-silver precipitate, he dehydrated and then imbedded his tissue in paraffin, fixed the sections with albumin fixative and then stained them with Heidenhain's hematoxylin and counterstained them in eosin), has obtained results which deserve especial mention. His conclusions are " that the neurogliar tissue of the white substance of the human cord consists of (1) cells and (2) fibrils. The cells possess numerous processes, some of which are branched and which run in part transversely and in part obliquely; the majority of

G. Carl Huber 47

them, however, run vertically, that is, parallel to the nerve-fibers. These processes are well stained by the chrome-silver method. The fibrils differ morphologically, physically and chemically from the cellprocesses. They are, however, developed from the protoplasm of the cells and lie partly in and partly on the protoplasm and have a direction which in the main is opposite to that of the cell processes. For the most part these fibrils, the length of which is unknown, are emancipated from the cell-bodies. The fibrils differ in thickness and probably do not anastomose. These are the fibrils which are so clearly brought out by the Weigert method."

Erik Miiller's " Studies on Neuroglia " may also be mentioned in this connection. His observations were made on tissues taken from amphioxus, myxine, acanthias, and from teleosts; they were fixed by the Golgi method and then stained after Heidenhain's iron-lack-hematoxylin. In all of these forms he finds the neuroglia made up of cells and fibers, which are, however, in such relation to each other that all the fibers may be regarded as processes of cells.

As may be seen from the above brief summary of neuroglia literature giving accounts of observations made with the chrome-silver method, all observers who have' used this method, even when such method has been subjected to special modifications, have reached the conclusion that the neuroglia is composed of cellular elements and cell processes— neuroglia-cells and neuroglia-fibers, the latter being the processes of the former. It is true that Andriezen found certain astrocytes in which the neuroglia fibers passed through the cell-body, and that V. Kolliker regards himself justified in describing a cell-plate with cellprocesses, which may under certain conditions become separated from the nucleated portion of the neuroglia cell, and that Reinke believes that he has harmonized the conflicting views by his discovery of two varieties of neuroglia fibers, the one being processes of neuroglia cells and the other developed from the protoplasm and in part at least emancipated from such protoplasm; yet a careful study of the account and figures given by Andriezen and v. Kolliker does not reveal sufficient evidence to indicate that these observers regard the neuroglia fibers as other than processes of the neuroglia cells, and Eeinke's statement will need further confirmation with methods other than a chrome-silver precipitate before they can be accepted as established. At this date it does not seem necessary to enter into a consideration of the chromesilver method as such, as its advantages and disadvantages have been the subjects of frequent consideration and are now very generally known; suffice it to say that we agree with Taylor when he states that

48 Studies on the Neuroglia

"A method which colors by precipitation is a priori incapable of giving US the information which we require ; it must of necessity be confusing in its pictures of structural detail."

We may now turn our attention to the observations of investigators who have employed a differential chemical stain in their study of neuroglia. Weigert, whose comprehensive work gave a new impetus to the study of neuroglia, summarizes his results in the following statements: " The neuroglia fibers, which have been hitherto regarded as processes of Deiters' cells, differ chemically from the protoplasm of these cells. This difference in the chemical constitution of the ' cell-processes ' is apparent 'in the immediate vicinity of the cell nucleus, as well as at some distance from it. The majority of the so-called cell processes are not cell processes since two such apparent processes form a continuous fiber, which is in no way interrupted by the cell body, as would be the case were they true processes arising individually from the cell body. In a word, there is no question here of cell processes or cell extensions, but of fibers which are fully differentiated from the protoplasm. Weigert describes two types of nuclei of neuroglia cells — large, vesicular nuclei the chromatin of which has a granular appearance, and smaller ones in which the chromatin forms a deeply-staining, homogeneous mass, with transition forms between the two varieties. The large vesicular nuclei often show a definite relation to the neuroglia fibers, in that they form centres, over which and around which the neuroglia fibers pass, simulating in a most characteristic manner the astrocytes of other investigators. Numerous free neuroglia cell nuclei, notably of the smaller, deeply-staining variety, which bear no special relation to the neuroglia fibers, are also found,

Mallory, working with his own method, obtained results which were practically identical with those obtained by Weigert. Although these 'two investigators worked independently, their results were published almost simultaneously. Pollack in a short note confirms Weigert's statements and calls attention to the importance of his differential neuroglia stain and the results obtained. Aguerre, after discussing the recent neuroglia literature and calling attention to the superiority of Weigerfs neuroglia stain and corroborating his observations, discusses at some length the shape and structure of the neuroglia cell nuclei as found in the human spinal cord, stating that these nuclei vary much in shape and size and are often polymorphous. He classifies the neuroglia cell nuclei according to their size as follows: (1) small nuclei, 3// to 4/^, to which variety the small deeply-stained nuclei belong; (2) medium-sized nuclei, G/x to S/i, the smaller nuclei of the

G. Carl Huber 49

vesicular variety; (3) large nuclei of the vesicular variety, often polymorphous and measuring as high as 14^^. Krause and Aguerre, in another communication, describe at some length the distribution of the neuroglia tissue in the human spinal cord. Attention should also be drawn to the fact that these two observers have been able to stain the neuroglia in apes and half-apes, after having slightly modified the Weigert method. Krause has given a full account of the neuroglia in the spinal cord of the ape, in which he describes small, deeply-staining nuclei of neuroglia cells and larger often polymorphous, vesicular nuclei, having only a small amount of chromatin. He finds the majority of the neuroglia fibers fully difi'erentiated and many of them relatively thin. Enrich in his last publication places himself in accord with Weigert's view on the structure of the neuroglia and advances theoretical reasons for accepting the same. It would lead beyond the limits of this paper to do more than mention the observations of Taylor, who worked with the Mallory method, and Storch and Bonome, who used the Weigert method in their study of glioma and gliosis and of the behavior of the neuroglia in certain pathological conditions of the central nervous system of man. As pertains to the structure of the fully-developed neuroglia tissue, their published results confirm in the main the views expressed by Weigert and Mallory and others who have used these methods. Yamagiwa has recently described a new stain for neuroglia which consists of a modification of Strobe's differential axis-cylinder stain. As a result of observations made with this method, he is led to conclude that the neuroglia fibers are differentiated intercellular structures, which are, however, not entirely or not in all instances completely separated from the neuroglia cells. Mention may also be made of a somewhat crude method described by Whitwell, by means of which he aims to differentiate the neuroglia fibers. Sections from hardened tissues are treated for a few seconds with a hot concentrated solution of caustic potash, are then rinsed in water and allowed to desiccate on the slide. When dry, the sections are covered with a cover glass. In such preparations examined with a moderate magnification, but with good illumination, a dense feltwork of fibrils may be seen. Whitwell regards these fibrils as neuroglia fibers and states that " they show no evidence of being direct processes of cells and do not appear to branch and form a complete basket network for each element in the nervous tissues, including the blood-vessels." Benda has recently described a differential neuroglia stain which will be given fuller consideration presently and will therefore receive no further mention at this time. From the foregoing account it may be seen that those

50 Studies on the Neuroglia

observers who have used differential neuroglia stains in their study of this tissue agree in regarding the neuroglia fibers, not as cell processes, since they are readily differentiated from the protoplasm of neuroglia cells, from which they differ in chemical constitution and physical properties, as is shown by these staining methods, but as an intercellular substance emancipated from the protoplasm of the neuroglia cells.

One of the disadvantages of both the Weigert and Mallory neuroglia stains is the fact that these stains can be used only on human tissue; furthermore, owing to the fact that the neuroglia fibers break down very readily, as was pointed out by Virchow many years ago, it is necessary to have at one's disposal very fresh tissue in order to obtain satisfactory staining of the neuroglia. Krause and Aguerr^, as has been previously stated, were able by careful manipulation of the Weigert method to obtain successful differential staining of the neuroglia in apes and half-apes; this, however, is the only instance, so far as I have been able to ascertain, in which a successful differential staining of the neuroglia in vertebrates other than man has been obtained. Weigert and Mallory both admit that their neuroglia staining methods are useful only on fresh human tissue. The difficulty of obtaining very fresh human tissue no doubt accounts for the fact that we have so few confirmatory observations of the views of Weigert and Mallory concerning the structure of the neuroglia. The fact that it is often difficult to obtain fresh human tissue and the further fact that these methods cannot be used to stain the neuroglia of animals no doubt explains in part the apparent hesitancy to accept their results in place of the results obtained by the chrome-silver method, by means of which, as is well known, neuroglia tissue may be stained in the central nervous system of all vertebrates, whether embryonic or adult tissue is used. The reason why the Weigert and Mallory stains are selective for human neuroglia only is difficult to explain and must be ascribed to some slight difference in the chemical composition of the neuroglia fibers of man and other vertebrates, in which case we may look upon these staining methods as so highly differential as to be applicable to the staining of human neuroglia only. In the hope that by modifying these methods a procedure might be found by means of which the neuroglia of animals might be stained, I spent much time in experimentation. All of my endeavors, however, proved fruitless so long as I confined my attention to the Weigert and Mallory methods. Much more satisfactory results were, however, obtained after the writer became familiar with the Benda differential neuroglia stain, published in September of last year. Benda was led to continue his endeavors to discover a satisfactory stain for

G. Carl Huber 51

neuroglial- tissue, partially interrupted by the appearance of Weigert's large monograph, because the Weigert method did not seem to possess the certainty ascribed to it by its discoverer. I am not aware that Benda is familiar with the fact that his method may be used as a differential staining method for the neuroglia of animals. I find, however, no reference to this fact in his account of his method. This fact seems to me, however, of sufficient importance to warrant my giving his method somewhat in detail, since it does away with one of the obstacles to the use of the Weigert or Mallory method— namely, the necessity of using fresh human tissue in order to study the neuroglia with a differential stain.

The method used by me in my study of the neuroglia of animals is essentially the same as the first of the three methods given by Benda. I have not been successful in staining the neuroglia of animals by the other methods given by him.

The method as used by me is as follows : '

1. The tissues, which should be in small pieces, not more than 0.5 cm. in thickness, are fixed and hardened for two to four days in a 4^ or 10^ solution of formaldehyde (10 parts or 25 parts respectively of formalin in 100 parts of water). A large quantity of the solution is used and, during the hardening process, the tissues rest on several layers of filter paper placed in the bottom of the dish.

3. The tissues are then placed for two to four days in Weigert's chrom-alum mordant, used in his neuroglia stain, the solution being kept in the warm oven at 38° C. during this step.

3. Wash in flowing water for 24 hours.

4. Place tissues for two to four days in a 0.5;^ aqueous solution of chromic acid.

5. Wash for 24 hours in flowing water.

6. Dehydrate in graded alcohol. It is necessary to dehydrate the tissues very thoroughly.

7. Imbed in parafiin. It is necessary that this procedure be very carefully carried out. After dehydration the tissues are placed in xylol for 24 hours, renewing the xylol several times; then place them for 12 hours in toluol and again 12 hours in benzole. Eenew the benzole and add an equal quantity of melted soft paraffin and place in the warm oven. At the end of 24 hours the mixture of benzole and paraffin is replaced by soft paraffin and in three to four hours by hard paraffin (58° C. melting point, Grlibler); after three hours' stay in this, they may be imbedded.

52 Studies on the Neuroglia

8. Cut sections and fix to slide or cover glass with albumin fixative. To facilitate the application of this step, I may say that sections from paraffin-imbedded tissues of the central nervous system are most readily made by placing the knife at an angle of about 30° and placing a layer of distilled water on the knife, renewing it constantly as necessity requires. I have found no difficulty in cutting 3// to 5^« sections of the spinal cord or even of the medulla of animals ordinarily used in the laboratories. The sections are then caught on a small brush and floated on distilled water contained in a small evaporating dish. When 40 to 50 sections have been cut and floated on the distilled water, the evaporating dish is placed over a flame and the water is gently heated until the sections flatten out, care being taken not to melt the paraffin. The sections are then caught on cover glasses smeared with a thin layer of albumin fixative and placed for 24 hours in the warm oven.

9. Eemove paraffin and bring sections through alcohol into distilled water.

10. The sections are now placed for 24 hours in a mordant consisting either of a 4^ solution of ferric alum or of a solution of liquor f erri tersulphatis, made by adding one part of this to two parts of distilled water.

11. Sections are rinsed in two tap waters and one distilled water and placed for 24 hours in a solution of sodium sulphalizarate, made by adding to distilled water a sufficient quantity of a saturated solution of sodium sulphalizarate in 70^ alcohol to give the distilled water a sulphur-yellow color.

12. Einse the sections in distilled water and dry between filter papers.

13. Sections are now stained for 15 minutes or longer in a 0.1;^ solution of toluidin blue, which should be heated, after the sections are in the stain, until the solution steams. Allow the stain to cool and rinse sections in distilled water.

The sections are next rinsed in a slightly-acidulated solution. For this purpose Benda recommends primarily a 1^ aqueous solution of glacial acetic acid, in which the sections remain for five to ten seconds and are then dried between filter papers, hastily washed in absolute alcohol and placed in creosote. This step was found necessary in staining the neuroglia of the frog, tortoise and dove. Benda further recommends the use of acidulated alcohol, made by me by adding six drops of hydrochloric acid to 100 ccm. of 70fo alcohol. This was found more useful in differentiating the neuroglia of mammalia (dog, cat, rabbit), and experience showed that the proper degree of washing was usually

G. Carl Huber 53

attained by dipping the sections into this solution as many times as there were micra in the thickness of the section. Tne further procedure is as above.

15. The sections are differentiated in creosote. Benda states that the sections are properly differentiated after they have remained in the creosote about ten minutes. In my work, however, the time varied from about ten minutes to several hours. Sections washed in acidulated alcohol are usually differentiated in about ten minutes ; sections washed in the dilute acetic acid solution require a much longer time, varying from one to several hours. It is therefore necessary at all times to control the differentiation under the microscope.

16. After differentiation in the creosote the sections are dried between filter papers, washed in several xylols and mounted in xylolbalsam.

To the naked eye, preparations well differentiated should have a bluish-red or brownish-red color, large masses of neuroglia showing as blue areas. Under the microscope, the neuroglia fibers appear stained deeply blue and stand out very distinctly. The chromatin of the neuroglia cell nuclei presents a purplish-blue color; the remainder of the nucleus is brownish-red and the protoplasm is of the same color, but of a lighter hue. The myelin and neuraxes of the nerve fibers are of a brick-red or brownish-red color, the neuraxes staining much more deeply than the myelin. The nerve cells are of a brownish-red color, the chromophile substance staining more deeply, and the nucleoli are a deep purplish-blue. The fibrous connective tissue stains a pink-red, its nuclei a purplish-blue. The red-blood cells are of a dark greenishblue. The colors here given are those seen in a well-differentiated preparation, especially after washing in acidulated alcohol (step 14). In case the acetic acid wash is used, the myelin and neuraxes retain some of the blue color, giving them a reddish-purple or even a bluish tinge, the axis-cylinders staining more deeply; even in such preparations, however, the neuroglia fibers may be clearly made out by reason of their deep blue color. It is necessary to add that it is not always possible to obtain a clear differentiation, as the method while often reliable, is not without its whims. It is my custom to fix as many sections to one cover glass as I can, and nearly always some of the sections, if not all, will show the desired result. In over-differentiated preparations, the neuroglia fibers have a brownish-red color, but may usually be clearly seen. Such preparations may, after removal of the creosote and xylol, be brought into water and then stained again in toluidin blue, the further treatment being as above described.

54 Studies on the Neuroglia

For this investigation material was obtained from the following vertebrates :

Mammalia — Dog, cat, rabbit.

Birds — Dove.

Eeptilia — Tortoise (Emys meleagris).

Amphibia — Frog (Eana Catesbiana and Kana halecina).

It is my purpose at this time to give a brief descriptive statement of observations made on the neuroglia of the spinal cord of the abovementioned vertebrates, differentially stained after Benda's method; I shall deal only with the structure of the neuroglia tissue of adult animals and not with its origin nor its distribution. A much more extended treatment of this subject is contemplated and many of the statements here made will then be substantiated by figures. This will appear on the completion of work now in progress.

Dog. In the dog, the neuroglia of the spinal cord consists of neuroglia cells and neuroglia fibers. With the method used, the nuclei of the neuroglia cells stain a purplish-blue color the protoplasm a brownish-red and the neuroglia fibers a deep blue. The nuclei of the neuroglia cells vary greatly in shape and structure, but may be described under two general types with transition forms. The majority of the neuroglia cell nuclei are vesicular with the chromatin arranged in fine granules. The nuclei belonging to this type vary greatly in shape and size. In the smaller varieties, which are generally of round or oval shape, the chromatin is in the form of numerous small granules; in the larger varieties, which are round, oval, or polymorphous, the chromatin is found in the form of one or several granules, the nucleus being otherwise homogeneous in appearance. The nuclei of the other type, which are not numerous, stain diffusely and usually quite deeply and are usually of round or oval shape and generally quite small. The Benda method has the advantage of staining the protoplasm of the neuroglia cells. The amount of protoplasm seen in connection with the dift'erent neuroglia nuclei varies greatly. In connection with the small vesicular nuclei with numerous chromatin granules, it is often difficult to make out any protoplasm, many of them appearing as free nuclei; now and then, however, a thin layer of protoplasm is made out, either surrounding the whole nucleus or appearing at only one side of it. The large vesicular nuclei with one or several larger chromatin granules are usually associated with larger masses of protoplasm, in which case distinct neuroglia cells are distinguished. In cross sections of the spinal cord, such cells present a variety of appearances, their shape depending more or less on the space occupied; they may be triangular or quadran

G. Carl Huber 55

gular or somewhat spindle-shaped, with a varying number of processes (usually not more than four or five), which may be traced for short distances between the nerve fibers; such processes are, however, not always made out and when present give the cell a very characteristic appearance. These cells are easily recognized in longitudinal sections of the spinal cord by reason of the structure of their nuclei. The cell protoplasm is often clearly brought out, the cells presenting a rectangular or irregularly oval shape with now and then a few short processes. The small, round or oval deeply-staining nuclei are often associated with a small amount of protoplasm, which usually stains somewhat deeply, often making it difficult to distinguish between protoplasm and nucleus.

The neuroglia fibers stain deeply blue. They vary somewhat in size, but the majority are relatively fine and only here and there does one find coarser fibers. The relation of the neuroglia fibers to the neuroglia cells or free nuclei is generally easily ascertained, in both cross and longitudinal sections of the cord. In the majority of instances, the neuroglia fibers are seen passing over or under the neuroglia cells or nuclei and appear independent of them. Other fibers are seen, however, in close proximity to cells and are seen to lie against them. The large neuroglia cells with protoplasmic branches are particularly interesting in this connection. In such cells it is often possible to trace one or several neuroglia fibers along the side of a protoplasmic branch to the cell body and then either directly across the cell and away from it, or along the side of some other protoplasmic branch and then away from the cell. Three, four or more neuroglia fibers may thus be traced over or under or along the borders of one of these branched neuroglia cells. Usually also numerous small blue dots, cross sections of neuroglia fibers, are seen in close proximity to such cells and now and then in the peripheral portion of their protoplasm; this latter fact must be interpreted as showing that some of the neuroglia fibers pass through the protoplasm of the neuroglia cells. Longitudinal sections of the cord give a better idea of the course of the neuroglia fibers than can be gained from cross sections. In longitudinal sections it will be seen that the neuroglia fibers run parallel to the nerve fibers, at right angles to them and obliquely across them. The relation of the neuroglia fibers to the deeply-staining cells above mentioned is often more difficult to make out, since the cell protoplasm of such cells and the neuroglia fibers now and then stain more nearly the same color than is the case with other cells. In such cells, the neuroglia fibers often appear as processes of the cells, especially when a cross section is studied

56 Studies on the Neuroglia

and when a section is not completely differentiated. In well-differentiated sections and more easily in longitudinal sections, it may usually be made out very clearly that the neuroglia fibers pass over or under or in close proximity to such cells and are not interrupted by them.

Cat. — The neuroglia of the spinal cord of the cat presents essentially the same appearances as those described for the dog. Nearly all of the neuroglia cell nuclei are of the vesicular variety with very little chromatin. In many of the cells, very little protoplasm can be made out, the nuclei appearing as free nuclei. In others, the protoplasm, staining a brownish red color, is readily made out, such cells often appearing distinctly branched, with three, four or five protoplasmic branches, varying in thickness and length and recognized between the cross-cut nerve fibers. The neuroglia fibers of the spinal cord of the cat are somewhat coarser than those found in the dog. They are clearly differentiated and may be traced over or under or around the neuroglia cell nuclei or neuroglia cells. The statements made concerning the relation of the neuroglia cells and nuclei and neuroglia fibers, as seen in the dog, are equally applicable for the cat and need, therefore, no further repetition.

Rabhit. — The neurogliar tissue of the spinal cord of the rabbit is not so easily stained differentially as that of the dog and cat. This is due to the fact that the protoplasm of many of the neuroglia cells shows a greater affinity for the toluidin blue than seems to be the case in the dog and cat and, as a consequence, one often finds neuroglia cells with protoplasm and nucleus staining a purplish color, sometimes of a lighter and sometimes of a darker shade. A longer differentiation in the creosote bleaches not only such cells but also to some extent the neuroglia fibers. The right degree of differentiation is therefore somewhat difficult to obtain. The majority of the nuclei of the neuroglia cells are of the vesicular type, varying greatly in size and shape and in the character and amount of chromatin contained. This may be present in the form of numerous fine granules or as one or several larger granules. Small nuclei, staining deeply, are found, but are not numerous. Many of the nuclei appear as free nuclei and show no or very little protoplasm surrounding them. The large vesicular nuclei, with one or several granules, are generally found in masses of protoplasm which are readily made out. Such cells are usually branched and when stained are purplish-blue in color; the protoplasmic branches can be very readily traced between the nerve fibers. The neuroglia fibers of the rabbit are relatively fine and stain a deep blue color and, in the great majority of cases, it can be readily seen that the fibers are independent of the nu

G. Carl Huber 57

clei and protoplasm of the neuroglia cells. In case of the branched neuroglia cells, when the protoplasm is stained a purplish-blue, it is now and then difficult and in certain cells quite impossible, in cross sections of the cord, to differentiate between neuroglia fibers and protoplasmic branches of neuroglia cells. It is often possible to trace with the utmost clearness a neuroglia fiber along the side of a protoplasmic branch of a neuroglia cell, along the side of or over the cell and by the side of some other protoplasmic branch and away from the cell. At other times, however, when a neuroglia fiber appears cut near the cell body of a neuroglia cell or near one of its protoplasmic branches, it appears as if the neuroglia fiber terminated in the cell. In longitudinal sections of the spinal cord of the rabbit, the relation of the neuroglia fibers to the cells under discussion is more readily made out. In such sections, the neuroglia fibers can be readily traced over, under, and around the neuroglia cells, even though at times the color of the neuroglia fibers is similar to that of the protoplasm of the cells.

Dove. — I have had more difficulty in staining the neuroglia of the spinal cord of birds than was experienced with the other vertebrates studied. This is due to the fact that with the method used the neuroglia fibers seem to bleach out at about the same time as the neuroglia cells and nerve fibers. When well stained, the color of the neuroglia fibers is a light blue and, even in the most successful preparations, they do not stand out as clearly as in the mammalia studied. In cross sections of the cord, the great majority of the neuroglia cells of the white matter appear as branched cells with relatively large vesicular nuclei, containing numerous chromatin granules. The protoplasm of such cells often stains a reddish-blue or purplish-blue color. Here and there in the white matter and more generally in the grey matter, free neuroglia cell nuclei are seen of vesicular structure and containing numerous chromatin granules, or similar nuclei surrounded by very little protoplasm.

The neuroglia fibers in the spinal cord of the dove are relatively fine. In longitudinal sections, it can be seen that they are independent of the cell protoplasm of the neuroglia cells; in cross sections of the cord, it is more difficult to see this, but in favorable sections, one can usually trace the neuroglia fibers over or along the borders of the neuroglia cells and gain the conviction that they are not the processes of these cells.

Tortoise. — The neurogliar tissue of the spinal cord of reptilia is quite easily stained by the Benda method, the neuroglia nuclei and cells staining a brownish-red and the neuroglia fibers a deep blue. The nuclei of

58 Studies on tlie Neuroglia

the neuroglia cells are generally vesicular and show usually only one round or oval refractive granule, which stains deeply and of a purple color. The amount of protoplasm found with these nuclei varies and oftentimes is scarcely made out. Protoplasmic branches, when present, are slender and short. Here and there are found nuclei, which stain reddish-blue with protoplasm of the same color; such cells usually present clearly-marked protoplasmic branches. The neuroglia fibers vary much in thickness, stain a deep blue and are readily differentiated from the protoplasm of the neuroglia cells and may be traced over and along the borders of the neuroglia cells, often following the protoplasmic branches when these are present.

Frog. — In the frog, the greater proportion of the nuclei of the neuroglia cells are large, round, oval, or polymorphous and vesicular in structure. They are often stained a purplish-blue color, in which case they present a homogeneous appearance; when more bleached, they show numerous small chromatin granules. Here and there, smaller nuclei, staining deeply are also found and are more clearly seen in longitudinal sections. Many free nuclei or nuclei with very little protoplasm are found. The amount of protoplasm found varies greatly with the different cells. The protoplasm usually stains a reddish-blue or purplish-blue color. Branched neuroglia cells are found in the white matter of the cord.

The neuroglia fibers of the spinal cord of the frog are relatively very large and are stained a deep blue color. The greater proportion of these fibers run at right angles to the nerve fibers and in cross sections may often be traced for relatively long distances. In cross sections, not many fibers — usually not more than three or four — are seen in relation with any one neuroglia cell or nucleus. The relation of the neuroglia fibers to free nuclei and neuroglia cells is generally very clearly made out; the neuroglia fibers being so large and of such definite course, we are enabled in both longitudinal and cross sections to differentiate between these fibers and other structures. The neuroglia fibers can usually be traced over or by the side of neuroglia cell nuclei or neuroglia cells and are in no way interrupted by the protoplasm of such cells.

Conclusions. These observations seem to warrant the following conclusions: 1. The neuroglia of the spinal cord of the dog, cat, rabbit, dove, tortoise and frog consists of neuroglia fibers and neuroglia cells. The neuroglia fibers differ chemically from the protoplasm of the neuroglia

G. Carl Hiiber 59

cells— as shown by differential staining — but this difference is not equally well marked in all the forms studied. In the animals studied, this chemical difference between the protoplasm of neuroglia cells and neuroglia fibers is most marked in the dog, cat and tortoise, less so in the rabbit and frog and least in the dove.

II. The neuroglia fibers may be regarded as intercellular structures, as they bear no constant relation to the great majority of the cell nuclei or neuroglia cells observed.

III. By reason of the fact that the protoplasm of the neuroglia cells is stained by the Benda method, this method has been helpful in showing that there are certain neuroglia cells, usually possessing protoplasmic branches, the neuroglia fibers of which are not completely separated from the protoplasm, but are in continuity with it or even pass through it. That such neuroglia fibers are not simply processes of the cells is usually clearly shown by their chemical reaction — behavior toward stains — and by the fact that such fibers may generally be traced over, under, or along the sides of such cells without suffering interruption. That such cells are normal constituents of neurogliar tissue is shown by the fact that they occur in the spinal cord of the four classes of vertebrates studied.

IV. These observations present no evidence which would go to confirm V. Kolliker's hypothesis concerning the structure of neuroglia cells — namely a nucleated cell body with differentiated cell-plate, from which arise processes, the neuroglia fibers. Cross and longitudinal sections of the spinal cord of the animals studied show no such relation of neuroglia fibers and neuroglia cells. These observations cannot be used in confirmation of Eeinke's views of the structure of the neuroglia. The branched neuroglia cells seen by me bear no resemblance to the astrocytes or astroblasts, as seen in the chrome-silver preparations with which I am familiar, nor do they resemble the figures given by Eeinke. In my own preparations, the neuroglia fibers very generally follow the course of the protoplasmic branches of the neuroglia cells.

V. The observations here presented strengthen materially the position held by Weigert and Mallory as to the structure of neuroglia tissue, as well as by others who may have used their methods or modifications thereof in the study of neuroglia, and extend their observations to cover the more important vertebrate classes.

Finally the following quotation from Pollack's article may serve to emphasize the further advantages of the method used by me: " Angesichts des Umstandes aber, dass wir des Thierexperimentes nicht entrathen konnen, da wir ja am Menschen keine Versuche mit experi

60 Studies on the Neuroglia

menteller Degeneration anstellen konnen, erscheint mir die Anwendbarkeit auf das thierische Nervensystem als das nachste Postulate dieser hochbedeutsamen Methode." This postulate, it seems to me, has to a large extent been met by the introduction of the Benda differential neuroglia stain. I may add that experimental work on cats and dogs, undertaken with a view of studying the behavior of the neuroglia under certain })athologic conditions has been begun and will form the subject of some future contribution.

In conclusion I wish to thank Mr. August Henry Eoth, student in medicine, for much valuable assistance given in the preparation of the sections on which these observations are founded.


1900. J. A. Aguerre. — Untersuchungen iiber die menschliche Neuroglia.

Archiv f. Mik. Anatom. und Entwicklungsgescli., Vol. LVI, p. 509. 1893. W. Lloyd Andriezen. — The Neuroglia Elements in the Human Brain.

Brit. Med. Journ., Vol. II, July-Dec., p. 227.

1900. C. Benda. — Erfahrimgen iiber Neurogliafarbungen und eine neue

Farbungsmethode. Neurologisches Centralblatt, Vol. XIX, p. 786.

1901. A. BONOME. — Bau und Histogenese des pathologischen Neuroglia Gewebes. Virchow's Archiv, Vol. CLXIII, p. 441.

1897. F. W. EuRiCH. — Studies on the Neuroglia. Brain, Vol. XX, p. 114.

1898. F. W. EuRiCH. — Contribution to the Comparative Anatomy of the

Neuroglia. Journal of Anatomy and Physiology, Vol. XXXII, p. 688.

1899. E. Krause. — Untersuchungen iiber die Neuroglia des Affen. Anhang

z. d. Abh. der Konigl. Akad. der Wissenschaften zu Berlin. 1899.

1900. K. Krause and J. Aguerre. — Untersuchungen iiber den Bau des

menschlichen Riickenmarkes mit besonderer Beriicksichtigung der Neuroglia. Anat. Anzeiger, Vol. XVIII, p. 239.

1893. A. V. KoLLiKER. — Handbuch der Gewebelehre des Menschen, 6 Aufl.,

Vol. II, pp. 136 to 193. 1895. F. B. Mallory.— Centralblatt f. Allg. Pathol, u. path. Anat., Vol. VI,

p. 753; also Method of Fixation for Neuroglia Fibers, pp. 532-3,

Journal of Exp. Med., Vol. II, 1897.

1899. Erik Muller. — Studien iiber Neuroglia. Arch. f. Mik. Anatom. und

Entwicklungsgesch., Vol. LV, p. 11. 1897. B. Pollack. — Einige Bemerkungen iiber die Neuroglia und Neurog liafilrbung. Arch. f. Mik. Anatom. und Entwicklungsgesch., Vol.

XLVIIT, p. 274. 1897. Fr. Reestke. — Ueber die Neuroglia in der vs^eissen Substanz des Eiick ensmarks vom ervi^achsenen Menschen. Arch. f. Mik. Anatom. und

Entwicklungsgesch., Vol. E, p. 1.

G. Carl Huber 61

1899. E. Storch. — Ueber die pathologish-anatoinischen Vorgange am

Stiitzgerlist des Centralnervensystems. Virchow's ArcMv, Vol. CLVII, p. 127.

1897. E. W. Taylor. — A Contribution to the Study of Human Neuroglia.

The Journal of Experimental Medicine, Vol. II, p. 611. 1895. C. Weigert. — Beitrage zur Kenntniss der normalen menschlichen Neuroglia. Festschrift, Frankfurt a M.

1898. J. E. Whitwell. — On the Structure of the Neuroglia. British Med,

Journ., Vol. I, Jan.-June, p. 681.

1900. K. Yamagiwa. — Eine neue Farbung der Neuroglia. Virchow's Archiv,

Vol. CLX, p. 358.





Assistant Professor of Pathology^ Pathological Laboratory, University of Michigan.

In the prevertebral fat of the bullock and sheep there are found in large numbers glands varying in size from a pin-point to a large cherry and of a deep-red or chocolate color. Because of their number and striking color these glands stand out with much greater prominence than the pale lymph glands of these anipials, but despite this fact they have been strangely overlooked and their histology and function investigated by but few observers. The earliest mention made of them that I have been able to find is by Leydig (Lehrbuch der Histologic der Menschen und der Thiere, 1857, pages 434, 429). This writer speaks of glands occurring along the thoracic aorta in many mammals, particularly in the hog, which from their color might be mistaken for accessory spleens in case they were found in the immediate neighborhood of the spleen. The cut surface of many of these glands he describes as perfectly resembling that of the spleen, presenting a darkred pulp in which lie white masses of cells corresponding to the Malpighian follicles. Leydig noticed further that transition-forms exist between these glands and ordinary lymph-glands, but his observations were confined entirely to the gross appearances, and the histological structure of these glands was not investigated by him.

In 1884 H. Gibbes noticed the presence near the renal vessels of the human body of glands differing from ordinary lymph-glands in that they possessed sinuses containing blood in place of lymph. This important discovery was not carried further and the observation was lost sight of, the next mention of these organs being made by Eobertson (Lancet, Nov. 29, 1890) who, apparently unaware of Gibbes' discovery, made the first histological study of these structures and gave them the name of hemolymph glands (suggested by Dr. Russell under whom Eobertson was working). He found these glands to be present in the sheep, bullock and human body, but his histological descriptions are based chiefly upon their structure as found in the sheep. His work 6

G4 The Normal Histology of the Human Hemolymph Glands

upon the human body was unsatisfactory because of the unfavorable conditions under which his autopsies were made. He was able, however, to determine the presence of these glands in man, and states that their structure in the human body is very similar to that in the sheep and bullock.

In 1891 Clarkson reported observations on " certain hitherto undescribed glands " found in the neighborhood of the renal vessels in the horse, sheep and goat. To these he gave the name of " hemal glands," and considered them to be a different variety from the hemolymph glands described by Eobertson though probably possessing the same function.

Gibbes in 1893 made a second report in which he states that Eobertson's description of these organs accords with the glands discovered by himself in 1884, and accepts the designation of hemolymph glands as an appropriate name for them. Finding his discovery thus confirmed he had made further investigations in the human subject, and found these glands to be constantly present near the renal vessels. He added nothing, however, concerning their histology or function.

In Clarkson's " Text-Book of Histology," 1896, several pages are devoted to the histology of the " hemal glands." Clarkson states that these organs have been found in the pig, horse, ox and sheep, but have not yet been observed in man. His descriptions are similar to those of Gibbes and Eobertson, and the hemal glands must be regarded as hemolymph glands.

Vincent and Harrison, in 1897, gave a detailed histological description of the hemolymph glands of the ox, sheep and rat. They noted also the occurrence of similar glands in the horse, dog, common fowl and turkey, and pointed out the histological resemblance existing be'tween hemolymph glands and the head-kidney of certain teleostean fishes. In several human cadavers examined they found no hemolymph glands, but Vincent in a later examination found them in the mesentery of a young boy.

In 1900 Drummond made a more thorough study of the histology ol: the hemolymph glands of the sheep, ox, rat and dog; and gave a very clear description of their structure. He confirmed the work of Eobertson and Vincent and Harrison; and added many important points concerning the distribution and structure of these glands in the lower animals.

In so far as I have been able to discover these are the only observations that have been made of the occurrence and structure of the hemolymph glands, and as seen above the chief part of these have been

Aldred Scott WartMn 65

of these glands as occurring in the lower vertebrates. Gibbes' discovery of their presence in the human subject, confirmed by Eobertson, the second report of the former and the solitary observation of Vincent represent the sum total of the published work upon the human hemolymph glands up to the publication of my article, " A Contribution to the ISTormal Histology and Pathology of the Hemolymph Glands" (Journal of the Bost. Soc. of Med, Sciences, April, 1901). In this paper I made a preliminary report of a study of the human hemolymph glands which has been carried on by me in this laboratory for several years. My attention was first drawn to these glands by certain cases in which some of the retroperitoneal lymph glands appeared to play a part independent of that of the other lymph glands of the body. In its earlier stages the study was confined to the retroperitoneal region, but later systematic investigation was also carried out of the cervical, thoracic and mediastinal glands. The material for this work has been obtained from my autopsy cases of the last 'five years, 94 in all. It was early evident to me that there were two distinct varieties of lymph glands, one containing blood sinuses aud apparently possessing distinct hemal functions. At that time the observations of Gibbes, Eobertson and Vincent were unknown to me so that the essential nature of the glands was also independently discovered by me.

Since the publication of my article in April of this year there has appeared in the Archiv f. Mikr. Anatomie (July) a paper by Weidenreich on the "Gefasssystem der Menschlichen Milz," in which he treats theoretically of the hemolymph glands, basing his conclusions upon the work of the English observers. The more important of his theoretical deductions are confirmed by my previously-reported observations.

The present paper will be devoted to a consideration of the normal histology of human hemolymph glands to a much fuller extent than in my preliminary report. With the exception of a few accident cases none of my autopsy subjects can be said to have been normal. The majority were chronic cases, many of which showed various stages of anemia and cachexia. In so far as I am able to divide the appearances observed by me into histological and pathological such classification is based upon the fact that only in cases showing extensive changes in the blood were conditions found in the hemolymph glands that could be regarded as being essentially pathological. In all other cases the structure of the glands was assumed to be histological because of their identity of structure with the glands found in normal individuals killed by accident, the general similarity of structure in all cases except certain blood cases, and finally their resemblance to the hemolymph glands of

66 The Normal Histology of the Human Hemolymph Glands

normal animals. As a result of this study the following conclusions regarding the normal histology of the human hemolymph glands have been reached.

Technique. — The thymus, anterior mediastinal and renal regions are examined in the usual order of the autopsy; the prevertebral tissues at the close of the autopsy. The neck-organs, thoracic and abdominal vessels, root of the mesentery, and other structures attached are stripped from the spinal column from above downwards and removed from the body for minute examination. When there is much prevertebral fat present or when the tissues are very opaque the search for hemolymph glands may be made much more successful by first fixing the tissues in mass in 4 per cent formalin. The color of the blood-sinuses is in this way brought out much more sharply, and the glands may be recognized when the search in the fresh tissue would have been negative. When it is desired to study the exact position and relations of these glands the prevertebral tissues should be dissected in place and not stripped from the spine. Since the sinuses of the human hemolymph glands partly collapse after death the recognition by color alone is not easy and at times impossible. It therefore becomes necessary to remove all glands found in a region for microscopical diagnosis and in many cases it is only in this way that the character of a gland can be definitely determined. In the search for these glands in the human body their proximity to large vessels should be borne in mind; they occur very frequently in the connective tissue between artery and vein.

OccuREENCE. — Taking the presence of a sinus containing blood in place of lymph as the essential feature of a hemolymph gland, such glands are found constantly present in the human body. They are apparently more numerous, at least are more easily recognized, in early adult and middle life than in infancy or old age. In new-born children they are discovered with difficulty, and usually can be found only after microscopical examination. In late life they become atrophic, the blood-sinuses being obliterated for the greater part by connective-tissue increase. No difference in their occurrence has been observed in the sexes. With the individual they apparently differ very much as regards their location, number and size, seldom being found under exactly similar conditions; but owing to the difficulty of recognizing them with the naked eye it is probable that these variations may be only apparent, resulting from imperfect technique.

They are found in greatest numbers in the prevertebral retroperitoneal and cervical regions, in the neighborhood of the adrenal and renal vessels, along the brim of the pelvis, in the root of the mesen

Aldred Scott Warthin 67

tery but rarely extending far out into it, still more rarely in the omentum and epiploica. In normal individuals they are rarely found in the mediastinal tissues or along the thoracic vertebrae, but in cases of a,nemia in which the hemolymph glands throughout the entire body are enlarged they may be found in large numbers in these regions making it appear probable that under normal conditions they are of such small size as to escape notice. In the cervical region they are usually found below and behind the lobes of the thyroid in association with the parathyroids.

Gross Appeaeances. — The human hemolymph glands are not nearly so easily recognized by the naked eye as are those of the steer and sheep, because of the fact that their blood-sinuses are frequently collapsed and partly emptied after death. They usually lie deeply embedded in fat or connective tissue, and as a rule near the wall of some large vessel. As a rule only a few show distended sinuses, these glands are deep-red or bluish in color and may be easily mistalsen for hemorrhages, bloodclots, or deeply-congested lymph glands. The resemblance to spleentissue is often very close. In the transition forms which are partly hemolymph glands and partly ordinary lymphatic glands the bloodsinuses appear as red points or streaks. It is often difficult or impossible to distinguish these from congested lymph glands. The smallest hemolymph glands may often be found by stretching the tissue against the light, the blood-sinuses then appearing as red points or lines. Fixation of the tissues in formalin is of great advantage as the blood-content of the sinuses of these glands is brought out in sharp contrast to the lighter color of lymphoid tissue. When the blood-sinuses are small or few in number the gland cannot be distinguished on naked-eye inspection from an ordinary lymph gland. It is, therefore, safest in studying the occurrence of hemolymph glands to remove all apparent glandular structures and examine them microscopically.

On cross-section the blood-sinuses resemble spleen-pulp and contrast according to their blood-content more or less sharply with the whitish areas of lymphoid tissue. Small round masses of whitish lymphoid tissue often project into the pulp-like peripheral sinus suggesting splenic follicles. Partly collapsed and emptied sinuses appear as points and streaks of red. The presence of a peripheral red streak just beneath the capsule of the gland with red lines radiating toward the center of the gland is a very important point in the naked-eye diagnosis of these organs. Occasionally the capsular surface of a hemolymph gland is studded with small beaded elevations, giving it a raspberry appear

68 The Normal Histology of the Human Hemolymph Glands

ance; on section these are found to be small round masses of lymphoid tissue projecting into the peripheral sinus.

The size of the human hemolymph glands varies from that of a pinpoint to a large cherry or almond. The latter size is, however, uncommon, the usual size being that of a yellow mustard seed or pea. In the majority of cases their shape is round or oval, some are flattened, others elongated, while not infrequently glands of an almond shape are found, the larger end being somewhat recurved upon itself. They usually possess a distinct hilum into which vessels enter. Their consistency is somewhat softer than that of ordinary lymph glands depending upon the amount of blood in the sinuses. If these are large and dilated the capsule of the gland may be so stretched that it is easily ruptured and the gland pulp have the appearance and consistency of a fresh bloodclot; and these glands are undoubtedly many times mistaken for such.

Attached to the gland there is usually a relatively large plexus of vessels, the veins in particular being large and prominent. Occasionally these remain dilated and filled with blood, and under such conditions are of great aid in making the naked-eye diagnosis. No lymph vessels can be demonstrated in the case of those glands, even the largest, which contain blood-sinuses throughout, but in the glands of mixed type, partly hemolymph and partly lymphatic, lymph vessels can be made out.

The number of hemolymph glands in the human body is exceedingly difficult of estimation. Since the ultimate diagnosis depends upon the microscopical examination it would be necessary in making an exact estimation to examine every lymphatic gland in the body. Moreover, since many of the hemolymph glands are very small and lie deeply embedded in fat and connective tissue, it is necessary to remove all the tissues in the regions where the glands are found and make serial sections of the entire tissue. The difficulty of this is evident. The number of lymph glands visible to the naked eye in the retroperitoneal region varies from 200-500, and the prevertebral tissues contain also numerous nodes of lymphoid tissue too small to be seen on naked-eye inspection. In a number of cases the entire retroperitoneal tissue has been examined, both in the fresh state and microscopically, but the results are so much at variance that no definite statements regarding the number of hemol3^mph glands can be made. Ordinarily the relative proportion to lymphatic glands is 1-20 to 1-50, but this statement is based upon very incomplete observations. It is very probable that the number of hemolymph glands is much greater than that expressed by these ratios, since in one case of pernicious anemia over sixty of the glands were removed

Aldred Scott Warthin 69

from the cervical, thoracic and retroperitoneal regions alone ; in another case of pernicious anemia, over thirty from the retroperitoneal region alone ; and forty from the same region in a case of leukemia. Vincent's finding of hemolymph glands, about fifty in number, in the mesentery and gastro-colic omentum may also be remembered. In the human body many of these glands appear to be in a resting state, and are not easily distinguished from ordinary lymph glands, but in certain conditions, particularly blood diseases, they become enlarged and prominent. A new-formation of these glands in compensation for spleen or bone-marrow is also possible and undoubtedly takes place under certain pathological conditions. The use of formalin in fixing the tissues and rendering more prominent the blood-content of hemolymph glands may be mentioned again in this place as being of great service in the estimation of their number.

Microscopical Examinatiox.

Technique. — Alcohol fixation does not give the best results m the study of these glands except for the use of the tri-acid stain. It produces too much contraction of the reticulum and changes the red cells so that they do not stain well. Mercuric chloride, formalin and Zenker's fluid are the best fixing agents in the order named. With all of these there is less shrinkage, and the red cells are better preserved, staining well with eosin, etc., so that the course of the blood-sinuses is well outlined by the blood yet remaining in the meshes of the reticulum. Mercuric chloride fixation is especially recommended in the study of their finer structure, since all stains, including the tri-acid, give good results after this fixation, the celldivision figures and the red-blood cells are well preserved, and there is little shrinking. Hematoxylin and eosin, the tri-acid stain, etc., are used according to the object sought. Mallory's reticulum stain is essential for the study of the reticulum of the blood-sinuses, and is particularly valuable in tracing the course of these. Kresylechtviolett is also very valuable in the study of mast-cells, etc.

For the study of the cells of these glands it is also advisable to make cover-glass smears from the freshly-cut surface. This should be done as soon as possible after death. The smears are made in the usual manner, fixed by heat or alcohol and ether, and stained as desired. In this connection kresylechtviolett is also recommended as of value in the study of cellgranulation.

' Microscopical Appearances. — The lymph glands of the human body may be divided broadly into two groups: those containing only lymphsinuses, ordinary lymphatic glands, and those possessing blood-sinuses, hemolymph glands. Between these two groups intermediate forms exist — a gland may contain both blood and lymph-sinuses— the pres

70 The Normal Histology of the Human Hemolymph Glands

ence of a blood-sinus, however small, is sufficient warrant for the classification of a gland as a hemolymph gland.

The microscopical study of the lymphoid nodes containing bloodsinuses reveals a most striking variety of structure in so far as the relative size, number and arrangement of the blood-sinuses, lymphoid tissue, etc., are concerned. It is possible, however, to divide the different forms into two distinct types, to which I have given the names splenolymph and marrowlymph gland as indicating their structure and probable functions. Between these two there is every possible transition-form, just as there is also between the spleen, hemolymph glands and ordinary lymphatic glands. It is not by any means intended to replace the designation, hemolymph gland, by these names, as the latter should still be used as a collective term.

Splenolymph Glands.

The great majority of hemolymph glands correspond to this type. They are found chiefly along the abdominal aorta, vena cava, adrenal and renal vessels, in the neighborhood of the solar plexus, cervical region, occasionally in the omentum, mesentery, epiploica, mediastinal and thoracic prevertebral regions. These glands are usually round, but also frequently almond-shaped, varying in size from a pin-point to a large cherry. As a rule they possess a distinct hilum into which numerous vessels of large size enter. These are also found penetrating the capsule at many points. Very often the gland appears to be surrounded by a plexus of vessels, sometimes arterial, at other times venous. Their gross appearances correspond with those given above for hemolymph glands in general. Their resemblance to the spleen is sometimes so great that they may be mistaken for accessory spleens, aild undoubtedly many of the so-called accessory spleens belong to this type of hemolymph gland. This fact was dimly recognized by Haberer in his recent article "Lien Succenturiatus und Lien Accessorius" (Arch. f. Anat. u. Phys., March, 1901). Apparently unaware of the existence of hemolymph glands this observer concluded that many glands regarded as accessory spleens were in reality peculiar types of lymph glands representing intermediate forms between spleen and lymph glands. •

Capsule. — The splenolymph glands possess a capsule of connective tissue which may be very thick in proportion to the size of the organ or very thin and delicate. It contains a varying amount of unstriped muscle, and very little yellow elastic tissue. Adipose tissue surrounds the capsule. The latter is frequently pierced by many obliquely-pene

Aldred Scott Warthin 71

trating blood-vessels. Occasionally the blood-spaces and vessels in the capsule are so numerous as to give it the cavernous structure so frequently seen in the capsule of these glands in the steer; but on the vrhole the splenolymph glands of man are distinguished from those of the lower animals by the less vascular structure of the capsule. From the external capsule trabeculse of similar tissues run into the gland dividing it into irregular lobules. Accompanying the trabeculas are the communicating blood-sinuses and between them lies the lymphoid tissue.

Blood-sinuses. — Immediately beneath the external capsule there is a blood-sinus which sometimes extends entirely around the periphery of the gland, but more frequently only for portions of the way, being frequently interrupted by masses of lymphoid tissue which reach to the external capsule. In the great majority of cases this sinus is much smaller and less prominent than in the hemolymph glands of the steer and sheep. Glands are, however, occasionally seen in man with the peripheral sinus dilated and 'containing as much blood as in any gland from these animals. From the peripheral sinus branches pass in with the trabeculae toward the centre of the gland or toward its hilum, increasing in size and becoming confluent towards these points until they are often very large and prominent. These radiating sinuses frequently commiinicate with each other, and so divide the lymphoid tissue into irregularly-shaped islands apparently surrounded on all sides by a blood-sinus. Serial sections, however, show that these islands are not entirely cut off from each other in the majority of cases, but at some point or other are connected by an isthmus of lymphoid tissue of varying size. The number and size of the blood-sinuses as well as their general arrangement vary greatly, so that scarcely any two glands exactly resemble each other in these respects.

The lumen of the peripheral as well as of the communicating and central sinuses is traversed by a coarse reticulum through the meshes of which the blood circulates. The amount of this reticulum and the size of its meshes vary in different 'glands; frequently the central sinuses are open, possessing but a scanty reticulum or none at all. The reticulum of the sinuses is probably lined throughout with flattened endothelium, but it is sometimes difficult or impossible to make this out, so that the blood appears to be in direct contact with fibres of the reticulum. A wide sinus is often abruptly narrowed by a constriction of coarser reticulum suggesting a valve-like arrangement, but this point is yet to be worked out. The course of the sinuses is clearly shown in sections by the lighter staining nuclei of the reticulum in contrast

72 The Normal Histology of the Human Hemolymph Glands

to the more deeply-stained lymphoid tissue and the red cells lying in the reticular meshes.

The central sinuses frequently form the most striking feature of the splenolymph glands. Though usually partly or even wholly emptied of blood they often remain dilated. Scanty reticulum may extend either across or only for a short distance into the lumen of the larger sinuses. At other times the central sinuses may contain as much reticulum as either the peripheral or communicating ones. Red-blood cells and large phagocytes containing red cells and blood pigment are found in the reticular meshes along the sides of these sinuses. In some glands the central and communicating sinuses take up the greater part of the central portion of the gland, so that microscopical sections resemble a much-congested spleen-pulp.

Lymplwid Tissue. — The lymphoid tissue lying between the sinuses resembles very much that of an ordinary lymph gland. It varies very much in amount, sometimes forming a mere network between the sinuses, while in other cases it may form 'the chief part of the gland. Usually the greater mass of lymphoid tissue is toward the periphery, forming the inner border of the peripheral sinus, but it also frequently extends to the capsule interrupting the peripheral sinus. The arrangement of the lymphoid tissue also varies much in different glands. It is usually cut up into irregular areas or lobules which are for the greater part surrounded by the blood-sinuses. Small round collections of lymphoid cells are often seen, resembling splenic follicles. These occur more frequently at the periphery, where they may be partly or wholly surrounded by the blood-sinus, but they may also be found toward the central portion of the gland. Serial sections show that they are almost perfectly round. In the majority of cases they possess no arterial relations as in the case of the splenic follicles, but occasionally a small capillary is found in them which under certain pathological conditions may become gradually converted into a small arteriole with thick walls. The resemblance in this case to the splenic follicle is complete.

The cells of the lymphoid tissue are for the greater part small lymphocytes. These vary greatly with respect to the relative size and staining power of the nucleus and relative amount of protoplasm. Next to the small lymphocyte the large mononuclear cell is the most common form present. These also va^y much in size, form and staining power. Transitional and polymorphonuclear leukocytes are also present. A small number of basophile and mononuclear eosinophiles is usually present, mast-cells are rare in the majority of cases, but occasionally a

Aldred Scott Warthin 73

gland is found, as in the steer and sheep, in which the majority of the cells of the central portion of the gland are mast-cells.

Eed-blood cell^^ lie free in the meshes of the reticulum. The small blood-vessels and capillaries of the lymphoid tissue are usually filled with red cells and leukocytes, the large mononuclear form appearing to predominate. Throughout the reticulum there is usually present a varying amount of blood pigment, partly free and partly contained within large mononuclear phagocytes. Eed cells in various stages of disintegration are also found in these cells. Scattered areas of a hyaline substance which stain pink with eosin, red with fuchsin, and blue with Mallory's reticulum stain are often found throughout the lymphoid tissue, but are most numerous toward the periphery of the gland. Small hyaline, highly refractile spherules of varying size, usually about the diameter of a red-blood cell, are frequently seen in small groups lying free in the reticular meshes and are also found in the mononuclear phagocytes. They stain intensely with eosin and fuchsin, retaining the latter with Mallory's reticulum stain. These hyaline bodies are evidently the products of the destruction of red-blood cells, as all stages of their formation can be found. They may contain iron, especially those found in the phagocytes, the reaction being absent from many of the free bodies. In some cases these spherules can be seen partly extruded from the phagocyte.

Beticulum. — The reticulum of the lymphoid areas resembles that of ordinary lymphatic glands. That of the blood-sinuses is much more abundant and of a coarser mesh-like structure than that of the lymphsinuses of lymph glands. It differs from the reticulum of the spleen-pulp in the same respects. It stains blue with Mallory's reticulum stain, and consists of branching fibres and stellate or spindleshaped cells arranged in a coarse mesh-work, the surfaces of which are covered with flattened endothelial cells. Small fibrillge of yellow elastic tissue and occasional unstriped muscle-cells may be scattered through it. In its meshes there are found constantly large mononuclear phagocytes containing disintegrating red cells. These cells are always more abundant in the reticulum of the sinuses than in the lymphoid tissue. Under certain conditions they are so greatly increased in number as to almost entirely fill the spaces of the sinus. Normally their number i^ individual glands varies greatly, suggesting a possible cyclical function of hemolysis. Glands containing many of these cells may be found side by side with others whose reticular spaces contain but few. The same appearances are found in the hemolymph glands of the lower animals, particularly in those of the dog -and rat, Multinuclear giant

74 The Normal Histology of the Human Hemolymph Glands

cells, eosinophile, basophile and mast-cells may at times be found also in the reticular meshes. The origin of the phagocytes has not yet been definitely determined; they may arise either from leukocytes or from the endothelial cells lining the reticulum.

Vascular System. — The exact manner of circulation in these glands has not yet been worked out, but it seems probable that the arteries entering the hilum quickly divide into small branches which, passing toward the periphery, empty into the blood-sinuses from which the blood is again gathered into veins which pass out at the hilum or obliquely through the capsule. As in the lower animals, there are great individual differences in the number and mode of branching of the blood-vessels. Occasionally the entering arteries pass along the trabecule and do not divide until near the periphery. In well-stained sections the course of the blood-sinuses and vessels is well shown by the blood contained in them which serves the purpose of an injection. The exact manner of communication between arterial and venous systems cannot, however, be made out by this means. Injections have not yet been attempted in the human subject, but in the lower animals they have been unsatisfactory. The circulation in the sinuses is of the type known as sinusoidal, only the endothelium separating the blood from the cells contained in the reticular meshes. The current in these spaces must be extremely slow and a long period of time must be required for the complete circulation through the intricate meshes of the reticulum crossing the sinuses.

In the splenolymph glands containing blood-sinuses throughout, afferent and efferent lymph vessels are not found, small lymphatics alone being present in the capsule. In glands of mixed type containing lymph-sinuses lymphatic vessels are also found. Whether there is any communication between the lymphatic and blood-systems in these glands remains yet to be shown. In the round masses of cells lymphspaces or capillaries are probably present as in the splenic follicles, since under certain pathological conditions these may become edematous, the cells being separated by an accumulation of fluid in the intercellular spaces. These lymph spaces probably empty directly into the blood-sinuses and convey leukocytes into the circulation.

Varieties. — As already mentioned transition forms exist between the splenolymph glands and the spleen on one hand and ordinary lymphatic glands on the other. The significance of these intermediate forms is not yet apparent.

Development. — No work has yet been done on the development of these glands. They are present as fully-developed organs in the new

Aldred Scott Warthin 75

born child, and have been found at an early period in the foetal calf. They are without doubt individual organs whose early stages of development are probably parallel with those of the lymph glands. Under certain pathological conditions it is possible that they may be developed from ordinary lymphatic glands or even in adipose tissue in compensation for the spleen or bone-marrow.

Function. — A discussion of the probable functions of these glands is beyond the limits of this paper. Briefly stated, it is probable that their function is chiefly one of hematolysis. The great variation in appearances in different glands suggests a cyclical function. They are also leukocyte-forming organs. Under normal conditions no evidences of red-blood cell formation have been discovered in them. It may also be possible that in the glands containing both blood and lymphsinuses there is communication between the two systems and these glands may serve as the means of the return of lymph into the bloodstream. This supposition must, however, be regarded as purely hypothetical.

Differential Diagnosis. — To the inexperienced observer the hemolymph glands may at first be taken for deeply-congested or hemorrhagic lymph glands, even on microscopical examination. This mistake has probably occurred many times. The essential features of these glands as given above are, however, easily seen and, when once known, a glance at a section is sufficient for their recognition. The differences in reticulum, lymphoid tissue, sinuses, etc., together present a picture entirely distinct and characteristic from that of a congested or hemorrhagic lymph gland. Care in the treatment of the autopsy material, perfect fixation and good staining are points of technique which are of great service in the recognition of these organs.


The second form of hemolymph gland to which I have applied the designation of marrowlymph gland is of very much less frequent occurrence. They are much more prominent in certain pathological conditions than they are in the normal body, suggesting the possibility of resting glands or of new formations. In many cadavers I have been unable to find them, but this may have been due to a lack of time for a thorough examination. They have been found only in the retroperitoneal region, along the spine and brim of pelvis, always in close proximity to the large vessels, vena cava, abdominal aorta, adrenal and renal vessels and common iliacs. They are present most frequently

76 The Normal Histology of the Human Hemolymph Glands

along the vertebrae behind the aorta or between it and the vena cava. These glands are flattened and long in proportion to their breadth, their greatest dimension lying parallel to the axis of the neighboring vessel. They may have a distinct hilum, bnt the number of vessels entering is not so great as in the case of the splenolymph glands. Lymph vessels may also be found in connection with these glands. The latter vary greatly in size, but are sometimes found as slender cylinders several centimeters long embedded in adipose tissue. They are white or pinkish in color with fine red lines corresponding to the blood-sinuses. Their consistence is very soft and on section they present an almost homogeneous surface.

They possess a thin capsule which contains but little luistriped muscle and yellow elastic tissue. Delicate trabeculge run from this toward the centre of the gland. Beneath the capsule there is a bloodsinus of small size which usually runs entirely around the periphery and from this there are narrow branching sinuses accompanying the trabeculge toward the centre of the gland. All of the sinuses are filled with a coarse reticulum through the meshes of which the blood circulates. Dilated sinuses like those of the splenolymph glands are not present. The course of the sinuses is shown by the lighter staining nuclei of the reticulum and by the presence of red-cells. Lymph sinuses are also present in some of these glands. Between the sinuses lies the lymphoid tissue arranged in irregular islands or lobules and is in much greater amount than in the splenolymph glands. Collections of cells resembling follicles are not found in the pure type of this gland. Throughout their central portions large numbers of fat cells are usually present.

The reticulum of the lymphoid tissue is more delicate and contains but little elastic tissue. The lymphoid areas are richer in cells and these present a much greater variety than in the splenolymph glands. Mononuclear eosinophiles and mast-cells are more numerous, and multinuclear as well as large mononuclear forms with deeply-stained knobbed nuclei also occur. Giant-cells resembling those of the bone-marrow are also occasionally found, and in certain pathological states of the blood may be very numerous. No nucleated red cells have been found in normal conditions. The lymphocytes and large mononuclears show a much greater diversity of form and staining power, and there is also a great variety of cells in the reticulum of the sinuses. Phagocytes containing red-cells pigment, fuchsinophile hyaline bodies and leukocytes occur to a much less extent than in the splenolymph glands. Eed

Aldred Scott Warthin 77

blood cells are found scattered throughout the reticulum of the lymphoid tissue.

Transition-form? between spleno- and marrowlymph glands are found and also between the latter and ordinary lymphatic glands. The function of the marrowlymph glands is not clear. They are evidently leukocyte-forming organs, and the presence of giant-cells and many mononuclear eosinophiles suggests the bone-marrow and the formation of red-cells. In certain forms of anemia and leukemia these glands are greatly enlarged and come to resemble the lymphoid marrow, containing nucleated red cells, neutrophile and eosinophile mononuclears, and many giant-cells exactly similar to those of the marrow. It can hardly be doubted that in these cases these glands are centres of red-cell formation perhaps compensatory for the marrow. Under normal conditions they may be in a resting state in so far as their function is concerned. Their part in hemolysis is much less marked than that of the splenolymph glands. In old age they become atrophic, their sinuses obliterated, large deposits of hyalin occur in them, and finally they cannot be distinguished from atrophic lymphatic glands. The study of these glands from the pathological side promises much more than from the normal.


Our conceptions of lymphoid tissue are greatly broadened by the study of the hemolymph glands. The intimate relations existing between the different organs of this class, the transition-forms, the possibility of compensation o£ function, etc., throw new and important light upon both histological and pathological problems. The various lymphoid organs might be compared as follows:


Hemal gland.

Blood-forming function.

Sinusoidal organ.

Afferent and efferent blood-vessels.

No afferent lymph-vessels.


H«molymph gland.

Hemolytic function.

Sinusoidal organ.

Afferent and efferent blood-vessels.

No afferent lymph-vessels.

Efferent lymph-capillaries.

78 The Normal Histology of the Human Hemolymph Glands


Hemolymph gland.

Hemolytic function.

Sinusoidal organ.

Afferent and efferent blood-vessels.

No afferent lymph-vessels.

Efferent lymph-capillaries.


Lymph gland.

Lymphatic function.


No blood-sinuses.

Lymph and blood-systems separate.

With respect to the relations between blood and lymphatic systems the red marrow might be considered the most primitive type of lymphoid structure, and the ordinary lymphatic gland the most highly developed, the hemolj'-mph glands and spleen occupying intermediate positions. If viewed from a broader standpoint regarding both general structure and functions the relations of the organs might be theoretically represented in the following manner:

Lymphatic Gland.

/ \

Splenolvmpli Gland Marrowlymph Gland


Spleen. Lymphoid Marrow.

In conclusion, the field of the human hemolymph glands has barely been entered and its most important problems remain unsolved. Much is .to be hoped from experimental work upon the lower animals in whom these glands are larger and more numerous than in man. Work along this line has already been begun in this laboratory.

Note. — The limits of this article have prevented me including in the brief review of the literature given above references to observations made on lymph-glands with 'blood-containing sinuses by a number of writers who apparently unaware of the existence of hemolymph glands interpreted their findings as hyperemic or hemorrhagic lymph-glands. Eindfleisch, Weigert, Neumann and Orth are among those who have made such observations. Especial attention is, however called to the article by Saltykow (Ueber bluthaltige Lymphdriisen beim Menschen, Zeitschr. f. Heilkunde, 1900). In an examination of 60 cadavers this

AldreS Scott Warthin 79

observer found blood-containing lymph-glands in 91.66^ of the cases. Saltykow considered these glands to be only ordinary lymphatic glands in which there had been hemorrhage or a backward flow of blood into the lymph-vessels. Eeferring to the hemolymph glands as described by GibbeS;, Robertson and Clarkson he concludes that these writers were in error in their assumption that these were glands sui generis. I have already pointed out the close resemblance between hemorrhagic lymph glands and hemolymph glands and some of the more important differential points between these structures. I regard Saltykow as being wholly in error in so far as his conclusions regarding the nature of these glands are concerned.


Clakkson. — British Med. Jour., July 25, 1891.

Text-book of Histology, 1896.

Drummond. — Jour, of Anat. and Phys., 1900. GiBBES. — Microscopical Journal, Vol. XXIV, 1884.

Amer. Jour, of Med. Sciences, 1893.

Habeker. — Arch. f. Anat. u. Phys., March, 1901.

Leydig. — Lehrbuch der Histologie d. Menschen u. d. Thiere, 1857. Robertson. — Lancet, 1890.

VmcENT AjfD Harrison. — Jour, of Anat. and Phys., 1897. Warthin. — Jour, of the Bost. Soc. of Med. Sciences, April, 1901.

Jour, of Med. Eesearch, July, 1901.

Weidenreich. — Arch. f. Mikr. Anatomic, July, 1901,


CHARLES SEDGWICK MINOT, LL. D. From the Embryological Laboratory of the Harvard Medical School.

With 14 Text Figures.

The term "pineal region'" is used here in a descriptively topographical sense, the observations reported concern the epiphysis, paraphysis, vehim transversum, and the superior and posterior commissures chiefly in dog-fish embryos of from 11.5 mm. to 86.0 mm. The sections studied form part of the Harvard Embryological Collection, the general plan of which I have briefly described in an earlier article.' To facilitate confirmation of the observations the number of the embryo and of the section, as catalogued, is given for each of the illustrations.

Our previous knowledge of the pineal region in Elasmobranchs is based on the observations of Balfour {Worls, Vol. I, p. 399 ff.) and Ehlers, 78.1, on the development, and of Ehlers and Cattie, 81.1, on the adult anatom3^ Cattie reviews the earlier literature quite thoroughly. Gaupp's resume, 98.3, in the " Ergehiisse " is invaluable for the comparative study of the parts. The account of the development of the epiphysis proper, given by Balfour and Ehlers is essentially correct. They both saw the velum transversum, but did not specially study it. It has been more accurately figured by W. His, 92.1, p. 361, Fig. 14. Balfour figures only the superior commissure, which he identified as the posterior, and appears not to have observed the true posterior commissure at all. Ehlers, 78.1, has indicated both commissures in his Fig. 8, very clearly, but gives no description of their development in his text. Neither Ehlers nor Balfour mention the paraphysis, which was not recognized as a morphological constituent of the brain until much later (Selenka, 90.1), but Balfour has recorded several of the changes in the paraphysal region. Balfour's descriptions of the pineal region are incomplete, and owing to the defective methods of the time, often inexact as to details.

In embryos of Squalus acanthias of 11.5 mm. in length " the changes

1 Anatomischer Anzeiger, Bd. XVIII, pp. 138-139.

'^ The measurements of lengths are from the preserved specimens in alcohol.


Ou the Morphology of the Pineal Kegion

in the roof of the diencephalon are just beginning. Fig. 1 represents a sagittal section, the plane of which is not true since it passes through the epiphysal anlage about in the median plane, but in the region of the hind brain passes considerably to one side so that it strikes the jugular vein, Ve, the first aortic arch, Ao, and the three " head cavities," the praemandibular, 1, the mandibular, 2, and the hyoid, 3. Miss Piatt's " anterior cavity," 91.2, 201, is also shown, and is connected in sections 88 and 89 of this series with the praemandibular cavity. In our transverse series, No. 206, section 120, the two cavities are just separated. It seems, therefore, probable that the " anterior cavity " arises

Fig. 1. Embryo of 11.5 mm. Sagittal series, No. 208, section 93. (For explanation of the lettering see at end of the article.)

X 30 diams.

from the praemandibular as van Wighe found to be the case in Galeus. It may be noted that the tip of the notochord joins the wall of the praemandibular cavities, which at this stage are still united across the median line.* The fore-brain is already subdivided into the wider anterior prosencephalon and the narrower posterior diencephalon. The roof of the latter has two well-marked arches, of which the posterior, Ep., is the anlage of the epiphysis. In connection with these arches appear on the inside of the brain wall in the section three points; of

3 The condition of the head-cavities is described as it offers a convenient means of fixing the stage at which the dififerentiation of the pineal region begins.

Charles Sedgwick Minot 83

thesC;, the first, V, is the anlage of the velum; the second, which marks the boundary between the two arches, is the site for the future commissura superior; the third, which forms the posterior boundary of the epiphysal anlage is the site for the future commissura posterior — compare Fig. 5. The curve in front of the velar anlage I propose to name the paraphysal arch, on account of its subsequent differentiation; the curve between the velum and the epiphysal anlage may be termed the post-velar arch. We thus distinguish six fundamental morphological divisions in the median line of the diencephalic roof :

1. Paraphysal arch.

3. Velum transversum.

3. Post-velar arch.

4. Superior commissure.

5. Epiphysis.

6. Posterior commissure.


Fig. 3. Embryo of 11.5 mm. Sagittal series, No. 308, section 93. x 30 diams.

The homologues of all these parts exist probably in all vertebrates.

In the next stage, which I have, an embryo of 13.0 mm.. Fig. 2, the brain is larger, and the outline of the fore-brain has been considerably modified, chiefly owing to the growth of the infundibular region. The " anterior " cavities in this embryo (No. 224, sections 99-100) seem to have no longer any connection with the praemandibular cavities, but lie further laterad. The relations are well demonstrated by transverse sections — thus No. 223, section 99, shows both the " anterior cavity and the median connection of the praemandibular cavities. In both


On the Morphology of the Pineal Eegion

series the anterior end of the notochord is bent ventralwards at a sharp angle, Fig. 2, nch; it tapers off gradually, and has a rounded termination, which lies close against the wall of the praemandibular cavity. The roof of the diencephalon has advanced slightly as compared with figure 1. The velum, V, projects further into the cerebral cavity and appears more clearly as a fold of the brain wall; in another embryo of the same length, No. 235, section 63, the projection is more marked. The epiphysal anlage has begun to deepen, Ep., and immediately behind it the brain wall shows a thickening where the posterior commissure is to appear.

As shown in the figure the narrower diencephalon is marked externally by a depression between the mesencephalon, M. B. and prosencephalon, F. B. This depression can still be easily traced in embryos of 40-45 mm, but gradually disappears.

Fig. 3. Embryo of 1.5 mm. Sagittal series, No. 238, section 51. x 30 diams. Fig. 4. Embryo of 18 mm. Sagittal series, No. 204, section 100. x 30 diams.

In embryos of 15 mm.. Fig. 3, the epiphysal evagination, Ep., has grown, as has also the velum, Y, which now has distinctly an anterior and posterior surface; the ependymal coverings of the two surfaces lie close together so that there remains only a minimal space between the two ependymal layers. In this specimen the brain-wall is thicker in the pineal region than in both the younger and the older stages, which I have examined. Similarly the walls of the epiphysal anlage. Fig. 5, Ep., are thicker than in both the older and younger stages of Figs. 4 and 6. I think these variations are not important morphologically.

The next. Fig. 4, is from an embryo of 18 mm, and shows a decided advance of the differentiation. The velum, Y, is now a well-marked fold of the ependyma, with a narrow middle layer of mesenchyma; the fold extends across the fore-brain from side to side, and according to the current interpretation is regarded as marking the boundary between the diencephalon and the prosencephalon. The epiphysal anlage is more distinctly an evagination than before. Immediately in front of it has appeared the small but well-marked superior commissure, s. c,

Charles Sedgwick Minot


and immediately behind it has appeared the much larger posterior commissure, p. c, the position of which — compare also Figs. 5-10 — indicates that it belongs rather to the mid-brain than to the fore-brain. Opinion on this point must be reserved however until determined by an investigation that will fix the boundary between the two primary cerebral vesicles. Our present information seems to me insufficient to decide with finality this point. Both commissures are developed in the " edoglia." This term I propose as the equivalent of the " Randschleier " of His, to designate the outermost of the three primary layers of the medullary wall.^ In stained sections the commissures are con-' spicuous owing to the absence of nuclei, in contrast with the adjacent

Fig 5. Embryo of 23 mm. Sagittal series, 231, section 79. x 30 diams.

Fig. 6. Embryo of 28.0 mm. Sagittal series, 233, section 122. x 30 diams. A, approximate plane of the section represented in Fig. 11 ; B, approximate plane of the section represented in Fig 12.

tissue of the brain-wall, which is densely crowded with nuclei. The cross sections of the transverse nerve fibers are sharply marked in the sections.

Embryos of 22 mm. — 4 mm. longer than the last — show a decided growth of all the parts, and the growth is downward, i. e. towards the interior of the brain. The ectoderm, Ec, is still a thin layer, and the mesoderm between it and the brain-wall has increased a little in thick

Upon the fundamental morphological importance of these three layers I have insisted in my Human Embryology, p. 616. The entire structure of the adult brain and cord should be stated in terms of these three layers.

86 On the Morphology of the Pineal Eegion

ness; accordingly the outer surface of the mid-brain and of the forebrain, F. B., also the summit of the epiphysis, Ejp., are all nearly as close to the outer surface of the head as before, while on the contrary the orifice of the epiphysis, the posterior commissure and the superior and the edge of the velum are all much more remote from the surface than before. Comparison of the successive stages shown in Figs. 4-10 will suffice to demonstrate that increasing downward extension is the method of development by which all these parts elongate. This comparison will also demonstrate that the summit of the epiphysis remains quite close to the ectoderm, while the continued development of the mesoderm is increasing the distance between the epidermis and the mid-brain and fore-brain both, so that the epiphysis comes to project more and more above the level of the brain. Two further characteristic features are illustrated by Fig. 5, first the small growth of the post-velar arch, so that the distance between the base of the velum and the orifice of the epiphysis is barely more than sufficient for the superior commissure. It is probable that the small size of the post-velar arch is a special characteristic of the elasmobranch type. In other vertebrates the post-velar arch has considerable extension in the sagittal direction, as in Accipenser according to Kupffer, and in ISFecturus, Fig. 13, and Gallus, Fig. 14. Second, the ependyma on the posterior side of the velum has grown thinner, except that towards the inferior edge of the velum it thickens and is then reflected over the edge onto the anterior surface. Finally it must be noted that up to this stage the velum has elongated more rapidly than the epiphysis, after this stage on the contrary the epiphysis elongates much more than the velum.

In embryos of 28.0 mm. the permanent relations are already clearly indicated. The most important advance has been the thickening of the wall of the fore-brain, except as seen in the sagittal sections towards the velum, where the wall corresponding to the area of the paraphysal arch remains thin. The differentiation of this arch therefore occurs quite late, and may perhaps be best described as resulting from an arrest of the histological development, which just in front of the arch progresses rapidly, there causing the brain wall to thicken, and to change into nervous tissue proper. Owing to the continued downgrowth of the parts a deep fold or cleft is formed between the midbrain, M. B., and fore-brain, F. B. In and near the median plane the space of the cleft is almost filled by the epiphysis, as shown in the figure — the wall of the epiphysis being almost in contact with the wall of the mesencephalon behind, and with the paraphysal arch in front. Laterally the cleft between the two vesicles is filled only with mesen

Charles Sedgwick Minot


cliyma, as can be seen iu Fig. 11. Comparison with Fig. 5 shows that the velum has elongated, but comparatively much less than the epiphysis. Both commissures have grown, causing each a local thickening of the brain-wall. Our notions of the structure may be completed by the examination of cross sections, compare below Figs. 11 and 12.

In embryos of 34.0 mm. we find that there has been an obvious growth of all the parts, and in addition we note: — 1, that the paraphysal arch is more accentuated, and has changed in form so that it has now be


Fig. 7. Embrj'o of 34.0 mm. Sagittal series, 363, section 173. Fig. 8. Embryo of 40.5 mm. Sagittal series, 370, section 169.

gun to bend upwards, so as to reach above the level of the fore-brain in front of it; 2, the ependyma on the anterior side of the velum has thinned out, so that it is about the same as the ependyma upon the posterior side; 3, the epiphysis, Ep., is clearly differentiated into a terminal enlargement and a narrower stalk.

In an embryo of 40 mm., Fig. 8, we may point out the general growth, which has continued in all the parts, and besides must direct attention to the following special points: — the marked thickening of the nervous portion of the fore-brain wall, the upward protuberance of the paraphysal arch; the decided curvature of the velum, and its


On the Morphology of the Pineal Kegion

peculiar insertion, which causes it to appear almost (in these sections) like an appendage of the epiphysal stalk; the growth of the post-velar arch between the base of the velum and the superior commissure, s. c; and finally the well-marked thickening of the mesencephalic wall.

I have examined also sagittal sections of the pineal region of embryos of 50 and 60 mm., but, deeming it superfluous to present pictures

Fig. 9. Embryo of 70 ram. Sagittal series, 431, section 293. x 30 diams.

of them, will pass at once to Fig. 9, taken from an embryo of 70 mm. The nervous tissue proper of the mid-brain and fore-brain has grown very much so that these parts now form a striking contrast owing to their increased thickness with the ependymal covering of the pineal region, that is to say of the paraphysal arch, P, and velum, V, and the epithelium of the epiphysis, which, however, is considerably thicker than the velar and paraphysal epithelium. Around the superior com

Charles Sedgwick Minot 89

missure, s. c, there is also a considerable accumulation of differentiated nervous tissue, indicated in the figure by shading with lines. This material does not extend across from side to side but is interrupted by a deep and rather narrow cleft in the morphological median plane — see section 299 — as can be readily observed in transverse and frontal sections of this and earlier stages. The section figured passes through the mouth of the epiphysis but does not pass through the cleft mentioned, hence the illustration gives a somewhat false impression. The thickenings in question are continuous with the wall of the fore-brain; they were termed tuhercnia intermedia by Gottsche, 35.1, walls of the thalamencephalon by F. M. Balfour, and by recent writers are often referred to as the ganglia liahenulo'. I have not pursued the study of these structures further. I will only remark in passing that the term ganglion hahenuJce does not appear to correspond with any morphological conception sufficiently clear to be valuable in comparative anatomy. During the earlier stages the mesodern between the epidermis, Ec, and the brain has been steadily growing and has now reached considerable proportions, but the summit of the epiphysis, Ep., still lies relatively near the outer skin and the organ consequently projects far above the brain; it has begun to curve forward preparatory to its elongation rostrad. As regards the superior commissure, s. c, it is noteworthy that it is now nearly as large as the posterior. As we ascend the vertebrate series the posterior commissure increases in size and importance, but the superior commissure is persistent occurring even in mammals. The velum, V, has distinctly the character of a choroid plexus, being rich in blood vessels, and bearing irregular villous outgrowths, the beginnings of which can be seen in Fig. 8. The velar villi are much more developed laterally and their formation is spreading forward around the sides of the paraphysal arch, a fact wliich I regard as of fundamental importance for our final morphological interpretation.

The last stage I have been able to investigate is an embryo Acanthias of 86.0 mm. which is in a somewhat imperfect state of preservation, the epidermis being in part lost, entirely so in the part drawn, although indicated in the figure; the brain had shrunk a little; and as the ependyma had withdrawn from the mesenchyma on both sides of the velum that structure appears abnormally thick, but there seem to be no important distortions. The anlage of the skeleton, sic, is now clearly defined in the mesenchyma on the same level as the upper vesicular end of the epiphysis, Ep.; the stalk of the same is bent and has begun to elongate forwards above the brain thus making a distinct approach towards the adult condition described by Ehlers and Cattie. The para


On the Morphology of the Pineal Kegion

physal arch forms a distinct evagination, P, with thin walls and its top is irregular as if the walls had formed the anlages of new outgrowths. I am thus led to believe that the arch has not at this stage yet completed its differentiation, but on the contrary is about to form the true paraphysis, or paraphysal gland as a local development of the arch proper. My efforts to obtain more advanced stages having failed I must leave the decision to future observations. My belief, or better said — supposition, is confirmed by the fact that in amphibians and birds the

Fig. 10. Embryo of 86 mm. Sagittal series, 436, section 293. x 30 diams.

})araphysal gland is an appendix of the arch — see below. The velum, V, has now distinctively the character of a choroid plexus being very irregular in the form of its surface, rich in blood vessels, covered by a thin ependyma and projecting far into the cavity of the brain. Laterally the projections from its surface are much more developed, and as the organ has grown forward alongside the median paraphysal arch, it has produced what we can now easily identify as the plexus of the lateral ventricles. These plexuses are therefore to be interpreted morphologically as secondary modifications or appendages of the primary

Charles Sedgwick Miuot 91

velum transversum — compare below, Fig. 12. The superior commissure, s. c, is now in the area of its cross section fully equal to the posterior commissure, p. c, and must be regarded as of great morphological importance. The nervous portion of the wall of the fore-brain shown in the figure in front of the paraphysal arch is very thick.

The ependymal covering of the velum early develops a superficial coat, which appears lightly colored in stained sections. In embryos of 22 mm. (stage of Fig. 5) this border is easily seen on the anterior surface of the velum, but not on the posterior surface. In embryos of 28 mm. the border is conspicuous, but is thinnest at the base of the velum, and gradually thickens toward the inferior edge. The nature of the border is uncertain; the appearance is probably not due to cilia, but suggests rather the formation of secretory spherules, such as I have recently discovered in the cervical glands of the human uterus, the Wolffian body of the pig, and the kidney of the frog. These spherules are formed from the free ends of the cells, and resemble those which, as Mingazzini has shown, are formed on the inner ends of the absorbing epithelial cells of the mammalian intestine. I wish, however, not so much to offer a definite interpretation of the border, as to suggest a line of investigation.

In order to render clearer the relations of the velum Figs. 11 and 12 have been added. These are both from a series of transverse sections of an embryo of 28 mm., or of the same length as the embryo from which Fig. 6 is taken. The plane of Fig. 11 is approximately that of the dotted line A in Fig. 6. It passes, therefore, through the mid-brain, M. B., and fore-brain, F. B. Next the former is the oval section of the stalk of the epiphysis, Ep., crowded into the space between the two commissures. The posterior commissure, p. c, apparently belongs wholly to the mid-brain, as can be seen in the sagittal sections also. The development in Kecturus, in the chick, and in the rabbit and pig also indicates that the posterior commissure belongs to the mesencephalon. When it is further remembered that its fibres in man are of mesencephalic origin, we must conclude that the traditional description of the commissure as situated in the roof of the third ventricle is erroneous, since the commissure does not belong to the diencephalon. Keturning to Fig. 11, the superior commissure, s. c, runs in the cerebral tissue of the post-velar arch — compare Fig. 6 — between it and the velum proper, V, appears the narrow space of that arch; laterally this space curves towards the mid-brain. The velum, V, stretches from side to side of the brain, it consists of a thin central layer of mesench-\Tna continuous laterallv with the mesenchyma surrounding the


On the Morphology of the Pineal Kegion

brain, and of two layers of ependyma, of which the posterior is the thinner.

The plane of Fig. 13 is approximately that of the dotted line B in Fig. 6. The velum, V, belongs to the fore-brain, dividing the cavity thereof into a large anterior (in Fig. 13 lower) chamber and the much smaller chamber of the post-velar arch, which is extended far out laterally, the lateral cavities being slit-like; the lateral post-velar slits are bounded in front (below) by a thin ependyma, and posteriorly (above) by the thick brain wall of the tuhercula intermedia in which one readily


Fig. 11. Embryo of 28.0 mm. Transverse series, 232, section 130. The plane of the section is indicated by the dotted line A in Fig. 6. x 30 diams.

Fig. 12. Embryo of 28.0 mm. Transverse series, 232, section 163. The plane of the section is indicated by the dotted line B in Fig. 6. x 30 diams.

distinguishes the lateral prolongations, s. c, of the superior commissure. As the tubercula belong to the fore-brain their junction with the midbrain, which is marked externally by the apex of a deep furrow, indicates the division line between the first and second primary cerebral vesicles. From such a section as Fig. 13 one might be easily led to interpret the velum not as the division between the diencephalon and prosencephalon, but between mid- and fore-brain, and further to interpret the tubercula as appendages of the mid-brain. Attention should be paid to the two lateral projections, L. cli., of the ependyma on the

Charles Sedgwick Minot


anterior surface of the velum, because these projections not only fix the lateral boundaries of the paraphysal arch but also are the anlages of the choroid plexus of the lateral ventricles. These anlages from this stage on rapidly increase both in size and in complication of form.

I wish now to add certain comparisons of the structures described with those of other vertebrates, particularly with a view of determining the homologies of the paraphysis, velum, and superior commissure. Since Selenka's original brief announcement, 90.1, of the recognition of the paraphysis as a distinct vertebrate organ, little has been added to our morphological conception of it. It is completely ignored by the standard German and English text-books, and Prenant in his Traite


Fig. 13. Necturus maculatus of J 8 mm. X 30 diams.

Sagittal series, No. 23, section 90.

d'Embryologie is the only author, known to me, who has attempted a systematic analysis of the scattered observations.

The paraphysis is a gland developed by a local evagination of the epithelium of the paraphysal arch, and so far as known never is differentiated as a sensory organ.

As I have found no paraphysis in Acanthias embryos up to 86 mm., we must have recourse to other types. I will mention first Necturus, Fig. 13, and the chick. Fig. 14, as on these two forms I have made original observations. In a Necturus of 12.0 mm. (Sagittal series 49, section 60) the stage of the pineal region corresponds more or less to that of acanthias shown in Fig. 3; the epiphysis is evaginated, and there is

94 On the Morphology of the Pineal Region

a well defined paraphysal arch in front of the velum and post-velar arch behind it. In a jSTecturus of 15.0 mm. (Sagittal series 79, section 84) the paraphysis is a narrow elongated evagination from the arch, and so appears again in embryos of 18 mm.. Fig. 13. In embryos of 21 mm. the wall of the paraphysis is irregular, and in a larva of 26.0 mm. the anlages of the paraphysal gland tubes are easily recognized (Sagittal series 377, section 12G).

In the chick the paraphysis is developed very late and at seven days is a small nearly hemispherical evagination. Fig. 14, Par., formed from the paraphysal arch, P, quite near the rudimentary velum, V. A day later the epithelium is thicker and irregular as if the glandular tubules were beginning to form.

In Petromyzon Burckhardt figures a small evagination, which is probably the paraphysis, but his figure does not show the limits of the paraphysal arch. In various forms it is known that the paraphysis arises as a small evagination, which appears just in front of the velum and rather late in development as a local outgrowth. See for example Kupffer's observations on Accipenser; the observations of de Graaf, Burckhardt, Eycleshymer and others on Amphibia, those of Dendy and Burckhardt on reptiles. In all these cases there is a comparatively wide paraphysal arch, and a small paraphysis. The two things have heretofore not been distinguished so that there is considerable confusion in the descriptions.

The existing observations render it probable that the paraphysis is a true gland, the main evagination serving as the duct, while the secondary tubules are the secretory portions. Certainly the type of organization is that of a gland and not of a sense organ, and whenever the adult paraphysis has been found and studied it has presented the same plan of structure. Its secretion must of course pass into the cavity of the brain, so that functionally it is comparable to the glandular epiphysis in birds, and the infundibular gland of all vertebrates. One may suppose that these three glands supply some substances, which are useful to the nervous system, and that they are somewhat comparable from the physiological standpoint to the ductless glands at least in respect to the fact that their secretion has no direct open channel of escape from the body.

The velum probably is characteristic of all vertebrates. In elasmobranchs the post-velar arch remains small, hence the velum seems to arise later very close to the mouth of the epiphysis. In ganoids the post-velar arch is well developed, hence the velum is inserted quite far in front of the epiphysis. As regards the teleosts the data for a satis

Charles Sedgwick Minot


factory interpretation seem to me lacking. In amphibia (as in necturus) there is at first a well-defined velum ; the mesenchyma within the velum increases greatly in amount and converts the velum into a broad structure, Fig. 13, V. I have been unable as yet to follow the growth of the velum in sufficient detail, but it is probable that, as the velum expands, the post-velar arch is incorporated in it and disappears as a separate region. The enlarged velum projects backward, extending

Fiq. H

Fig. 14. Chick embryo of about 7.0 days. Sagittal series, 3.54, section :204. x 30 diams.

even into the mid-brain, and meanwhile expands laterally around and in front of the paraphysal arch, until its wings meet across the median line, Fig. 13, pi. x, to form the anlage of the anterior plexus. The entire expanded velum gives rise to the choroid plexus (supra-plexus of American writers), which accordingly surrounds the paraphysis. In the adult the paraphysis has become a gland of complex structure, as has been so well described by Francotte, but by most writers has been conftised with choroid plexus. In Bana haleciiia I find the adult

96 On the Morphology of the Pineal Region

paraphysal gland very clearly distinguishable by, 1, the character of its epithelium, 2, its tubular structure and 3, its apparently sinusoidal circulation, from the choroid plexus proper, in the midst of which lies the orifice of the gland. In reptiles the velum has been identified and its morphological importance for the class emphasized by Burckhardt (Anat. Anzeiger, IX, 320). In birds, Fig. li, the velum, 7, is almost rudimentary in its median portion, though very broad, and it merges without recognizable boundary into the very broad post-velar arch, which can already be identified as the anlage of the tela choroidea. As regards mammals further investigation is necessary, both to determine the history of the velum and of the paraphysis, if that organ is present in the placental, as Selenka states it to be in marsupial, mammals. In both birds and mammals the lateral portions of velum, i. e. the choroid plexus of the lateral ventricles is highly developed. It thus appears that as we ascend the vertebrate series there is first a broadening of the velum, and an increase of its lateral development, then occurs a further reduction and flattening out of the velum, and a much greater growth of the lateral plexus.

The superior commissure is a remarkably constant structure in the vertebrate brain, and must, since it persists in all vertebrate types, be regarded as a fiber tract of fundamental morphological importance. H. F. Osborn, 84.1, 268, was the- first to demonstrate the homologies, wide occurrence and topographical relations of the tract, and to apply the name " supra commissura." So far as I am aware, it has not yet been recorded in birds, but a more thorough search will perhaps lead to its discovery in that class. It has been found in representative types of all other vertebrate classes, including mammals, for I have observed it in embryos of rabbits, pigs and cats. In all cases it develops later than the posterior commissure — in mammals much later, and is at first ' much smaller than the posterior commissure. It acquires a large size in acanthias and perhaps other fishes, it attains less size in amphibians and is proportionately smallest in mammals. Its position is very constant as it is always situated immediately in front of the orifice of the epiphysis and at the outer surface of the brain wall.

The posterior commissure belongs morphologically to the mid-brain, not to the fore-brain. For the present the epiphysis may be accepted as marking the posterior limit of the fore-brain.

The pineal region develops a series of structures, which, from their anatomical characteristics, appear to be directly concerned in the formation of the fluid in the cavities of the brain. We may assume that the choroid plexus supplies the main bulk of the fluid, but the gland-like

Charles Sedgwick Miiiot 97

"organizatiou of the epiphysis and of the parapliysis indicates that they supply by secretion special chemical substances to the encephalic fluid. It remains, therefore, for the physiologist to investigate these glands and to determine how far their action is indispensable to the brain, and for the pathologist to investigate the possible relations of diseases of these glands to cerebral disorders. The pathological study of these organs seems to me all the more important, because I regard as reasonable the anticipation that the paraphysal gland will be discovered in man.


Ao., aorta.

Chi., optic chiasma.

Ep., Epiphysis.

Ec, Ectoderm.

F. B., fore-brain.

H. B., hind-brain.

Ily-, hypophysis.

Inf. g., infundibular gland.

L. cli., lateral choroid.

M. B., mid-brain.

M., mouth.

ncJi., notochord.

Oc, oesophagus.

P., paraphysal arch.

Par., paraphysis.

p. c, posterior commissure.

Plx., choroid plexus.

sk., anlagc of skull.

s. c, superior commissure.

v., velum.

Ye., jugular vein.

1, praemandibular head-cavity.

2, mandibular cavity.

3, hyoid cavity.

98 On tlie Morphology of the Pineal Region


Cattik, J. T., 81.1. — Verglijkend-anatoniische en histologische Onderzo kingen van der Epiphysis cerebri der Plagiostomi, Ganoidei en

Teleostei. Transl. Arch. Biol. Ill, 101-194, Pis. IV-VI. Ehlers, 78.1. — Die Epiphyse am Gehirn der Plagiostomen. Zeitschr. wiss.

Zool., XXX, Snppl., 607-634, Taf. XXV-XXVI. Galpp, Ernst, 98.3. — Zirbel, Parietalauge und Parapliysis. Ergebn. Anat.

Entwick-ges. VII, 208-285. GoTTSCHE, C. M., 35.1. — Vergleichende Anatomie des Gehirns der Griiten fische. Miiller's Arch., p. 453. His, WiLHELM, 92.1. — Zur allgenieinen Morpholog-ie des Gehirns. His'

Arch., 1892, 346-383. His, WiLHELM, 92.2 — Die Entwiekelung der nienschlichen imd thierischen

Physognoniien. Arch. Anat. Physiol., Anat. Abth., 1892, 384-424. OsBORN, He3«ry P., 84.1. — Preliminary observations upon the brain of

Menopoma. Proc. Acad. Nat. Sci., Philadelphia, 1884, 262-274. PI.

VI. Platt, Julia B., 91.2.— A contribution to the morphology of the vertebrate

head, based on a study of Acanthias vulgaris, Journ. Morphol.,

V. 79-112, PI. VI.

Selenka. Emil, 90.1.— Das Stirnorgan des Wirbelthiere. Biol. bbl. X, 323-326.



Aissista7it Professor of Histology and Embryology^ Cornell University.

With 4 Plates.

The work of which this paper is the outcome, was undertaken in the winter of 1896. Necturus maculatus was the form first chosen, but it was soon abandoned for the salamander Desmognathus fusca, which from its abundance, availability at all seasons of the year, and the size and structure of its testis, seemed a more suitable object for the work in hand. A preliminary note on the divisions of the spermatocyte was published in 1899. In November, 1900, the drawings, photographs and the larger portion of the preparations covering what would have been the first part of this article were destroyed by the burning of the histological laboratory of Cornell University. The plates (published herewith) and the portion of manuscript discussing the divisions of the spermatocyte were saved, and it has seemed best to publish that portion without waiting to repeat the work of the first part, which would take several years.'

When this investigation waf- begun, the need for careful work on the spermatogenesis of Ampjiibia was quite apparent. Eesearch on the spermatogenesis of Amphibia had been largely confined to the European salamander, Salamandra maculosa, and to that form alone had a monographic treatment been accorded, in the papers of Flemming, 87, vom Rath, 93, and Meves, though his final paper did not appear until 1897. Since this work was undertaken, however, McGregor, 99, has published a monograph on the spermatogenesis of Amphiuma, and Eisen, 00, has brought out an extensive article on Batrachoseps, another American salamander.

The results of Flemming and vom Rath have been reviewed by Meves, 97, and need not be discussed here in any detail. Flemming's paper,

1 The writer wishes to acknowledge gratefully the help which has been given him with the manuscript and preparations by S. H. Gage, head professor of the department, and by M. Hempstead and C. F. Flocken, students in the laboratory.

100 The Spermatogenesis of Desmognathus Fusca

which appeared iu 1887, is classical, not only as a contribution to tlie knowledge of spermatogenesis, but of mitoses in general. In Salamandra Flemming recognized the period of multiplication of the spermatogonia, followed by the period of growth to form the spermatocyte, which he believed divided twice, forming medium-sized and then small cells. The latter he apparently thought divided again,^ there being thus three generations of cells resulting from the division of the si3ermatocytes. In these divisions were recognized and named two divergent types of mitosis, " homotypic " and " heterotypic " — terms and types widely known in spermatogenesis work. The divisions of the spermatogonia were found to be homotypic, those of the spermatocyte of the first order characteristically heterotypic, though homotypic divisions also occurred; the division of the spermatocyte of the second order was believed to be heterotypic and homotypic equally.

In the interval between the appearance of Flemming-^s paper, 87, and vom Eath's, 93, Weismann, 91, had published his prophecy that in the process of spermatogenesis and oogenesis a " reducing " division would be found to take place.

The results of Haecker, Henking, Eiickert and vom Eath appearing soon after, brought an apparent confirmation of the correctness of this prediction, in the forms investigated by them. Yom Eath approached the investigation of Amphibian spermatogenesis (Eana, Salamandra) fresh from his results in Gryllotalpa, in which he had described tetrad formation. To the three generations described by Flemming he added a fourth in which tetrads were formed and which were distributed in two more divisions, as he believed to be typically the case, making six generations in all. Vom Eath's investigation was evidently undertaken to see if tetrad formation did not also obtain in Airiphibia, and he was led to suspect the correctness of this view by certain descriptions and figures 'of Flemming in which tetrad formation was apparently shown, though regarded as abnormal.

Moves' published work on the spermatogenesis of Salamandra began in 1891, his main paper appeared in 1897, followed the next year by a paper on the transformation of the spermatid into the ripe spermatozoon.

2 The statements of Flemming on this point do not seem to leave his interpretation entirely clear. In his schematic table of the cell generations, only two generations are given as descendants of the spermatocyte, and the following words convey the same impression " Mehr Tochtergenerationen der grossen Zellen als zwei scheinen mir den vorlindlichen Zellengrossen, nicht vorzukommen" (p. 401). Nevertheless, to Meves, Flemming personally stated that he had believed there were three generations of cells.

B. F. Kingsbury 101

His results did not sustain those of vom Eatli; no tetrad formation was found to occur, and there were but three cell-generations instead of six. On the other hand, the divisions of the spermatocytes he found to be equation divisions, the first heterotypic in character, the second homotypic, not mixed as Flemming had thought; furthermore, the spermatocytes of the third generation did not divide as Flemming seems to have supposed.' His paper, 97, dealt with the entire spermatogenesis, the structure and divisions of the spermatogonia, as well as the growth and divisions (two) of the spermatocyte, involving a consideration of reduction. The papers of McGregor and Eisen, which will be considered in the body of this article, supported the results of Meves (as far as the purposes of this paper are concerned). These five papers include the more exhaustive investigations of Amphibian spermatogenesis. Minor papers contributing to our knowledge but not dealing with the question of reduction need not be considered here, as also the work of older writers of historical interest in the analysis of the structure of the Amphibian testis. Of the publications on Amphibian oogenesis, the only one that is of impoi-tance for the purposes of this article is the paper of Carnoy and Lebrun.


The form chosen for this investigation is one of the most abundant salamanders in this locality (Ithaca, New York, U. S. A.), and in eastern United States generally; it is likewise the most abimdant species of the genus. In 1866 the Desmognathidce were recognized by Cope as a distinct family, represented by a single genus, to which have since been added the genera Thorius, Typhlotriton, and finally, Leurognathus.

Desmognathus, in habits is semi-aquatic, living at the edge of swiftly running brooks, especially near or at springs. It chooses concealment under stones at the edge of the stream, or among the pebbles of coarse gravel, saturated with water; dry ground, and also deep water in the bed of the stream, it apparently avoids. If confined in an aquarium of still water, it will crawl up the sides so that its head and a portion of its body are out of water; if prevented from thus gaining access to the air, it speedily dies.

It is said to be nocturnal in its feeding habits. Wilder, 99, wandering forth in search of its food, which consists of small insects (beetles, etc.) and their larvae.

Of its breeding habits and development very little is known. The

3 See note 2, p. 100.

102 The Spermatogenesis of Desmognathus Fnsca

eggs are laid some time in the summer. Wilder has found eggs laid in captivity, June 1st; Sherwood, 95, on the other hand, has found eggs from July to October, so that there is probably considerable individual variation in the time of ovulation. Desmognathus eggs have been found at Ithaca during July and August. Eecorded observations are insufficient for a more exact determination of the time of ovulation. The development of the form is known only in its grossest outline, but the very interesting statement has been made that cleavage in Desmognathus is meroblastic. Wilder, 99, and this statement has also been guardedly made of a member of an allied family, Autodax luguhris, Eitter, 99. The eggs are laid under stones near the stream in a hollow in the mud and are connected together by albuminous cords, uniting them in a group. The female remains with the eggs, which lie in a mass at her side, according to my observations, not wrapped about her body as Wilder has stated to be the case.

Mating habits. — Nothing definite is known of the mating habits of Desmognathus. The investigations, especially of Zeller and Jordan, have made us well acquainted with these phenomena in the genus Salamandra and in the genera of the allied family of the Pleurodelidce, where they consist in the deposition of a spermatophore by the male, over which the female passes while her cloacal lips, coming in contact with the mass of spermatozoa, either actively grasp it, or to them the spermatozoa adhere and enter later by their own activity. Within the cloacal chamber the spermatozoa become ensconced in tubules constituting spermathecae, in which they remain until the time of ovulation. Fertilization in Salamandra (Triton and Diemyctylus) takes place in the spring, and is preceded by a mating. In Triton and Diemyctylus, at least, there is occasionally an autumnal mating, the entire winter intervening before ovulation.

As compared with what is known of the mating habits of these forms, our knowledge of the same phenomena in Desmognathus is scanty, indeed. I know of no published observation on this form which, from its retiring mode of life and (presumably) nocturnal habits, is difficult to observe. The probability is strong, however, that the same mode of mating occurs in this form, since in the male Desmognathus are present in a well developed condition the same three cloacal glands found in forms whose mating is known and which undoubtedly produce the secretion that constitutes the body of the spermatophore. Likewise, the spermatheca is present in the cloaca of the female, and is a much more highly specialized organ than in the Salamandridce and Pleurodelidce. In specimens of Desmognathus taken in the fall, winter, spring and summer, it was

B. F. Kingsbury 103

foimd to be quite full of spermatozoa. This, of course, affords no due to the time of mating or ovulation. Examination of the testis shows that the mass of spermatozoa leave it in the early fall, so that it contains but few ripe spermatozoa. But the ducts are well filled and in the spring the cloaca as well contains a mass of spermatozoa upon a jelly-like base, presumably a spermatophore. This suggests a spring mating with quite probably a fall one also.

Nomenclature and Spermatogenetic Cycle.

It is perhaps hardly necessary to define the meaning of the terms that will be used in this paper as applied to the generations of cells in spermatogenesis, so universally have La Valette St. George's, 76, names of (a) spermatogonia, (b) spermatocytes of the first order, (c) spermatocytes of the second order, (d) spermatids and (e) spermatozoa, been used in all recent work; and so well recognized are the corresponding periods which they characterize— (a) a 'period of multiplication, (b) a period of growth or maturation, (c) a reduction period, sometimes spoken of as the maturation period, and (d) the period of transformation, sometimes also called the maturation period. Meves has further divided the spermatogonia into large and small spermatogonia, a division which, as a matter of convenience, seems to be helpful, though McGregor's terms of primary and secondary spermatogonia are preferred by the writer.

In Salamandra, as Flemming early pointed out, the spermatogenesis forms an annual cycle in which in adaptation to the breeding habits of the animal, the stages succeed each other in chronological order and characterize certain periods of the year. After mating in the spring a multiplication of the residual spermatogonia takes place which continues during the months of April, May and June. During late spring and early summer occurs the period of growth and maturation of the spermatogonia to form the spermatocytes; during June, July, and also into August, the reducing divisions are taking place, followed by the transformation of the spermatids into spermatozoa in August and September. The testis in the winter is filled with ripe spermatozoa, which are emitted in the following spring, when another yearly cycle begins.

In Desmognathus the cycle is shifted but slightly, so that the characteristic periods occur at nearly the same times as in Salamandra. The testis of animals killed in the autumn (October, November) contain none or but few ripe spermatozoa; the spermatogonia are, however, dividing and the secondary spermatogonia have entered upon their

104 The Spermatogenesis of Desmognathus Fusca

period of growth to form the spermatocytes. Mature and dividing spermatocytes occur sporadically, but seem to be those left over from the preceding summer. During the winter a sluggish division of the spermatogonia is taking place and a few lobules containing spermatozoa or spermatids are usually encountered. There does not seem to be much activity in the organ during the cold months, and there is practically no increase in size, but in the spring mitoses become numerous and more cysts of spermatogonia are found in stages of cell division. Growth to form the spermatocytes also begins anew, continuing well into July. During June and July, especially in July, divisions of the spermatocyte are taking place, and multiplication of the spermatogonia has practically ceased. The transformation of the spermatogonia into spermatocytes stops in June, while the transformation of spermatids into spermatozoa, beginning in June or July, extends through August into September. Most of the spermatozoa leave the testis in the early fall and the cycle begins again.

The spermatogenetic cycle of Desmognathus corresponds closely, therefore, with that of Salamandra, differing in the extrusion of the mass of the spermatozoa in the fall, so that the cycle may be said to begin then instead of in the spring, though there is no great activity until spring. The months when the processes of spermatogenesis are actively taking place are the spring and summer months. During April, May and June transformation of the spermatogonia into spermatocytes is predominant; the divisions of the spermatocyte characterize July, and the transformation of the spermatid, August. The processes, however, overlap widely with doubtless considerable seasonal and individual variation.

The Testis.

' This organ is quite elongated and attached to the dorsal wall of the abdominal cavity by a mesorchium in which are the vasa efferentia, blood-vessels, etc., as in other Amphibia. It is highly pigmented, as is also the spermatic duct, the pigmentation diminishing with the expansion of the organ as spermatogenesis proceeds.

The structure of the Amphibian testis has been investigated by several writers, among them von Wittich, Leydig, Bidder, Spengel, La Valette St. George, 76, and Hoffmann, 78, all of whose papers appeared prior to 1880. Of these the papers of Spengel and Hoffmann appear most valuable. Spengel, in his analysis of the structure of the organ, recognized " capsules " (containing sperm-producing cells), connected with a collecting duct by branch-tubules. According to the relations and ar

B. F. Kingsbury 105

rangement of the capsides in regard to the collecting duct, three general types of structure were found: (1) with a longitudinal collecting duct in the center and the capsules radially arranged, (2) with the longitudinal canal superficial and the arrangement of the capsules fan-like, (3) the capsules short (more spherical) and terminating the divisions of the richly branched collecting duct. Between these extreme types, however, are transitional forms even in different parts of the same testis, as, for example, the central longitudinal canal becoming superficial. Hoffmann extended Speu gel's work, adding also to the knowledge of the development of the organ. La Valette St. George, 76, in his wellknown paper, carried the analysis of the structure a step farther. The testis is made up of " tubules " (capsules of Spengel), which may be hollow or not, and these " tubules " are made up of " follicles " enclosing " spermatocysts "—clusters of cells formed from a single original cell by division.

Meves recognizes the occurrence of the " cysts," which are arranged together in the form of a " thick- walled vesicle enclosing a central cavity. . . . Therefore, it is not proper to speak of tubules (referring to La Valette) here." These vesicles again are the tubules of La Valette, the capsules of Spengel.

Far more appropriate seems the designation of " lobule," which will be the term employed in this article. These divisions or lobules, separated from each other by connective tissue, are the structural units of the organ, and each is connected with the central collecting duct by a short tubule. Further, their homology with the lobules of the mammalian testis seems probable. Hoffmann has found that the collecting duct with its branches is developed from the tubules of the mesonephros, suggesting, therefore, their homology with the rete testis and vasa recta. The lobules of the Amphibian testis are not differentiated into tubules, though in some forms (e. g., Bufo) the structure, of the tubule of the mammalian testis is suggested.

The testis of Desmognaihus combines the first and second of Spengel's three types, there being a longitudinal collecting duct, which is centrally located save at the ends of the organ, where it becomes nearly or quite superficial. About this collecting duet the lobules are placed radially, each connected with it by a short cord of cells whose arrangement in the form of a tubule is more or less evident. The form of the testis varies greatly with the season of year, due to the changes in the spermatogenetic cycle. In general, it is enlarged in the center and tapering at both ends, the enlargement and shape of any part being caused by the state of development and therefore size of the component lobules.

106 The Spermatogenesis of Desmognathus Fusca

These begin as hollow vesicles, as Moves has described them in Salamandra, in which the wall is composed of the primary spermatogonia in a single row, each enwrapped by one, or possibly two or three follicle cells. By division of the primary spermatogonia, the wall of the vesicle becomes thicker and of several layers of cells, but the descendants of each spermatogonium remain enclosed by its follicle cells and are thus separated into a group or cyst. By increase in size of the cysts, the cavity of the lobule is soon obliterated and the lobule becomes a solid mass of cells. Each secondary spermatogonium of the last generation undergoes a period of growth to form the spermatocyte, nearly doubling in size during the process. Each spermatocyte divides twice to form the spermatids which become transformed into the spermatozoa. As a result of this increase in the number and size of the component cells, the lobule, and therefore the testis as a whole, becomes greatly enlarged. During these changes the cysts are still evident, each encompassed by its follicle cells, the descendants of the cells surrounding the primary spermatogonia. When the spermatids begin their transformation, the cysts — as cysts — become disorganized, and the follicle cells assume a new function, or (probably) the old function is modified to suit the new conditions. The protoplasm increases in amount and is accumulated especially upon one side of the nucleus. In this protoplasm are inserted the heads of the maturing spermatozoa, as is the case in the organ in the higher vertebrates. AVhen the spermatozoa are fully mature, they lose their relation to the follicle cells and accumulate in the center of the lobule ready for expulsion, while the follicle cells occupy and form the wall of the lobule. The spermatozoa finally pass from the lobule through the short tubule into the collecting duct, and thence by means of the vasa efferentia into the spermatic duct.

The follicle cells now undergo a degeneration and disappear, so that the lobule, as such, eeases to exist. Each lobule, therefore, has a life cycle of a year; develops, reaches maturity and degenerates, and during this time increases greatly in size, finally to shrink and disappear.

While the mass of the cells of the lobule undergo the changes of spermatogenesis, a few primary spermatogonia in the apex of the lobule (i. e., near the collecting tubule) remain unchanged and persist throughout the succession of stages through which the other cells pass. When the lobule is greatly distended with the maturing spermatozoa, they become so flattened as to be rocognized with difficulty. After the spermatozoa have been extruded and the lobule has collapsed, these residual spermatogonia, surrounded by their follicle cells, again round out and present their original appearance and structure. These, by their later

B. F. Kingsbury 107

multiplication, may furni&h the cells for a new formation of the lobule in a succeeding season. McGregor, 99, was inclined to believe that the cells which replenish the lobule came from the cells, forming the ducts of the lobules. This seems hardly probable, since the collecting duct and its branches appear to be developed from the mesonephros, Hoffmann, 86, and not from the germinal epithelium. I believe therefore that residual spermatogonia occur in Amphiuma as they do in Desmognathus, but were not recognized as such. In other salamanders, as Spelei'pes, regeneration of the lobule begins before the spermatozoa leave it. This does not seem to occur in Desmognathus, so that when the lobule is regenerated, it is as a new formation.

In Desmognathus, the spermatogenetic wave passes over the testis in a forward direction, that is, from the caudal to the cephalic end. There are encountered in an organ at the proper season of the year, a succession of lobules containing ripe spermatozoa, maturing spermatozoa, spermatids, speimatocytes, maturing and in division — second growing spermatocytes (of the first order), secondary spermatogonia, and finally primary spermatogonia in the more filiform cephalic end to which the organ tapers. At the opposite end the organ also becomes constricted, general!} more abruptly, where it passes into the mass of interlobular connective tissue, which became more prominent when the lobules shrank and disappeared. The testis accordingly has a rather fusiform shape, tapering at both ends to a filament. The accompanying diagram indicates the succession of regions in the testis of a specimen

Fig. a. Diagram of a longisection of the testis of Desmognathus during July, to show the succession of regions, a. primary spermatogonia ; 6. secondary spermatogonia; c. growing spermatocytes; d. zone containing dividing spermatocytes of the tirst order and spermatocytes of the second order, resting and dividing; e. spermatids; /. transforming spermatids and immature spermatozoa ; ^. ripe spermatozoa; h. " degenerated" lobules.

killed in July, as seen in longisection. The shape and size of the organ, as is evident, is variable, depending on the time of year, being larger in the summer than at other seasons, and of a correspondingly different shape. Occasionally, and perhaps usually, it consists of two, three or even four enlarged portions in which the spermatozoa are

108 The Spermatogenesis of Desmognathus Fusca

developing, these enlargements being connected by more constricted portions. The enlarged parts are identical in structure, each with a succession of stages, from the primary spermatogonia at one end to the degenerated lobules at the other. Their occurrence evidently means that there have been 2, 3 or 4 centers of growth, either primary — in the original cord of germ cells; or secondary — by the division of residual spermatogonia left when the lobule, as such, disappeared. There seems to be no absolute correlation of this condition with other structural features of the salamander, save that the presence of two or more enlargements occurs more often, in fact, quite constantly, in large animals. A similar division of the testis into "lobes" occurs in other salamanders with an elongated body, and has been noted in Amphiuma and Spelerpes. The segmented condition of the organ in Coecilians is perhaps to be associated likewise with the elongated form of body. In the European salamander, Meves, 96, and others have noted a division of the testis into lobes, whose size, shape, and possibly existence, are dependent upon the stage of development of the spermatozoa. Of these lobes, 4-5 in number, the more caudal contain ripe spermatozoa, the cephalic lobe, the cells that will form the spermatozoa, and they are so arranged that different stages follow one another in cephalo-caudal succession. In the cephalic tip, which is prolonged into a thread, the spermatogonia are located, caudad of which are the spermatocytes in successive stages of maturity or division. The lobes also possess different color according to the contents, the spermatozoa imparting a yellowwhite, the cells, a transparent blue-gray. With these lobes, which are not well marked in Desmognatlius, must not be confounded the succession of enlargements spoken of above, which are in effect independent testes.

In the spermatogenetic cycle of Desmognathus degenerations seem to occur quite constantly and normally. These are: (a) a degeneration of spermatozoa left over from the preceding year. This occurs in the organ in the spring, and even, it would appear, in the winter and preceding fall; (b) likewise spermatids undergo degeneration in the spring; (c) when the spermatogonia cease to undergo transformation into spermatocytes in the summer, the last cysts of spermatogonia apparently undergo a chromatolysis and solution, and the boundary between the spermatocytes which are to form spermatozoa that season and the spermatogonia remaining over until the next summer, is thus well marked. After the expulsion of the spermatozoa and the collapse of the lobule, the follicle cells degenerate and disappear. These degenerations, together with the divisions and structure of the spermatogonia

B. F. Kingsbury 109

and a more detailed discussion of the structure of the Amphibian testis, the writer hopes to consider at some future time.

The Geowth and Division or the Spermatocyte.

As is well known, from the spermatogonia are formed, by a process of growth, the spermatocytes of the first order. Eoughly speaking, this transformation involves an increase in the size of the nucleus and the amount of cytoplasm, together with chromatic changes resulting in the formation of twelve chromosomes. The character, however, is given to the cell at the beginning of the period of growth, so that in this article the name of spermatocyte will be applied to it during this time of development, a distinction being made when necessary by applying to it the qualifying word " immature." The growth is evidently slow and takes some time for completion. In the fall and early winter the number of nearly mature spermatocytes is small. The mass of spermatozoa formed from the spermatocytes of the preceding summer, as has been said, have been largely expelled from the testis, though a few lobules usually still retain them. A few lobules likewise may contain spermatids and immature spermatozoa. During the fall, on the other hand, the spermatogonia are undergoing evidently rapid division, so that entire cysts are often found in stages of mitosis, and this process continues at a much retarded rate through the winter, to become very active again in the spring. The transformation of spermatogonia into spermatocytes, beginning in the fall, continues up to about the middle of the summer when it ceases. Divisions of the spermatocyte of the first order are but rarely found during the fall, winter and early spring, though occurring sporadically, especially in the early winter. But during late spring and early summer the divisions are abundant, continuing up to August (about), when they have nearly ceased.

A secondary spermatogonium of the last generation, small spermatogonium of Meves, 96, possesses a round or slightly oval nucleus of' medium size. The chromatin is in the form of an apparent network of irregular shape and distribution. One or two small nucleoli are present. The amount of cytoplasm is small and is especially accumulated on one side surrounding the idiozome (to use the name introduced by Meves). Within the idiozome two centrosomes may usually be distinguished.

In the transformation into the spermatocyte, the network of chromatin becomes changed into a thread which is, I believe, at this time already segmented into the twelve chromosomes which are easily distinguishable a little later in their growth. The spirems are at first very fine and

110 The Spermatogenesis of Desmognathus Fusca

intricately interwoven, the only indication of the segmentation being afforded by apparently free ends protruding on the side toward the idiozome.

Synapsis. — The beginning of the growth period of the spermatocyte is evidently a time of great importance in the process of spermatogenesis. The spermatocyte possesses chromosomes of one-half the number normally occurring in the mitoses of that species, and this pseudoreduction has generally been located as occurring in the growth of the spermatocyte. The view, too, that the reduction in number of the chromosomes is only an apparent or pseudo-reduction, is generally accepted. That the chromosomes of the spermatocyte are bivalent and are two joined together end to end, is of course well known and also seems quite generally accepted. The terms introduced by Moore to indicate this pseudo-reduction and the corresponding period, " synapsis " and "synaptic phase," have been used in rather a confusing way. As used by Moore, 95, in his work on the spermatogenesis of Elasmobranchs, synapsis is equivalent to Riickert's, 93, pseudo-reduction, though Moore apparently does not assume that the chromosomes of the spermatocyte necessarily represent two joined together and therefore bivalent, which pseudo-reduction does assume. At the time that this reduction was believed to take place, there occurred in the forms studied by Moore (Scyllium and Torpedo), a peculiar contracted condition of the chromatin, causing it to be massed upon one side of the nucleus, and giving the appearance of an artifact. This Moore belieyed characteristic of the synaptic period and to be of general occurrence, and to this phenomenon of chromatin contraction, by a species of m.etonymy, the term synapsis has been transferred by several investigators. For this Moore himself seems partly responsible, since in a later paper, in conjunction with Farmer, 95, he uses the term synapsis as equivalent to " the contraction figure." A distinction between these two uses of synapsis seems to be necessary. The first use is evidently the correct one and is followed in this article. The use of the term in my preliminary communication upon this subject is wrong. The contracted condition of the nucleus, Moore found in the elasmobranchs investigated by him and in Amphibia (the Triton). Brauor, 93, had figured and described the massing of the chromatin upon one side

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