This dissertation has been microfilmed exactly as received

HAYES, Thomas G., 1936- THE EMBRYONIC DEVELOPMENT OF THE ELLIPSOID SHEATH AS IT OCCURS IN THE DOG SPLEEN.

The Ohio State University, Ph.D., 1965 Anatomy

University Microfilms, Inc,, Ann Arbor, Michigan THE EMBRYONIC DEVELOPMENT OF THE ELLIPSOID SHEATH

AS IT OCCURS IN THE DOG SPLEEN

DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Thomas G. Hayes, B.A., A.M

*******

The Ohio State University

1965

Approved by

Adviser Sl Department of Anatomy ACKNOWLEDGMENTS

The author wishes to express his sincere appreciation and indebtedness to his adviser, Dr. John A. Eglitis, for his guidance, his interest, and his stimulation throughout the present investigation. The example he has displayed as a teacher and scientist will never be forgotten. To the faculty and members of the Department of

Anatomy for their many kindnesses shown during the course of his graduate studies, he is indebted. To his wonderful parents who helped make this goal possible, he is extremely grateful.

ii VITA

July 3, 1936 Born - Canonsburg, Pennsylvania 1958 ...... B.A. / Washington and Jefferson College, Washington, Pennsylvania 1958-1960. . . . Teaching Assistant, Department of Biology, Western Reserve University, Cleveland, Ohio

1960 ...... M.A., Western Reserve University, Cleveland, Ohio 1961-1962. , . . Teaching Assistant, Department of Anatomy, The Ohio State University, Columbus, Ohio 1963 ...... Assistant Instructor, Department of Anatomy, The Ohio State University, Columbus, Ohio 1964-1965. . . . N.I.H. Fellow. Predoctoral Fellowship awarded from the National Institutes of Health, Bethesda, Maryland 1965 ...... Instructor, Department of Anatomy, The Ohio State University, Columbus, Ohio

FIELDS OF STUDY Major Field: Histology Studies in Gross Anatomy. Professor Linden F. Edwards Studies in Histology. Professors John A. Eglitis and G. A. Ackerman Studies in Neuroanatomy. Professor James L. Hall

Studies in Embryology. Professor John Weston

iii CONTENTS

Page

ACKNOWLEDGMENTS...... ii

VITA ...... iii ILLUSTRATIONS...... v INTRODUCTION ...... 1

MATERIALS AND M ETHODS...... 8 OBSERVATIONS AND RESULTS...... 11 I. 35-Day Embryonic Dog S pleen...... 11 II. 40-Day Embryonic Dog S p leen...... 13 III. 45-Day Embryonic Dog S p leen...... 15

IV. 5 5-Day Embryonic Dog S p lee n...... 17 V. Adult Racoon and Woodchuck S p leen...... s 18 DISCUSSION...... 25 SUMMARY AND CONCLUSIONS...... 37 BIBLIOGRAPHY...... 39

iv ILLUSTRATIONS

Plate Page I 35-Day Embryonic Dog S pleen...... 44

II 35-Day Embryonic Dog S p leen...... 46

III 40-Day Embryonic Dog S pleen...... 48 IV 40-Day Embryonic Dog S p leen...... 50 V 40-Day Embryonic Dog S p leen...... 52 VI 45-Day Embryonic Dog S p leen...... 54 VII 45-Day Embryonic Dog Spleen...... 56 VIII 55-D ay Embryonic Dog S p l e e n...... 58

IX 55-Day Embryonic Dog S p leen...... 60 X 55-Day Embryonic Dog S p leen...... 62

XI 55-Day Embryonic Dog S pleen...... 64 XII Adult Racoon Spleen...... 66

XIII Adult Racoon Spleen...... 68 XIV Adult Racoon Spleen...... 70 XV Adult Racoon and Dog Spleen...... 72 XVI Adult Racoon Spleen...... 74

XVII Adult Woodchuck Spleen...... 76

XVIII Adult Woodchuck Spleen...... 78

XIX Adult Woodchuck Spleen...... 80

v INTRODUCTION AND HISTORICAL REVIEW

The present investigation was undertaken in order to determine the possible origin of the so-called ellipsoid or Schweigger-Seidel sheath (48) as it occurs in the dog spleen. In addition to the basic problem cited above, a purely histo­ logical description of the adult racoon (Procyon lotor) and woodchuck

(Marmota monax) spleens will be presented. Since the spleens of these two animals have not been adequately described in the literature, it is hoped that this presentation will add additional information to our com­ parative knowledge of the mammalian spleen. Anyone who has studied the spleen is well aware of the pre­ vailing controversies concerning the internal vasculature of this organ. This is particularly true of the so-called sheathed arteries. The vasculature of the spleen is unique in that it not only deter­ mines the distribution and inter-relationship of both the red and white pulp tissue but also determines the structural plan of the spleen as a whole. The arteries tend to be associated with the white pulp tissue; the veins form an intimate relationship with the red pulp tissue. The arteries enter the spleen at the hilus and follow the larger trabeculae into the organ as the trabecular arteries. When the latter vessel reaches a diameter of 0.2 mm., it leaves the trabeculae and enters the splenic parenchyma as the white pulp or central artery. The central artery while coursing through the follicle gives off capillary branches to all of the white pulp. Upon reaching a diameter of 50 micron. , the central arte­ ries lose their investment of white pulp and enter the red pulp. As the central artery enters the red pulp tissue, it arborizes into three to five long, straight, branches which, because of their resemblance to fine brushes, were called penicilli. The penicilli, in turn, are divisible into three successive segments: the red pulp artery, the ellipsoid artery, and the terminal capillary. The first segment, the red pulp artery, is the longest. This vessel courses within the red pulp tissue, finally dividing into two to three divisions. Each division, in turn, is provided with a spindle-shaped thickening, the ellipsoid sheath; this segment is the sheathed artery. The latter vessel gives rise to two to three branches as the terminal capillaries whose exact manner of ter­ mination is still disputed among investigators of the splenic vasculature. The ellipsoid sheath that surrounds the segment of the peni- cillar artery consists of an aggregation of reticular cells and reticular fibers arranged about the capillary vessel. The latter vessel is referred to in the literature as the Schweigger-Seidel sheath capillary, sheath artery, and the ellipsoid artery.

An ellipsoid or sheath artery was first noted by Billroth (1857)

(4) in the spleens of birds. He described them as iusiform-shaped bodies occurring along the terminal arteries. Six years later, Schweigger-

Seidel (1863) (48) gave a more accurate description of similar bodies that he observed in the pig spleen, calling them "hfllsenarterien." He also described these structures in the dog, cow, cat, and man but failed to identify them in the horse spleen which he studied. These structures, that bear his name, were proposed by him to be organ filters of the blood. The first comprehensive work on these fusiform bodies was carried out by Muller (1865) (37), who described them in various rep­ tiles, fishes, birds, and mammals. He is responsible for coining the term "ellipsoid." Muller observed that the ellipsoids are well developed in birds and carnivores but are rudimentary in man and rodents. The ellipsoids were described as endothelial lined channels consisting of a mass of protoplasm representing an adventitial thickening of the axial vessel. They served as a point of termination for the splenic nerves.

Kyber (1870) (23) maintained that the ellipsoid was a loosened adventitia infiltrated with lymphocytes resembling the splenic follicle in structure. The above investigator was the first to deny any communi­ cation existing between the capillary lumen and the sheath proper.

Bannwarth (1891) (2), having described the sheath in the cat spleen, considered it as a growth center for the splenic pulp, serving its principal function during the embryonic period. In 1894, Whiting (62) conducted a comparative study of the ellipsoid sheath. He observed that the ellipsoids are composed of a homogenous ground substance containing a few lymphocytes. He main­ tained that the ellipsoids contain smooth muscle fibers that formed an essential constitutent of the sheath. This latter view was held by von Ebner (1899) (10) and Janosik (1903) (18).

Kultschitzky (1895) (22) reported the sheath to be composed of a compact mass of leucocytes. In contrast, Carleir (6) in the same year described the sheath as being composed of reticular fibers infiltrated with connective tissue cells. It was Mall (1900) (29), who first proposed the theory that the sheath acts as a one-way valve, controlling the reflux of blood from the venous into the arterial side. Sabin (1910) (45), working with injected mammalian spleens, observed that during embryonic development of this organ the ellipsoids make their appearance very early. These structures developed before the splenic follicles and were considered by her to be important em­ bryonic structures serving their principal function at this time. She suggested that the ellipsoid acted as an early lymphatic organ, a view

similar to the one proposed by Bannwarth (2) in 1891. Comparative studies of the ellipsoid sheath by Li et al. (1929) (26) showed these structures, in all animals investigated, to be com­ posed of reticulo-endothelial cells arranged in a mesh of reticular fibers. These findings were similar to those of Solnitsky (1937) (53). By means of intravenous injections of various dyes, he demonstrated that the ellipsoid sheath possessed marked phagocytic properties. He considered the sheath a condensation of the splenic pulp, composed of these reticular fibers and intensely phagocytic reticular cells. Imai

(1947) (17), from his-.injection studies of the cat spleen, also showed the active phagocytic function to be associated with the cells com­

prising the ellipsoid sheath.

Zwillenberg and Zwillenberg (1962) (63) recently studied the

ellipsoid of the dog spleen by means of electron microscopy. They

found that the cells comprising the sheath contained an abundance of intracytoplasmic filaments about %80 in diameter. The sheath cells also demonstrated signs of phagocytosis, indicated by the presence of large, dense bodies within the cytoplasm of the reticular cell. Regarding the functional significance of the ellipsoid sheath, a number of hypotheses have been proposed. These may be divided into

.two general categories: those concerned with a mechanical function

and those with a biological function. A purely mechanical function includes the regulator theory,

the valve theory, and the protective capillary theory, whereas the bio­ logical functioning category includes the nervous apparatus theory, the

embryonic center theory, and the filter apparatus theory.

The valve theory was first advocated by Stieda (1862) (55), and, later, by Mall (1900) (29). By means of perfusion studies, both Stieda

and Mall were able to demonstrate that the spleen could not be perfused

from the venous side into the arterial side. These results were later disputed by the work of Mills (1927) (33) and Imai (1947) (17). According to their investigations, a reverse flow through the ellipsoids could be demonstrated in spleens which were in a state of dilation, thus dis­ proving the valvular theory. Hueck (1928) (16) also denied the exis­

tence of a one-way valve function attributed to the ellipsoid sheath, maintaining that the resistance to reverse flow was due to some other

factor, such as the compression of the capillaries and arterioles by the

venous sinusoids which are distended by the perfusing fluid.

Heidenhain (1928) (14) and Weidenreich (1901) (58) attributed a capillary regulator function to the ellipsoid sheath. They assumed

that when the spleen contracted, the ellipsoid sheath also contracted as a result of its natural tonus and thus acted to protect the splenic pulp from a sudden overflowing of blood cells and plasma during a sud­ den rise in blood pressure. A protective capillary theory was proposed by Reidel (1932) (41) for the function of the ellipsoid sheath. It protects the capillary from damage in the case of sudden contraction of the spleen. According to this investigator, the ellipsoids are best developed in those animals exhibiting an abundant musculature in the capsule and trabecular net­ work. Hypotheses of a strictly biological function have been advo­ cated by a number of investigators of the ellipsoid. The sheath was regarded as a growth center for the splenic pulp by Bannwarth (1891) (2) and Staemmler (1925) (54). According to this theory, the sheath contributed to and maintained the substance of the splenic pulp by its constant proliferation. The supporters of this theory were also con­ vinced that the sheath was important only during the embryonic period. Miiller (1865) (37) attributed a nervous function to the ellip­ soid sheath. He considered the sheath a nervous structure which re­ ceived the terminal arborizations of the splenic nerves. This theory was later abandoned because of the lack of histological and physio­ logical evidence for its support.

The filter hypothesis was first proposed by Schweigger-Seidel

(1862) (47) and has recently been emphasized once again by Robinson (1926) (43), Mills (1927) (33), and Solnitsky (1937) (53). These investi­ gators consider the sheath a part of the reticulo-endothelial system.

Solnitsky (53) has demonstrated that the constitutent cells of the sheath are characterized by a selective and intense property of phagocytosis of particulate matter, by their rapid mobilization into migratory, ameboid phagocytes under the influence of injected dyes, and by their remarkable capacity for proliferation and regeneration. MATERIALS AND METHODS

Animal Tissues The animal tissue employed for this investigation consisted of four date-mated female dogs of the fox-hound breed. All of the animals were obtained from the Animal Research Laboratories, The Ohio State

University, Columbus, Ohio. The last day of standing heat was taken as day one in calcu­ lating the desired period of gestation. Embryos from 35, 40, 45, and 55 days gestation were employed in the present study. The total number of specimens for each respective period plus their C/R measurements are as follows:

DAYS OF GESTATION TOTAL NUMBER C /R MEASUREMENT 35 6 15 mm. 40 4 20 mm. 45 5 90 mm. 55 5 . 130 mm.

Intravenous and intraperitoneal injections of five to ten mili- liters of Sodium Nembutal (Abbot Laboratories) were employed in sacri­ ficing the adult pregnant dogs.

The whole embryos from the early embryonic stages (35-40 days gestation) were placed in fixative; whereas, in the case of the later em­ bryonic stages only the excised spleen was processed for serial recon­ struction. Segments of the adult dog spleen were also removed for study.

8 9

The whole embryos were fixed in 10 percent Formalin, Bouin's and F orm al-sublim ate-acetic acid solution (FSA). R epresentative s e c ­ tions of the remaining specimens were each placed in one of the fol­ lowing fixatives: 10 percent Formalin, Zenkers-Formal Solution, and

Formal-sublimate-acetic acid solution (1). In addition to the above animal tissues, spleens were also ob­ tained from two racoons (Procyon lotor), three woodchucks (Marmota monax), two pigs (Sus domestica), and three humans (Homo sapiens).

The racoon and woodchuck spleens were obtained after shooting the animals; these spleens were immediately fixed in 10 percent Formalin and FSA solutions. The pig spleens were supplied by a local abattoir; fixed in a solution of Zenker's-Formal solution. The human spleens were obtained from various hospitals during post-mortem examinations and were fixed in a 10 percent Formalin solution. All of the above spleens were obtained from the adult animal.

All specimens studied were left in the fixative for the optimum time, washed, and then dehydrated through a series of absolute ethanol. All tissues that were used in the present study were processed by a double-embedding procedure. The blocks of tissue were removed from the absolute ethanol and placed into a graded series of 1 percent, 2 percent, and 4 percent parlodin in methyl benzoate solution every

24 hours. The tissues were then transferred through three changes of

benzene for 4, 8, and 12 hours respectively. Routine paraffin embedding at 57 °C completed the process.

The tissues were cut on a Spencer AO "820" microtome. Serial

paraffin sections of the whole embryo as well as the spleens from the 10 later embryonic stages were cut at 6 microns. A fraction series of every ten to twenty sections was employed in the case of all the adult spleens investigated. These latter sections were cut at 6, 8, and 10 microns.

The tissue sections were affixed to the slides with Mayer's egg albumin, diluted 1:10 with distilled water. The basic stains employed for all sections, with the exception of the very early embryonic stages (35 and 40 days gestation), consisted of Snook's Reticulum Stain (49) and a modification of M asson's Trichrome

Stain (1). The modification of the latter stain involved pre-heating of the phosphomolybdic acid at 45° C before the tissue sections were placed into the solution. The tissues were then incubated in this solu­ tion for ten minutes. The early embryonic tissues cited above were stained by a routine hematoxylin and eosin procedure (3). Representa­ tive sections of all specimens under investigation were also stained by the following: Heidenhain's Iron Hematoxylin (1), Toluidine Blue (1),

Periodic-acid-Schiff's-Hematoxylin-Orange G (27), and Weigert's Elastic Connective Tissue Stain (27). OBSERVATIONS AND RESULTS

A survey of the literature regarding the general development of the spleen is well documented; however, developmental studies of the vasculature per se warrants further investigation. This is particularly true concerning the origin of the ellipsoid or sheath artery. For the present study, dog embryos were employed because the sheath is very prominent and easily identified in the adult animal.

The results and observations are presented according to the various stages of embryonic spleens studied. As a supplement to the above problem, a purely histological description of the adult racoon and woodchuck spleens is presented.

I. Thirty-five day embryonic dog spleen stage The splenic rudiment, at this stage, presents a somewhat triangular-shaped structure within the left wall of the dorsal meso- gastrium ventral to the region of the developing pancreas (Figure 1).

Surrounding the splenic rudiment is the coelomic epithelium consisting of several layers of elongated cells exhibiting a distinct nuclear membrane and a prominent nucleolus. This layer of cells repre­ sents the definitive visceral peritoneum. It has been implied that these cells may contribute to the early anlage of splenic tissue (24). Silver impregnation studies reveal that these cells are separated from the underlying condensed mesenchymal cells by a delicate band of reticular

11 12 fibers (Figs. 2 and 3). In Figure 3# these same delicate reticular fibers may be identified within the area of the future pulp spaces. The vasculature of the embryonic spleen during this stage con­

sists of a capillary network extending throughout the rudiment. In Figure 4, the capillary vessels appear as irregular, narrow, channels possessing a distinct endothelial lining. Within the lumen of these channels an occasional red blood corpuscle may be encountered indi­ cating a definite circulatory pathway within the rudiment at this time.

The primordia of the future, splenic artery and vein are easily distinguished from one another, coursing within the dorsal mesogastrium. The former vessel is identified by the presence of a distinct region of developing smooth muscle fibers not evident in the accompanying vein.

The splenic artery upon reaching the rudiment gives off a number of vessels of capillary dimensions that are seen to penetrate the compact

mass of mesenchyme. During this stage of development, the arterial vessels cannot be distinguished from their venous counterparts within

the rudiment. Serial studies indicate that the venous capillaries, when

traced to the pedicle of the future hilar region, become continuous with the primordial splenic vein located within the dorsal mesogastrium

(Fig. 5). The cellular components of the early splenic rudiment con­

sists of large, round, compact cells representing a syncytial appearing

mass resulting from the condensation of the original mesenchyme. In Figure 6, the latter cells are represented within the dorsal mesogastrium presenting a looser arrangement than in the adjoining segment of the splenic rudiment where they assume a more compact configuration. 13

There is no evidence of distinct sinusoids or ellipsoid vessels within the splenic rudiment during this stage of development.

II. Forty-day embryonic dog spleen stage In the 40-day embryonic spleen stage, the splenic rudiment presents an elongated-shaped structure attached to the dorsal meso­ gastrium in the area of the future hilar region (Fig. 7). The overall appearance of the rudiment during this stage resembles the red pulp tissue found in the adult dog spleen with the exception that in the adult erythropoiesis is nearly or completely suppressed (Figs. 8 and 9). Within the same photomicrographs, the majority of the cellular com­ ponents present an ovoidr-shaped nucleus that exhibits a rather delicate chromatin pattern and prominent nucleoli. The capillary plexus forms a more elaborate network throughout the rudiment as compared with the previous stage of development. These endothelial: lined channels exhibit, for the first time, the pre­ sence of nucleated red blood corpuscles within their lumina (Figs. 9 and

10). The majority of the developing red blood corpuscles encountered are confined to the lumen in contrast to that of the adult dog spleen where they can also be identified within the intercellular spaces of the pulp cords. Some investigators (2), (24), (40), (45), interpret the con­ finement of these cells as evidence of a closed-type of circulation thought to prevail during the early embryonic stages.

The cells comprising the definitive visceral peritoneum no longer exhibit a columnar to cuboidal shape but tend to assume the shape of a squamous cell characteristic of the serous membrane 14 encountered in sections of the adult dog spleen (Figs. 10 and 11). A distinct capsule and trabecular system are not evident at this time; however, application of Weigert's elastic connective tissue stain re­ veals the presence of a few elastic fibers in the vicinity of the future capsular region.

The development of a more definitive arterial system commences during this stage of development. Differentiation of distinct arterial vessels from the primitive capillary plexus is apparent with the appea­ rance of smooth muscle fibers lying external to the endothelial cells that line the lumen of these primitive vessels. In the embryonic spleen of 40 days, the development of the ellipsoid sheath is initiated. The ellipsoid sheath consists of a con­ densation of mesenchymal cells around a vessel of capillary dimension.

On the inner surface of the capillary is an endothelium whose cells possess a scanty amount of cytoplasm and a large, darkly-stained nucleus that projects into the lumen. Occasionally, the projection of the nucleus is so pronounced that the diameter of the lumen is reduced considerably. The mesenchymal cells in the area of the developing sheath lose their ovoid-shape assuming an elongated, cigar-shape as they orient themselves about the lumen of the capillary vessel. The latter shape is typical of those cells that are found lying adjacent to the lumen of the vessel (Fig. 12). In Figure 13, the transformation of the ovoid-shaped cells of the preiphery to the irregular, elongated­ shaped cells nearer the lumen of the capillary may be observed. These irregular shaped nuclei exhibit a dust-like chromatin pattern and very pronounced nucleoli; however, distinct cell boundaries of the adjacent 15 cells are not readily identified (Figs. 14, 15, and 16). Following silver impregnation, very fine reticular fibers may be recognized between the cells comprising the sheath; in the peripheral portion of the developing sheath, coarse reticular fibers separate the sheath from the surrounding red pulp tissue. Additional cells are added to the primitive ellipsoid sheath by the transformation of the surrounding mesenchymal cells.

III. Forty-five day embryonic dog spleen stage The spleen during this stage of development closely resembles the general architecture found in the adult organ. The capsule consists of three to four layers of cells beneath the now flattened mesothelial cells. The mesothelial cells present numerous microvilli that project from the cytoplasm of the individual cells (Fig. 17). Application of the periodic-acid Schiff's reaction re­ veals the presence of a PAS positive membrane separating the mesother lial cells from the capsule proper; a slightly darker PAS band may be identified within the mesothelial cell just beneath the microvillus pro­ jectio n s. The cellular components of the capsule proper consist of elon­ gated fibroblast nuclei between which a scattering of smooth muscle fibers may be identified. The capsule is made up predominantly of collagenic fibers; however, silver impregnation and elastic connective tissue stain demonstrate the presence of reticular and elastic fibers respectively. The latter fibers tend to be confined to the innermost segment of the capsule. The trabeculae exhibit the same tissues as found in the 16 capsule with the exception that the smooth muscle fibers tend to pre­ dominate; the elastic fibers continuing into the trabeculae from the capusle tend to be confined to the periphery of the trabeculae. Figure 18 represents a section of the trabecular vein joining with the splenic vein. The tributaries of the splenic vein are surrounded by the thick bundles of smooth muscle fibers found within the trabe­ culae. These trabecular veins lack a distinct adventitial layer. The transition from the smooth muscle of the trabecular vein to a distinct adventitial layer of the splenic vein is apparent in this photomicro­ graph (Fib. 18). The arterial vessels have progressed to where they are recog­ nized as individual vessels. In Figures 18 and 19, the smooth muscle fibers of the media may be seen in cross and longitudinal section re­ spectively. An ellipsoid sheath may be identified just below the artery (Fig. 19). The development of distinct arteries is said to be respon­ sible for initiating the formation of the splenic white pulp (9). The lymphocytes, characteristic of the white pulp tissue, are evident for the first time in this particular embryonic stage. These cells are ran­ domly distributed around an arterial vessel forming a sheath-like pattern rather than a distinct nodular or follicular arrangement observed in the later stages of development. Sections stained with toluidine blue reveal that this periarterial sheath is composed predominantly of small lymphocytes. The ellipsoid sheath during this stage of development consists of three to five layers of cells concentrically arranged around a capil­ lary vessel (Fig. 20). A few of the mesenchymal cells in the vicinity of the sheath still may be seen to undergo the transformation characte­ ristic of the previous stage. In addition to this transformation, a num­ ber of mitotic figures were observed among the cells contained within the sheath. Mitosis occurring among these cells thus provides addi­ tional cellular components to its structure. This is most apparent in the stage that follows, 55-day embryonic spleen. The ellipsoid cells pre­ sent a delicate chromatin pattern with prominent nucleoli; however, the outline of the individual cell is not readily identified (Figs. 21 and 22).

IV. Fifty-five day embryonic dog spleen stage The mesothelial cells external to the capsule proper exhibit the same microvilli demonstrated in the previous stages.. These cyto­ plasmic projections are present where ever the mesothelial cells are located. The nuclei of these cells have a veryd&licate chromatin pat­ tern, prominent nucleolus, and a distinct muclear membrane (Fig. 23). During this stage of development the white pulp tissue pre­ sents evidence of organizing into a follicular arrangement characteristic of the adult organ. The primitive arterial vessels, found within the white pulp tissue are the so-called central arteries in the adult organ (Figs. 24, 25, and 26). Following silver impregnation, the general distribution of the reticular stroma may be seen in Figures 27 and 28. The reticular fibers present in the tunica media of the central artery continue into the remaining segments of the arterial tree enabling one to trace serially the distribution of these vessels within the red pulp tissue. Figure 27 represents a cross-sectional view of the central artery surrounded by the organizing lymphatic sheath of white pulp. 18 Coarse reticular fibers within the future follicle form a concentric pat­ tern in the meshes of which the cellular components of the white pulp

are h eld. In Figure 30, the ellipsoid sheaths appear as a lighter staining

area within the red pulp tissue. The reticular fibers within the ellip­

soid sheaths present a more delicate appearance than the corresponding

fibers found in the white pulp areas. The cross-sectional view of the

ellipsoid in this photomicrograph demonstrates the reticular fibers be­

tween the cellular components of the sheath. In the dog spleen, the red pulp artery bifurcates into two and sometimes three branches, each of which becomes surrounded by a typical ellipsoid sheath (Fig. 31). The cellular components of the sheath exhibit a remarkable number of mitotic figures. The photomicrographs of Figure 32 represent cross sections of the ellipsoid sheaths demonstrating the mitotic figures among the cells comprising this structure. The mitotic figures appa­ rently account for additional cells being added to the sheath in these later stages of development when the mesenchymal precursors are no

longer available.

V. Histological description of the adult racoon (Procyon lotor) and woodchuck (Marmota monax) spleens To our knowledge, the histology of the racoon and woodchuck

spleens have not been described adequately in the literature.

Schuhmacher, in 1900 (46), described the distribution of the elastic

fibers within the woodchuck spleen; however, a description of the racoon spleen was not available. We decided to investigate the above 19 two spleens with particular emphasis on the ellipsoid sheath, hoping to contribute additional knowledge to the comparative histology of this lymphatic organ. The racoon and woodchuck spleens examined present contras­ ting histological appearances unique to the respective animal.

Racoon Spleen The capsule of the racoon spleen, covered externally by a layer of mesothelial cells, consists of an outer layer of collagenic fibers and an inner layer of elastic fibers among which are scattered numerous smooth muscle fibers. The capsule forms an intimate con­ nection with the anastomosing system of trabeculae that project into the

splenic tissue from its surface. Histologically, the structure of the trabeculae is identical with the capsule; however, smooth muscle fibers are more numerous within the trabeculae than in the splenic capsule. The elastic fibers, within the trabeculae, assume a peripheral distribu­ tion (Fig. 35). ~ The majority of the trabeculae in the racoon spleen are of the vascular type although non-vasuclar trabeculae may also be observed. The latter trabeculae demonstrate a greater content of smooth muscle fibers than their vascular counterparts. In Figure 36, both the vascular

and non-vasaular trabeculae are demonstrated.

The white pulp consists of an abundance of nodular lymphatic tissue surrounding the eccentrically positioned central artery. A number of the primary nodules were found to exhibit secondary centers of the reaction type. In a hematoxylin and eosin preparation, a fairly wide 20 zone of lighter stained tissue immediately surrounding the follicle proper may be observed. This is the marginal zone (Fig. 37). The cellu­ lar components of this region were found to be reticular cells, lympho­ blasts, macrophages, and various blood-cell types. The marginal zone of the racoon is narrower than the same area in the woodchuck spleen.

The reticular fibers encountered in this region present a very fine web­ like pattern in contrast to the description of the same fibers encountered within the follicle proper. The marginal zone may be heavily infiltrated with adult red blood cells. It is postulated that these cells enter the marginal zone from the capillaries terminating in the region of the peri­ follicular space. The latter area lies between the primary follicle and the marginal zone. The nodular capillaries, which are branches of the central artery as it courses through the nodule, terminate within this perifollicular space.

Within the smaller primary follicle, reticular impregnated sections demonstrate the presence of coarse reticular fibers oriented in a concentric fashion; in the larger-sized follicles the concentric ar­

rangement of these same fibers are found only in the area of the follicle which lies adjacent to the marginal zone. The reticular fibers tend to be somewhat sporadically distributed in these larger follicles. The trabecular arteries in the racoon spleen do not remain con­

fined to the trabecular masses for any great distance. These vessels

depart from the trabeculae and become surrounded by a sheath of lym­

phatic tissue, the white pulp. In the racoon spleen the presence of the ellipsoid or

Schweigger-Seidel sheath is evident. The ellipsoid in the racoon 21 spleen is an oval-shaped structure. Avery characteristic distribution of these structures is noted around the primary follicle where they assume a concentric arrangement within the tissue of the marginal zone; others are found randomly distributed throughout the red pulp tissue (Fig. 38). This concentric distribution of the ellipsoid is the result of the penicillar branches which course within the red pulp tissue for a short distance only to reapproach the marginal zone of its respective nodule at which time it acquires an ellipsoid sheath.

The ellipsoid artery is in reality a typical blood capillary con­ sisting of reticular fibers and cells that tend to accumulate about the vessel. In an iron-hematoxylin preparation, the endothelial cells appear as elongated, darkly stained nuclei in contrast to the pale

staining reticular cell nuclei comprising the sheath proper (Figs. 39 and 40). The reticular cells of the sheath present a pale, vesicular nucleus with prominent nucleoli. The cytoplasmic outline of the indi­ vidual cells is not readily apparent in most histological preparations

(41); however, following silver impregnation, reticular fibers may be

identified between the reticular cells (Fig. 42). The penicillar artery usually exhibits a single ellipsoid sheath along its course but in the racoon and especially in the adult dog

spleens investigated, the red pulp artery was observed to bifurcate into two to three branches, each of which is surrounded by a characteristic

ellipsoid sheath consisting of the pale staining reticular cells within a delicate reticular network (Figs. 44 and 45). The sheathed artery con­ tinues as the arterial capillary. The exact manner of arterial capillary

termination was not readily observed in the present study. Silver 22 impregnation studies indicate that the termination of the arterial capil­ lary appears to enter the sinus wall in an oblique fashion while in another type of arterial termination, the capillary vessel tends to termi­ nate within the tissue spaces of the red pulp. Perhaps, both types of arterial endings may be present in the racoon spleen (50). The venous system originates as a series of anastomosing venous sinusoids within the red pulp tissue. In the walls of the venous sinusoids, the reticular fibers present a lattice network of ring-shaped and longitudinally arranged fibers (Figs. 47 and 48). These annular or ring fibers lie external to the reticulo-endothelial cells of the sinusoid.

Tangentially cut sections demonstrate the reticular fibers to be arranged as closely-set rungs of a ladder (Fig. 49). In areas where the sinusoids anastomose, the arrangement of the reticular fibers may be likened to that of a honeycomb pattern. Both the longitudinal and annular fibers become continuous with the reticular fibers within the red pulp tissue. The sinusoids communicate with the pulp or collecting veins. These latter vessels differ from the venous sinusoids in their larger diameter and the gradual acquisition of a small amount of connective tissue prior to joining with the trabecular veins.

Woodchuck Spleen

The capsule, with its characteristic outer mesothelial layer, consists of collagenic fibers and fibroblasts in the outermost segment whereas the innermost segment consists predominantly of smooth muscle fibers between which are found a scattering of elastic fibers. The anastomosing trabecular system is lacking in the woodchuck spleen. 23

The majority of the trabeculae that are present are of the non-vascular type. These trabeculae consist of bundles of smooth muscle fibers between which a few collagenic and elastic fibers may be identified. The trabecular arteries and veins are not readily observed as in the racoon spleen where they are found within the vascular type of trabe­ cu lae. The primary follicles are large, round structures that exhibit secondary centers. Immediately adjacent to the primary follicle is the lighter stained band of tissue known as the marginal zone. The latter area, in turn, is surrounded by large anastomosing series of venous sinusoids (Fig. 49). Silver impregnation reveals the outermost segment of the primary follicle to be delineated from the marginal zone by dense strands of circumferentially arranged reticular fibers. The reticular fibers within the marginal zone proper in contrast are more delicate in nature forming a web-like pattern (Fig. 50). In the woodchuck spleen, the branches of the splenic artery enter the hilus, exhibiting a true trabecular covering but tend to leave the trabeculae almost immediately to become surrounded by lymphoid tissue. A true trabecular artery as seen in the dog and racoon spleens investigated is not evident in the woodchuck spleen. Within the fol­ licle, the eccentric-positioned central artery gives rise to a number of vessels of capillary dimension that terminate in the region of the margi­ nal zone similar to the racoon spleen. The penicillar vessels enter the red pulp where they continue for a short distance as the red pulp arte­ ries. The terminal segment of the red pulp artery, the arterial capillary, is characterized by closely knit reticular fibers surrounding the 24 capillary endothelium. These vessels terminate within the pulp cords where the reticular fibers fray out and tend to blend with the reticulum of the red pulp. In the present study, the termination of the arterial capillary was not traced to a point where it directly opened into a venous sinusoid. These vessels appear to terminate, however, in close relation to the sinusoidal wall (Figs. 51 and 52).

The ellipsoid or Schweigger-Seidel sheath is not present in the woodchuck spleen. The red pulp tissue is abundant in the woodchuck spleen. It consists of plates of red pulp tissue between a vast anastomosing plexus of venous sinusoids (Figs. 53 and 54). The reticulo-endothelial cells lining the sinusoids are rod-like in shape with a nucleus that bulges into the lumen of the vessel. The annular and longitudinal reti­ cular fibers observed in the racoon spleen may also be identified in sections of the woodchuck spleen lying external to the cells lining the venous sinusoids (Fig. 55). Employment of the Periodic-acid-Schiff's- reaction reveals the outline of the anastomosing sinusoidal network as

a pink staining PAS positive band (Fig. 56). The sinusoids communicate with the large collecting veins con­ fined to the region of the red pulp tissue. The collecting veins acquire a connective tissue covering from the trabeculae only upon approaching the region of the hilus. This is the only area where a true trabecular vein is recognized. DISCUSSION

The most controversial problem encountered in investigations of the spleen is the disagreement regarding its internal vasculature.

The knowledge relating to the actual development of the vascular system in this lymphatic organ is less perplexing but still fragmentary (9), (25),

(39), (40), (45). In the present investigation, developmental studies were re­ stricted to one segment of the vascular tree, the ellipsoid sheath as it occurs in the dog spleen.

Our observations indicate that the cells comprising the ellip­ soid sheath of the dog spleen originate from a transformation of the mesenchymal cells that characterize the early splenic rudiment. These findings are in agreement with those of Ono (39) who carried out exten­ sive embryological studies on the vasculature of the human fetal spleen. He demonstrated that both the red pulp tissue and the ellipsoid sheath develop from the mesenchymal cells comprising the splenic anlage. Downey (9) reports similar findings from his observations of various developmental stages of pig embryos. In the 35-day embryonic dog spleen, the splenic anlage ap­ pears as a triangular-shaped structure in the region of the dorsal meso- gastrium. The spleen at this time is characterized by a primitive capil­ lary network with no apparent differentiation of distinct arterial or venous vessels. Sabin (45), from her studies of pig embryos, also

25 26 describes the primitive circulation of the spleen as a capillary network extending throughout the organ. This agrees with the general principal that in any organ the primitive circulation is in the form of a capillary network from which develops the definitive arterial and venous vessels. During this stage of development, the only vascular structures recog­ nized are the narrow, irregular, capillary vessels in which an occa­ sional normoblast may be identified. These primitive vessels exhibit a distinct endothelial lining that is easily identified by the staining of the cytoplasm and nucleus being more distinct than that of the surroun­ ding mesenchymal cells (2), (9), (45). The ellipsoid sheath or "Htilsenarterien," first described by Schweigger-Seidel (47), was not demonstrated in the 35-day dog embryo. In the dog spleen, the development of the ellipsoid sheath is initiated during the 40-day embryonic stage. During the latter stage of develop­ ment, the splenic rudiment becomes converted into a rather loose, mesenchymal structure consisting of blood sinuses containing nume­ rous red blood corpuscles within their lumens. The over-all appearance of the spleen at this time resembles the red pulp tissue characteristic of the adult organ; the exception being that blood cell formation is nearly or completely suppressed in the adult stage. The mesenchymal cells that comprise the splenic rudiment at this time assume a more rounded appearance in contrast to their characteristic stellate shape observed in the previous stage.

In an area of a developing ellipsoid sheath, the surrounding mesenchymal cells undergo a transformation from an ovoid-shaped cell to one that is elongated and cigar-shaped as they become oriented in a concentric fashion about the lumen of a capillary vessel. On the inner surface of the capillary lumen is a distinct endothelium. These endo­ thelial cells possess a small amount of cytoplasm and a fairly large- size nucleus. The nucleus tends to bulge into the lumen to such a degree that the lumen is often reduced in diameter. Weidenreich (58) proposed that the cells comprising the ellipsoid sheath were derived from these transformed endothelial cells. This observation is not in agreement with the present findings nor can it be substantiated by the works of previous investigators (9), (39). Downey (9), using a reti­ culum stain, demonstrated that the sheath cells were not derivatives of the dndothelial cells. This staining technique revealed the presence of reticular fibers between the cells comprising the early ellipsoid structures; however, these same fibers were not identified among the endothelial cells surrounding the lumen of the capillary vessel. The present findings are in agreement with the work of pre­ vious investigators (2), (45), who demonstrated that the ellipsoid sheath makes its appearance during the early embryonic stages of de­ velopment. These structures were found to precede the actual develop­ ment of the splenic follicles as well as the other segments of the penicillar artery. Following the initial appearance of the ellipsoid sheath, the splenic architecture of the ensuing embryonic stages re­ sembles that of the adult organ. It is speculated (45) that the ellip­ soid sheath may function in these early developmental stages as a primitive lymphoid organ. This apparent function of the ellipsoid sheath was not observed in the present study.

Further development of the ellipsoid sheath in the later embryonic stages investigated (45 and 55-days gestation) revealed numerous mitotic figures among the cells comprising the sheath. Addi­ tional cells are added to the sheath, in the absence of the mesenchymal elements that originally contributed to its formation, through the mitotic activity of these pre-existing cells. Several investigators (17), (53), have reported mitotic activity among the reticular cells comprising the adult ellipsoid sheath following injections of a colloidal dye. The reticular cells are transformed into phagocytic cells following the in­ jection of the dye. These cells then leave the ellipsoid structure and migrate into the surrounding red pulp tissue. All evidence pointed to the fact that the cells contributing to the re-formation of the ellipsoid sheath originated from mitosis occurring in the reticular cells that had not migrated out of the sheath. The ellipsoid sheath becomes a distinct structure in these later stages of development where it is seen to be composed of three to five layers of reticular cells and fibers investing the second segment of the penicillar artery — the ellipsoid vessel. The cells comprising the coelomic epithelium demonstrate variations in their shape as the splenic rudiment undergoes, progressive development. In the earlier embryonic stages, the epithelium consists of a layer of elongated columnar to cuboidal-shaped cells separated from the underlying mesenchyme by a delicate band of reticular fibers.

As development progresses, the elongated columnar-shaped cells are transformed into the typically flattened, mesothelial cells characteristic of the adult serous membrane. This transition is most evident between the 40- and 45-day embryonic stages. In the latter stage, the 2,9 mesothelial cells present a typical flattened appearance. The nucleus exhibits a distinct nuclear membrane, a prominent nucleolus, and a fine, granular, chromatin pattern. The free surfaces of the mesothelial cells were found to exhibit a microvillus border. This surfact modification is characterized by the presence of numerous protoplasmic processes resembling hair-like pro­ jections. These protoplasmic projections were observed by Cunningham

(7) on the serosal surface of the rat spleen following intra-abdominal injections of a dextrose solution. This surface modification of the mesothelial cells has been reported following electron microscopy studies (38), (34), but it has not been demonstrated very successfully with the light microscope. Electron micrographs reveal the microvilli of these mesothelial cells to be similar to those found in the tubules of the kidney and the epithelial surface of the small intestine (38). The presence of the microvilli on the free surface of these cells acts to in­ crease the surface area for the exchange of soluble substances between the cells and the peritoneal cavity (7).

These fine hair-like projections were first observed in sections of the 45-day embryonic dog spleen. Subsequent embryonic stages as well as sections of the adult dog and pig spleens also demonstrated the presence of this surface modification. Application of the periodic-acid-

Schiff's reaction demonstrates a definite PAS positive membrane between the mesothelial cells and the capsule proper. In addition, just beneath the protoplasmic projections within the cytoplasm of the mesothelial cell, a slightly darker PAS positive material was noted. This has been 30 described as the so-called "extraneous coat" representing an accumu­ lation of a glycoprotein material within the cell (11).

Racoon and Woodchuck Spleens The woodchuck and racoon spleens were found to possess characteristics unique to the respective animal. Following an extensive study of mammalian spleens, Snook

(51) proposed a classification based on the presence or absence of venous sinusoids within this organ. Spleens demonstrating the pre­ sence of venous sinusoids were termed "sinusal"; in contrast, those lacking these structures were termed "non-sinusal." Both of the above animals investigated demonstrate venous sinusoids and may be included iii Snook's classification of the "sinusal" type of spleens. The longest and widest sinusoids in both animals were observed surrounding the immediate area of the splenic follicle as the so-called paranodular

sinusoids. These paranodular sinusoids are broad, flattened structures differing from the cucumber or sweet potato-shaped sinusoids of the red pulp tissue (20). The paranodular sinusoids serve as a boundary zone between the marginal zone and the surrounding red pulp tissue. Electron microscopy studies reveal the presence of interruptions in the wall of these sinusoids thus permitting the direct passage of cellular elements as well as plasma constitutents from the area of the marginal zone into the lumen of the sinusoids (34).

The structure of the sinusoidal wall has been previously de­ scribed for various mammalian forms (5), (12), (19), (36), (58), (59). Electron microscopy studies have contributed additional information 3 0 a concerning the structure of these lining cells (60). The reticulo­ endothelial cells lining the sinusoidal wall were found to interdigitate with one another except in areas where cells were identified traversing the sinusoidal wall (34), (60). Sections of the racoon and woodchuck spleens, stained by the iron-hematoxylin technique, demonstrate the lining cells to be rod-shaped beneath which may be observed a dis­ continuous PAS positive membrane. This apparent discontinuity of the basement membrane as seen with the light microscope has recently been substantiated by electron microscopy studies of the rabbit spleen

(34), (60). The venous sinusoids when cut tangentially demonstrated an arrangement of reticular fibers closely resembling the rungs of a ladder.

In the racoon, the shorter sinusoids exhibiting frequent anastomoses demonstrate a honeycomb arrangement of the reticular fibers. This arrangement was also found in sections of the adult dog and human spleens examined. The reticulum external to these lining cells of the sinusoid has recently been shown to consist of a finely granular mate­ rial, possibly formed within the cytoplasm of the lining cells, plus distinct reticular fibers (13).

The ellipsoid sheath has received considerable study by various investigators of the spleen (28), (41), (51), (57). The mammalian

spleen may be classified on the basis of the presence or absence of an ellipsoid structure (5), (51), (57). These structures, when present in mammals, appear as swellings of reticulum surrounding the second segment of the penicillar artery. Histologically, the ellipsoid sheath in all mammalian forms examined consists of reticular cells and fibers. 31

These elliptical-shaped structures were first recognized by-

Billroth (4) and later described more extensively by Schweigger-Seidel

(47). The latter investigator examined these structures in the pig spleen describing them as oval-^shaped bodies surrounding the capillary vessel. He was unable to identify these structures in the spleens of the horse, guinea pig, and rabbit. These observations have since been confirmed by others (51), (60), (61), although the horse spleen has re­ cently been shown to possess an ellipsoid sheath (51).

The largest mammalian ellipsoid sheaths are found in the mole, pig, and dog spleens respectively (51). In the present study, the ra­ coon spleen exhibits well developed ellipsoid sheaths whereas this same structure is absent in the woodchuck spleen. Histologically, the mouse, rat, rabbit, and guinea pig spleens do not exhibit the presence of an ellipsoid structure (28), (32), (50), (54), (59), (60). The wood­ chuck may be included in the group of animals lacking an ellipsoid structure. In the dog and racoon spleens examined, the ellipsoid sheaths appear as pear-shaped structures organized about the capillary segment of the penicillar artery. In both of these animals, the ellip­ soid sheath consists of closely set reticular fibers. Immediately ad­ jacent to the capillary endothelium is a coarser arrangement of the reticular fibers comprising the so-called periendothelial network (50). The ellipsoid sheaths are separated from the surrounding red pulp tissue by very coarse reticular fibers.

In the racoon spleen, the ellipsoid sheaths exhibit a peri- nodular arrangement about the splenic follicle as well as an intimate relation with the venous sinusoids when identified within the red pulp 32 tissue. The penicillar artery upon entering the red pulp gives off branches that return to the immediate area of the respective nodule, each becoming surrounded by an ellipsoid sheath. Ellipsoids in the racoon and dog spleens are surrounded by the venous sinusoids and separated from one another by a small band of splenic pulp.

In the racoon and especially in the dog spleen, longitudinal

sections of the capillary vessel demonstrate a bifurcation into two or three divisions each of which becomes surrounded by a characteristic ellipsoid structure. These bifurcations have been described for the dog spleen as "bicornate" or "tricornate" structures (44).

Various investigators of the ellipsoid sheath are of the opinion that the endothelial cells lining the capillary vessel exhibit

small openings that provide an additional pathway for the passage of blood plasma into the surrounding pulp tissue (2), (33), (44), (56), (62).

Occasionally, red blood corpuscles may be encountered within the meshes of the sheath, indicating that corpuscular elements may also pass through these openings (2). The corpuscular elements were ob­ served within the sheaths of the pig spleen examined and only occa­ sionally in sections of the racoon and dog spleens. If patent openings

do exist between the endothelial cell nuclei and the ellipsoid sheath,

a shunt in the circulation of blood within this organ may occur at this

particular level of the vascular tree. The plasma and cellular elements

could be shunted from the capillary to the venous sinusoid by this in­

direct vascular pathway, exposing them to the potentially phagocyticr cells of the ellipsoid sheath. This property of phagocytosis, demon­

strated by the reticular cells, has recently been observed by electron 33 microscopy studies of the dog ellipsoid sheath (63). The cytoplasm of the sheath cells contained large, dense bodies identified as ingested material. If the above findings are characteristic of these cells, the filter hypothesis proposed by Schweigger-Seidel (48) may explain the presence of this particular vascular structure. The ellipsoid vessel, upon leaving the sheath, continues within the red pulp tissue as the terminal capillary. The exact manner of termination of this latter vessel constitutes the basis for the contro­ versy encountered in investigations of the internal vasculature of this organ. As far as the present study is concerned, it is not the purpose to present evidence for or against the various theories of circulation pertaining to this lymphatic organ. This has been adequately covered by previous investigators (21), (30), (31), (35), (43), (52). Snook (50) has demonstrated that several types of capillary terminations may exist within the same spleen. From his observations, the terminal capillary was found to end either as an ampullary dilation within the red pulp tissue or as a direct arteriolar-sinus connection. In addition to these capillary terminations, occasionally the terminal capillary was traced to the immediate area of the sinusoid whereupon the reticulum of the vessel frayed out to blend with the sinusoidal fibers. The latter means of termination was similar to that observed in the sections of the wood­ chuck spleens examined, although this same arrangement was not evi­ dent in the racoon spleen. It appears as though the terminal capillary of the rqcoon spleen ends within the red pulp tissue. Distinct ampul­ lary dilations were not observed in either of the above mammals examined. These ampullary segments of the terminal artery in the dog 34 spleen have been observed in the present study as well as in previous studies (8), (26), (29), (31), (51). The intermediate vessel providing the connecting link between the venous sinusoids and the trabecular veins arises within the red pulp tissue where it is designated the pulp or collecting vein. These latter vessels were found to be more prominent in the racoon than in the wood­ chuck spleen. In the former animal, the pulp veins differ from their sinusoidal tributaries by their wider diameter and straighter course they pursue within the red pulp tissue. As tributaries of the trabecular system of veins, these vessels gradually acquire a definite connective tissue layer in the immediate area of junction with a corresponding trabecular vein. The collecting veins, within the red pulp tissue, con­ sist of delicate reticular and elastic fibers (46) in both spleens exa­ mined. The capsule and trabecular system in the various spleens

examined demonstrate marked variation in their histological structure. Grossly, the capsule presents a tough, translucent coat covering the

entire organ. Histologically, it is covered externally by a layer of flattened mesothelial cells beneath which are bundles of collagenic

fibers. Among the latter fibers elastic and smooth muscle fibers may be observed. In the racoon, pig, and dog spleens, the capsule is com­

posed predominantly of smooth muscle fibers whereas these same fibers

are markedly reduced in the woodchuck and human spleens examined.

The itrabecular system, that serves as a framework for the splenic parenchyma, is reduced in the woodchuck spleen when compared to

sections of the racoon, dog, pig, and human spleens. The trabeculae 35 exhibit the same histological structure as found in the capsule. From studies of previous investigators (37), (42), (46), it appears that in rodent spleens, in general, the trabecular network is not well developed when compared with animals from other classes. Herroth (15) is of the opinion that the diversity existing in the trabecular pattern of various mammals may be responsible for differences in the storage function of the spleen. The woodchuck spleen with its reduced trabecular system may not possess the same storage capacity as the racoon whose trabe­ cular system was found to be well developed. In sections of the woodchuck spleen distinct trabecular arte­ ries are rarely observed. This is similar to the findings in the mole, mouse, and rat spleens (51). The arterial vessels entering at the hilus almost immediately become surrounded by sheaths of lymphatic tissue rather than entering a trabeculum where it would exist as a true trabe­ cular vessel. In contrast, the entering arterial vessels in the racoon spleen are accompanied into the interior of the organ by distinct tra­ beculae forming an easily identifiable trabecular vessel. In both the racoon and woodchuck the white pulp tissue appears as large, round nodules organized about the eccentrically positioned central arteries. A prominent marginal zone completely surrounds the follicle separating it from the red pulp tissue. The marginal zone is thought to function similarly to a secondary center of a primary nodule (34).

The red pulp tissue of the woodchuck spleen is represented by thick plates of tissue existing between the venous sinusoids (46). This plate-like arrangement was not apparent in the sections of the racoon 36

spleen examined. The venous sinusoids contained within this pulp -tissue are regarded as an essential component of the red pulp. Depen­ ding upon the presence or absence of these vascular compartments, the venous sinusoids, the volume of red pulp tissue varies in various

species of mammals (35), (51), (57). The racoon and woodchuck spleens examined in the above study provide additional information relating to the comparative histology of this organ. These animals each possess characteristics unique to

the particular species and may be considered as representatives of the "sinusal" type of spleens. SUMMARY AND CONCLUSIONS

Embryonic Dog Spleens 1. The objective of this investigation was to determine the possible origin of the so-called ellipsoid sheath as it occurs in the dog

spleen . 2. We postulate that the cell responsible for the embryonic development of the dog ellipsoid sheath is of mesenchymal origin. 3. The development of the ellipsoid sheath is first initiated

at 40-days gestation. At this time, the mesenchymal cells within the

splenic rudiment undergo a transformation in shape and orient them­ selves about the lumen of a capillary vessel. 4. In the later embryonic stages examined (45 and 55-days gestation), numerous mitotic figures were observed within the ellipsoid sheath. Mitosis of these pre-existing sheath cells contribute addi­ tional cellular layers to the definitive structure of the sheath. 5. We found that the free surface of the mesothelial cells, external to the capsule proper, exhibit cytoplasmic projections of a microvillus nature. These surface modifications were first observed in sections from an embryo of 40-days gestation.

Woodchuck and Racoon Spleens

1. In our observations of the racoon and woodchuck spleens, the histological sections examined revealed characteristics unique to the respective animal. 37 38 2. We found that the racoon and woodchuck spleens both exhibit the presence of venous sinusoids within the red pulp tissue.

They may be included in the classification of the “sinusal" type of sp leen s. 3. The racoon demonstrates well-developed ellipsoid sheaths investing the capillary segment of the penicillar artery; however, these same structures are absent in the woodchuck spleen. 4. A prominent marginal zone surrounding the individual fol­ licles is well demonstrated in both animals examined. 5. Our observations indicate that the trabecular system is markedly reduced in the woodchuck spleen when compared with sec­ tions of the racoon and adult dog spleens. 6. The red pulp of the woodchuck spleen is represented by thick plates of tissue between the venous sinusoids. This plate-like arrangement of red pulp tissue was not apparent in the sections of racoon spleen studied. 7. The exact manner of arterial termination in neither the racoon nor the woodchuck was determined by the technique employed in the above study. BIBLIOGRAPHY

ARMED FORCES INSTITUTE OF PATHOLOGY. M anual of H istologic and Special Staining Technics. New York: McGraw Hill Book Company, Inc., 1960. BANNWARTH, F. "Untersuchugen ueber die milz," Arch, f. Mikr. Anat.: 38, 1891, 345-350. BENSLEY, R. R. and S. H. BENSLEY. Handbook of Histological and Cytological Technique. Chicago: University of Chicago Press, 1938. BILLROTH, T. "Beitraege zur vergleichenden histologie der milz," Arch, f. Anat. u. Physiol.: 12, 1857, 88-108. BjORKMAN, S. E ., "Splenic circulation with special reference to function of the splenic sinus walls," Acta. Med. Scandinav.: supp. 191, 1947, 1-89. CARLEIR, E. W. "Note on the minute structure of the reticulum in the cat's spleen," I. Anat. and Physiol. : 29, 1895, 479-486. CUNNINGHAM, R. S. "The physiology of the serous membranes," Physiol. Rev. : 6., 1962, 242-280.

DOGGETT, T. H. "The capillary system of the dog's spleen," Anat. Rec. : 110, 1951, 65-81.

DOWNEY, H. and G. A. THIEL. "The development of the mammalian spleen, with special reference to its hematopoietic activity," Am. T. of Anat. : 28, 1920-21, 279-333. v. EBNER, V. "Ueber die wand der kapillaeren milzvenen," Anat. Anz. : 15., 1899, 482-486.

FAWCETT, D. “O bservations on the fine structure of hepatic c e lls ," T. Appl. Phys. ; j)4, 1953, 221-282.

FOOT, N. C. "On the reticulum of the human spleen," Anat. Rec. : 36, 1927, 79-87.

GALINDO, B. and J. A. FREEMAN. "Fine structure of splenic p u lp ," Anat. Rec. : 47, 1963, 25-42. 40

14. HEIDENHAIN, M. "Ueber die kapillarnentile der milz," Muenchen. Med. Wchnschr. : 75, 1928, 381-392. 15. HERRATH, E. "Bau und funktion der m ilz," Zeitschr. Zellforsch. u. Mikrosh. Anat. : .23, 1935, 375-430. 16. HUECK, W. "Die normale menschliche milz als blutbehaelter," Verhanal. d. Deutsch. Path. Ges. : 22, 1928, 6-12. 17. IMAI, T. "Reticular framework of the Schweigger-Seidel sheath of the spleen," Hukuaka Acta. Med. : 38, 1947, 1-5. 18. JANOSIK, J. "U eber die blutzirkulation in der m ilz ," Arch, f. Mikr. Anat. : 62, 1903, 580-589. 19. KLEMPERER, P. The Spleen. In Downey's Handbook of Hematology. New York: Paul B. Hoeber, Inc., 3, 1938, 1591-1754.

20. KNISLEY, M. H. "Microscopic observations on circulatory sys­ tems of living transilluminated mammalian spleens and parturient uteri," Proc. Soc. Exp. Biol, and Med.3 2 : 1934, 212-214. 21. ______. "Spleen studies: 1. Microscopic observations of the circulatory system of living transilluminated mammalian spleens. 2. Microscopic observations of the circulatory sys­ tem of living traumatized spleens and of dying spleens," Anat. Rec. : 65, 1936, 23-50, 131-148. 22. KULTSCHITZKY, N. "Zur fraqe ueber den bau der m ilz,11 Arch, f. Mikr. Anat. : .46, 1895, 673-680. 23. KYBER, E. "Ueber die milz des menschen und einiger saeugetiere," Arch, f. mikr. Anat. : 6., 1870, 540-547. 24. LAGUESSE, G. E. “Le tis s u splenique et son d eveloppem ent," Anat. Anz. lahrg. : 6^, 1891, 131-134.

25. LEWIS, O. J. “The development of the circulation in the spleen of the foetal rabbit," T. Anat. : 90, 1956, 282-289. 26. LI, P. L ., H. S. D. GARVEN and R. H. MOLE. “The microscopic anatomy of the vascular system of the dog's spleen," I. Roy. Microsc. Soc. : j^9, 1929, 110-119. 27. LILLIE, R. D. Histopathologic Technic. Philadelphia: The Blakis- ton C o., 1948.

28. MacKENZIE, D. W. Jr., A. O. WHIPPLE and M . P. WINTERSTEINER. "Direct observations on the circulation of blood in transillu­ minated mammalian spleens," Proc. Soc. Exp. Biol. and Med.: 44, 1940, 139-142. 41

29. MALL, F. M. "The architecture and blood vessels of the dog's spleen," Zeit. f. Morphologie u. Anthrop.1_, 1900,: 1-43.

30. ______. "The circulation of bldod in the dog's spleen," Am. T. Anat. : 2 , 1902, 30-40. 31. ______. "On the circulation through the pulp of the dog's spleen," Am. T. Anat. : _2, 1902-03, 315-332.

32. McNEAL, W. J. "The circulation of the blood through the splenic pulp, " Arch. Path. : 7., 1929, 215-227.

33. MILLS, E. S. "The vascular arrangement of the mammalian spleen," Quart. Tour. Exp. Physiol. : L6, 1927, 301-319.

34. MOORE, R. D ., V. R. MUMAW and M. D. SCHOENBERG. “The structure of the spleen and its functional implications," Exp. and Mol. Path. : J3, 1964, 31-50.

35. MOLLENDORFF, W. Handbuch der mikropischen Anatomie des Menschen. Berlin: Julius Springer, 1930, Vol. 6..

36. MOLLIER, S. "Ueber der bau der kapillaren der milzvene (Milzsinus)," Arch, f. Mikr. Anat. : 76, 1910-11, 25-40.

37. MILLER, W. "Ueber den feineren bau der milz." Milz. In Strieker, S. : Hanbuch der Lehre von den Geweben. Leipzig: Englemann, 1865. 38. ODOR, L. D. "Observations of the rat mesothelium with the electron and phase microscope," Am. T. of Anat. : 54, 1954, 433-465.

39. ONO, K. "Untersuchungen ueber die entwicklung der menschlichen milz," Ztschr. f. Zellforsch. u. Mikr. Anat. : 10, 1930, 573-640.

40. RADFORD, M . “The developm ent of the sp le e n ," J. Anat. and Physiol. : 42, 1908, 288-301. 41. REIDEL, H. "Vascular system of the cat spleen," Ztschr. f. Zellforsch. u. Mikr. Anat. : 1_5, 1932, 459-529.

42. REISSNER, H. "U ntersuchungen ueber die form des balken- geruestwerks," Ztschr. f. Mikr. Anat. Forsch. : 23, 1871, 95-102.

43. ROBINSON, W^L. “The vascular mechanism of the spleen," Am. T. Path. : 2, 1926, 341-356. 42

44. . "The venous drainage of the cat spleen," Am.T. P ath.: 2, 1926, 341-356. 45. SABIN, F. "The development of the spleen," in Keibl, F. and F.P. Mall, Manual of Human Embryology. London: Lippencott, 1910, 2 . 46. SCHUHMACHER, S. “Ueber die natur der zirkularen fasern der kanillaren milzvenen." Anat. Anz. : 18, 1900, 27-30. 47. SCHWEIGGER-SEIDEL, F. "Untersuchungen ueber die milz, " Virchows Arch. : 23^, 1862, 526-532. 48. ______. "Untersuchungen ueber die milz," Virchows Arch. : 27, 1863, 460-468. 49. SNOOK, T. "The reticulum stain," Anat. Rec. : 89^, 1949, 413-427. 50. . "The guinea pig spleen. Studies on the structure and connections of the venous sinuses," Anat. Rec. : 89, 1949, 413-427. 51. ______. "A com parative study of the v ascu lar arrangem ent in mammalian spleens," Am. T. of Anat. : 87, 1950, 31-61. 52. ______. "Arterial terminations in the red pulp of the rabbit's spleen," Anat. Rec. : 124, 1956, 249-260. 53. SOLNITZKY, O. "The Schweigger-Seidel sheath of the spleen," Anat. Rec. : j59, 1937, 55-64. 54. STAEMMELER, M. "Die bedeutung der Schweigger-Seidel capillar- hulsen der milz," Virchows Arch, f. Path. Anat. : 25, 1925, 585-598. 55. STIEDA, L. “Zur histologie der milz," Virchows Arch. : 24, 1862, 540-560. 56. TEITEL-BERNARD, A. "Ueber die blutzikulation in der hundemilz," Ztschr. f. Zellforsch. u. Mikr. Anat. : 12, 1931, 544-550.

57. WATZKA, M. "Study of the spleen of mammals, capillary sheaths, megakaryocytes, sinus endothelium, and plasma disentigra- tion," Ztschr. f. Mikr. Anat. Forsch. : 41, 1937, 452-498.

58. WEIDENREICH, F. "Das gefaess-system der menschlichen milz," Arch, f. Mikr. Anat. : 58, 1901, 247-376. 43

59. WEISS, L. "A study of the structure of splenic sinuses in man and the albino rat with the light microscope and the electron micro­ scope,M J^Bioghy^^nd^ioclien^Cxtol. : _3, 1957, 597-610.

60. ______- . "The structure of intermediate vascular pathways in the spleen of rabbits," Am. J. Anat. : 113, 1963, 51-92.

61. ______. "The white pulp of the spleen, " Bull, of the Johns Hopkins Hospital. : 115, 1964, 99-173. 62. WHITING, H. "On the comparative histology and physiology of the spleen," Exp. T. of Exp. Physiol. : 38, 1897, 253-316. 63. ZWILLENBERG, L. O. and H. H. ZWILLENBERG. "Electron micro­ scopic observations on sheated arterioles in the spleen of dogs," Expert entia. : 18, 1962, 136-137. PLATE I

Figure 1. Thirty-five Day Embryonic Dog Spleen Stage. The spleen presents a triangular-shaped structure attached to the dorsal mesogastrium. Masson Trichrome Stain. 100X. A-splenic rudiment; B-dorsal mesogastrium

Figure 2. Thirty-five Day Embryonic Dog Spleen Stage. This section was stained for the demonstration of reticular fibers. Snook's

Reticulum Stain. 100X. R-reticular fiber; C-capillary vessels

Figure 3. Thirty-five Day Embryonic Dog Spleen Stage. Higher magnification of the photomicrograph seen in Figure 2. Note the delicate reticular fibers in the area of the future pulp spaces. A band of reticular fibers may also be seen to separate the coelomic epithelium from the underlying mesenchyme of the splenic rudiment. 900X. R-reticular fiber; C-capillary vessels; E-coelomic epithelium.

44 * * 5

SllM L i

PLATE I PLATE II

Figure 4. Thirty-five Day Embryonic Dog Spleen. The irregu­ lar, narrow capillary channels are characteristic of the splenic rudiment at this stage. Hematoxylin and Eosin. 400X. C-capillary vessel;

E-endothelial cell nucleus

Figure 5. Thirty-five Day Embryonic Dog Spleen. This sec­ tion demonstrates the union of the capillary vessels of the rudiment with the primordial splenic vein within the dorsal mesogrstrium. Masson

TrichromeJ3tain. 100X. C-capillary vessel; V-splenic vein

Figure 6. Thirty-five Day Embryonic Dog Spleen. The mesen­ chymal cells within the dorsal mesogastrium, A, present a typical stellate appearance. The cells in B represent the condensed mesen­ chymal cells of the splenic rudiment. Hematoxylin and Eosin. 400X.

46 **7

PLATE II PLATE III

Figure 7. Forty Day Embryonic Dog Spleen Stage. General shape of the splenic rudiment during this stage of development. Hematoxylin and Eosin. 100X. S-splenic rudiment

Figure 8. Forty Day Embryonic Dog Spleen. The nucleated red blood corpuscles are present within the lumen of the capillary vessel. Hematoxylin and Eosin. 440X. C-capillary vessel

Figure 9. Forty Day Embryonic Dog Spleen Stage. The splenic rudiment presents a looser arrangement of the mesenchymal cells.

Hematoxylin and Eosin. 440X.

48 PLATE III PLATE IV

Figure 10. Forty Day Embryonic Dog Spleen Stage. Note the endothelial lined blood channels containing the nucleated red blood cells. Hematoxylin and Eosin. 440X. M-mesothelial cell

Figure 11. Forty Day Embryonic Dog Spleen Stage. The arrows indicate an area of a developing ellipsoid sheath. Hematoxylin and Eosin. 440X. M-mesothelial cells.

Figure 12. Forty Day Embryonic Dog Spleen Stage. Developing ellipsoid sheath. Notfe.the elongated shape assumed by the mesen­ chymal cells nearest the capillary lumen. Masson Trichrome Stain. 970X. L-lumen; S-sheath cells

Figure 13. Forty Day Embryonic Dog Spleen Stage. Photo­ micrograph representing a section of a developing ellipsoid sheath as outlined by the arrows. Hematoxylin and Eosin. 970X.

50 PLATE IV PLATE V

Figure 14. Forty Day Embryonic Dog Spleen Stage. Developing ellipsoid sheath. Hematoxylin and Eosin. 970X C-capillary lumen

Figure 15. Forty Day Embryonic Dog Spleen Stage. Developing ellipsoid sheath. Note the elongated cigar-shaped cells. Masson

Trichrome Stain. 970X. C-capillary lumen

Figure 16. Forty Day Embryonic Dog Spleen Stage. Developing ellipsoid sheath. Masson Trichrome Stain. 100X.

52 i 5 3

PLATE V PLATE VI

Figure 17. Forty-five Day Embryonic Dog Spleen Stage. This section represents the microvilli observed on the surface of the meso- thelial cells. Masson Trichrome Stain. 500X. M-microvilli

Figure 18. Forty-five Day Embryonic Dog Spleen Stage. Trabecular vein joining with the splenic vein. Hematoxylin and Eosin. 12OX. T-trabecular vein; S-splenic vein; A-trabecular artery

Figure 19. Forty-five Day Embryonic Dog Spleen Stage.

Longitudinal section of the trabecular artery. Hematoxylin and Eosin.

50OX. A-trabecular artery; E-ellipsoid structure

54 55

PLATE VI PLATE VII

Figure 20. Forty-five Day Embryonic Dog Spleen Stage.

Demonstration of the ellipsoid vessel as it appears during this stage of development. Hematoxylin and Eosin. 500X. E-ellipsoid sheath

Figure 21. Forty-five Day Embryonic Dog Spleen Stage. Same as the above. Hematoxylin and Eosin. 500X. E-ellipsoid sheath

Figure 22. Forty-five Day Embryonic Dog Spleen Stage. Oil immersion of the ellipsoid vessel seen in Figure 21. Hematoxylin and

Eosin. 1100X.

56 9T

PLATE VII PLATE VIII

Figure 23. Fifty-five Day Embryonic Dog Spleen Stage. Mesothelial cell with microvilli. Masson Trichrome Stain. 500X.

M-microvilli

Figure 24. Forty-five Day Embryonic Dog Spleen Stage.

General architecture of the spleen during this stage of development.

Hematoxylin and Eosin. 125X.

Figure 25. Fifty-five Day Embryonic Dog Spleen Stage. This section represents the development of the definitive white pulp tissue around the arterial vessels. Toluidine Blue. 125X. W-whitepulp tissue

Figure 26. Fifty-five Day Embryonic Dog Spleen Stage. General architecture of the spleen. Toluidine Blue. 125X. W-white pulp tissue; R-red pulp tissue; C-central artery

58 P L A T E VIII PLATE IX

Figure 27. Fifty-five Day Embryonic Dog Spleen Stage.

Reticular stroma of the white pulp areas. Snook's Reticulum Stain.

125X. W-white pulp tissue and reticular fibers

Figure 28. Fifty-five Day Embryonic Dog Spleen Stage.

General appearance of the spleen following silver impregnation. Snook's

Reticulum Stain. 125X.

Figure 29. Fifty-five Day Embryonic Dog Spleen Stage. Demon­ stration of the reticular figers within the white pulp tissue. Snook's Reticulum Stain. 50OX. W-white pulp tissue; R-reticular fibers;

C-central artery

60 P L A T E IX PLATE X

Figure 30. Fifty-five Day Embryonic Dog Spleen Stage.

Reticular fibers of the ellipsoid sheath. Snook's Reticulum Stain, 1100X. R-reticular fibers

Figure 31. Fifty-five Day Embryonic Dog Spleen Stage. Bifurcation of the red pulp artery into two ellipsoid vessels. The

arrows indicate the outline of the ellipsoid vessels. 480X.

C-capillary lumen; M-mitotic figure

62 PLATE X PLATE XI

Figure 32. Fifty-five Day Embryonic Dog Spleen Stage. Representative stages of mitosis as demonstrated within the ellipsoid sheath. A and B-Hematoxylin and Eosin; C-Masson Trichrome Stain.

50 OX.

Figure 33. Fifty-five Day Embryonic Dog Spleen Stage. Oil immersion of a mitotic figure within the ellipsoid sheath. Hematoxylin and Eosin. 1100X.

Figure 34. Fifty-five Day Embryonic Dog Spleen Stage. Oil immersion of photomicrograph C of Figure 32. Masson Trichrome Stain. 110 OX.

64 65

V *

PLATE XI PLATE XII

Figure 35. Adult Racoon Spleen. This section demonstrates the distribution of the elastic fibers within the capsule. Weigert's

Elastic Connective Tissue Stain. 480X. M-mesothelial cells;

C-capsule with elastic fibers

Figure 36. Adult Racoon Spleen. The presence of both the vascular and non-vascular trabeculae are evident in this photomicro­ graph. Weigert's Elastic Connective Tissue Stain. 120X. N-non-vascular trabeculae; V-vascular trabeculae with trabecular artery and vein.

Figure 37. Adult Racoon Spleen. Lymphatic nodule with the lighter staining peripheral marginal zone. Hematoxylin and Eosin. 480X. M-marginal zone; L-lymphytic nodule

66 P L A T E XII PLATE XIII

Figure 38. Adult Racoon Spleen. The distribution of the ellipsoid sheaths within the red pulp tissue. Iron-Hematoxylin. 12OX. E-ellipsoid sheath; C-central artery; T-trabecular vein

Figure 39. Adult Racoon Spleen. Bifurcation of an ellipsoid vessel. Note the dark staining endothelial cell nuclei in contrast to the pale staining nuclei of the priinitive reticular cells within the sheath proper. Iron-Hematoxylin. 980X. E-endothelial cell nucleus; R-reticular cell nucleus

68 PLATE XIII PLATE XIV

Figure 40. Adult Racoon Spleen. Longitudinal section of the ellipsoid artery demonstrating the basement membrane between the endothelial cells and sheath cells. Masson's Trichrome Stain. 980X.

B-basement membrane.

Figure 41. Adult Racoon Spleen, Ellipsoid sheath. The darker staining nuclei represent the endothelial cell nuclei; paler staining nuclei, the sheath cells. Masson Trichrome Stain. 980X.

Figure 42. Adult Racoon Spleen. Demonstration of the reti­ cular fibers following silver impregnation. Snook's Reticulum Stain.

R-reticular fibers within the sheath. 98OX.

70 PLATE PLATE XV

Figure 43. Adult Racoon Spleen. Bifurcation of the red pulp artery into two ellipsoid vessels. Snook's Reticulum Stain. 980X. R-red pulp artery; E-ellipsoid sheath

Figure 44. Adult Dog Spleen. Bifurcation of the red pulp

artery into two ellipsoid sheaths. Snook's Reticulum Stain. 980X.

R-red pulp artery; E-ellipsoid sheath

Figure 45. Adult Racoon Spleen. Longitudinal section of the ellipsoid vessel. Snook's Reticulum Stain. 480X. E-ellipsoid vessel;

S-sinusoids

72 73

PLATE XV PLATE XVI

Figure 46. Adult Racoon Spleen. Distribution of the reticular fibers in relation to the venous sinusoids. Snook's Reticulum Stain.

480X.

Figure 47. Adult Racoon Spleen. Same as the above. Note the lattice-like arrangement of the reticular fibers. Snook's Reticulum

Stain. 4 8 OX.

Figure 48. Adult Racoon Spleen. Same as the above. Snook's

Reticulum Stain. 480X.

74 75

PLATE XVI PLATE XVII

Figure 49. Adult Woodchuck Spleen. Primary follicle with the perifollicular sinusoids. Masson Trichrome Stain. 480X.

P-perifollicular sinusoids; M-marginal zone

Figure 50. Adult Woodchuck Spleen. Demonstration of the

marginal zone surrounding the primary follicle. Snook's Reticulum

Stain. 12OX. M-marginal zone

Figure 51. Adult Woodchuck Spleen. The reticular fibers

of the red pulp artery may be seen to fray out and blend with the reticular fibers of the red pulp tissue. Snook's Reticulum Stain. 120X.

Figure 52. Adult Woodchuck Spleen. Termination of the

arterial capillary within the red pulp cord. Masson Trichrome Stain. 980X. A-arterial capillary

76 7 7

Matisses

PLATE XVII PLATE XVIII

Figure 53. Adult Woodchuck Spleen. Anastomosing network of venous sinusoids. Snook's Reticulum Stain. 480X. T-trabecular tis s u e

/ Figure 54. Adult Woodchuck Spleen. Venous sinusoids.

Note the sporadic distribution of the trabecular tissue. Iron-

Hematoxylin. 12 OX.

Figure 55. Adult Woodchuck Spleen. Venous sinusoids demonstrating the distribution of the annular and longitudinal reticular fibers. Snook's Reticulum Stain. 980X. R-red pulp tissue;

S -sinusoid

78 79

PLATE XVIII PLATE XIX

Figure 56. Adult Woodchuck Spleen. Venous sinusoids.

Periodic-acid-Schiff's Reaction. 980X

80