THE ANATOMICAL RECORD 232:15-24 (1992)
Periarterial Lymphoid Sheath in the Rat Spleen: A Light, Transmission, and Scanning Electron Microscopic Study SHUNICHI SASOU AND TAMOTSU SUGAI Division of Pathology, Central Clinical Laboratory, Zwate Medical University, School Medicine, Morioka, Japan
of
ABSTRACT
The periarterial lymphoid sheath (PALS) in the rat spleen was studied by light, transmission, and scanning electron microscopy. The PALS was divided into three regions: the central region, peripheral region, and marginal zone bridging channel. In the central region, lymphocytes were easily washed away by perfusion. Large spaces were found between flat reticular cells or in large meshworks of stellate reticular cells; these may be deep lymphatic vessels. True lymphatic vessels were found in the central region near the hilus. In the marginal zone bridging channel, flat reticular cells surrounded the central artery in a circumferential pattern and formed channel-like spaces between the flat reticular cells. These spaces were connected with the meshwork of the red pulp reticular cells and may be a route for lymphocytes between the deep lymphatic vessels and the red pulp. In the peripheral region of the PALS, it was usually difficult to wash away free cells by perfusion, and free cells were found among the reticular cells. In places in the peripheral region, however, free cells were washed away. I t is suggested that the lymph flow may start from the region surrounding the PALS, and that the peripheral region of the PALS may also be another route for lymphocyte migration.
The white pulp of the spleen is usually divided into two parts, namely, the periarterial lymphoid sheath (PALS), which contains T lymphocytes, and the lymph follicle, which contains B lymphocytes (Waksman et al., 1962; Weiss, 1965; Muller-Hermelink, 1974; Veerman, 1974; Veerman and van Ewijk, 1975). The lymphocytes in the PALS are continually recirculating (Sprent, 1973). Intravenously (IV) injected B lymphocytes enter the PALS through the marginal zone and reticulin sheaths surrounding the terminal arterioles. Small numbers of injected cells migrate into the efferent lymphatic vessels (van Ewijk and van der Kwast, 1980). Deep lymphatic vessels are found around the central arteries (Fukuda, 1963; Janout and Weiss, 1972; Koshikawa et al., 1984) and arising from the follicles (Fukada, 1963). The lymph flow direction of deep splenic lymphatics runs counter to the blood flow (Janout and Weiss, 1972). It has been reported that there is a strong lymph flow from the red pulp to the deep lymphatic vessels in the PALS, and lymphocytes in the PALS are easily depleted by perfusion of the spleen via the splenic artery (Koshikawa et al., 1984). However, little study has so far been carried out on the basic structure of the PALS. In previous studies, we examined the perifollicular region, the arteries, and the blood distribution in human and r a t spleens, and reported the significance of the marginal zone and termination of the splenic artery (Sasou et al., 1976, 1980, 1982, 1986; Sasou and Satodate, 1979; Satodate et al., 1971, 1977, 1986). Just a little evidence concerning the white pulp was reported in a previous paper (Sasou et al., 1986). The aim of the present study was to clarify the basic structure of 0 1992 WILEY-LISS, INC
the periarterial region of the white pulp in the rat spleen, using light microscopy and transmission and scanning electron microscopy. MATERIALS AND METHODS
Five untreated adult male rats of the Wistar strain, each weighing from 200-300 g, were used. Heparin (2,500 IU, IV) was given 15 minutes prior to sodium pentobarbital anesthesia. The thoracic aorta, hepatic artery, and mesenteric arteries were ligated. The spleens were then immediately perfused with Ringer’s solution, via the abdominal aorta, at a pressure of 150 cm H,O. During perfusion the portal vein was closed off with tweezers until i t became maximally dilated, after which i t was opened. This procedure was repeated until the spleen became pale in color. The spleens were fixed by perfusion with a 2.8% glutaraldehyde solution adjusted to pH 7.4 with a phosphate buffer. They were then removed and cut into several pieces. Some pieces were further fixed in 20% formalin for paraffin sectioning, some pieces were fixed in a 2.8% glutaraldehyde solution, adjusted to pH 7.4 with a phosphate buffer, for 2 hours, for transmission electron microscopic examination, and the remainder of the specimens were fixed in a 2.8% glutaraldehyde solution, adjusted to pH 7.4
Received September 11, 1989; accepted June 25, 1991. Address reprint requests to S. Sasou, M.D., Division of Pathology, Central Clinical Laboratory, Iwate Medical University, School of Medicine, 19-1Uchimaru, Morioka 020, Japan.
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S. SASOU AND T. SUGAI
Fig. 1. Low magnification of the rat spleen near the hilus after perfusion. Lymphocytes in the central region (CR) of the PALS around the central artery (A) are decreased in number. The lymphatic
with phosphate buffer, for 24 hours, for scanning electron microscopic examination. Paraffin sections were cut a t 4-6 pm and stained with hematoxylin and eosin, the periodic acid-Schiff technique, and silver impregnation for reticular fibers (Gomori’s method). For transmission electron microscopic examination, the sections were dehydrated in graded alcohols and embedded in Epon. Sections were cut with a n LKB ultramicrotome (LKB 2080) and were stained with uranyl acetate and lead citrate. The specimens were examined and photographed with a transmission electron microscope (Hitachi H-700). For scanning electron microscopic examination, the specimens were postfixed in a 1%osmium tetroxide solution for 16 hours, dehydrated in a graded alcohol series, and broken into small pieces in liquid nitrogen. The broken specimens were immersed in isoamyl acetate for 20 minutes, then dried in CO,, using the critical point method, coated with carbon in a vacuum evaporator, and coated with platinum or gold by the ion-sputtering method. They were then examined by scanning electron microscope (Hitachi HSM-2B) at 20 KeV. Light microscopically, the white pulp was divided into PALS and lymph follicle. The PALS was further divided into the central region, the peripheral region, and the marginal zone bridging channel. These areas were also observed by transmission electron and scanning electron microscopy.
vessels (L) are seen as empty spaces in the PALS. The lymph follicles (F) and peripheral region (arrows) of the PALS are filled with lymphocytes. MZ, marginal zone. H.E. stain. x 130.
RESULTS Light Microscopic Observations
In the central region of the PALS, the lymphocytes had been washed away by perfusion and large spaces surrounded by reticular cells appeared. Lymphatic vessels were found in the central region at the hilus (Fig. 1). The lymphatic vessel was often difficult to differentiate from the spaces surrounded by reticular cells. Lymphocytes tended to remain in the peripheral region of the PALS and the marginal zone bridging channel after perfusion (Figs. 1 , 5 ) . In some areas of the peripheral region, however, lymphocytes were depleted a s much as in the central region (Fig. 2). In the lymphocyte-depleted areas of the peripheral region of the PALS, lymphocytes were often found in gaps and discontinuities of the reticular cells. In many lymph follicles, little change was caused by perfusion (Fig. 1). Reticular fibers were scant in the central region of the PALS but abundant in the peripheral region and marginal zone bridging channel (Fig. 3). Long reticular fibers appeared as lines that ran parallel with the rim of the PALS a t the peripheral region end marginal zone bridging channel (Figs. 3, 5 ) . Some of the lines of reticular fibers consisted of a mesh of fine reticular fibers (Fig. 4). Several layers of reticular fibers parallel to the PALS showed a stratiform pattern in longitudinal sections of the PALS and a circumferential pattern
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PALS IN SPLEEN
in cross section. Lines of reticular fibers in the peripheral region became continuous with lines of reticular fibers in the marginal zone bridging channel. Spaces between reticular fibers in the marginal bridging channel showed a channel-like structure (Fig. 5). Some layers of reticular fibers were found at the periphery of the lymph follicle. Very few reticular fibers were found in the central region of the lymph follicle. Transmission Electron Microscopic Observations
There were many reticular cells in the peripheral region and a few in the central region; large spaces appeared in the central region because of the depletion of free cells by perfusion (Fig. 6). In the peripheral region of the PALS, long and short thin reticular cells were arranged parallel with the rim of the PALS, and revealed a stratiform pattern. In places, they were connected with each other by their cytoplasmic processes. Stellate reticular cells were also found, especially in the central region of the PALS. The reticular cells enclosed reticular fibers (Fig. 6), but some reticular fibers were not enclosed by the reticular cells. Lymphocytes or macrophages were often found in discontinuities of the reticular cells (Fig. 6). The reticular cells connected with the pericytes of the central artery and arterioles. Almost all the free cells remaining in the PALS after perfusion were lymphocytes (Figs. 6, 7). A few macrophages, interdigitating dendritic cells, and plasma cells were also found. After perfusion, these free cells were very sparse in the central region of the PALS, but were dense among the reticular cells in the peripheral region (Fig. 6) and the marginal zone bridging channel (Fig. 11).Some red cells were found in the PALS. Some lymphatic vessels were found in the central region near the hilus (Fig. 7). Their walls consisted of three layers; that is, two layers of thin cells and a layer of reticular fiber-like substances between the cells, although some outer parts of the wall were not covered by these cells (Figs. 7, 8). It was often difficult to distinguish the wall of the lymphatic vessel from the long reticular cells enclosing the reticular fibers. In places, pinocytotic vesicles were found in the endothelium of the lymphatic vessels (Fig. 8). Free cells were rarely found in the vessels. In the marginal zone bridging channel (Fig. ll),the reticular cells were arranged circumferentially around the central artery and were continuous with the reticular cells of the peripheral region of the PALS. Spaces between the reticular cells formed a channel-like structure and free cells were found in these spaces. Discontinuities or gaps were found in the reticular cells, through which spaces of the marginal zone bridging channel were connected with the red pulp space (Fig. 11). Free cells were found in the gaps or discontinuities of the reticular cells. The lymph follicle was filled with numerous lymphocytes (Fig. 9).However, they were not tightly compacted after perfusion and some spaces were found between them. The follicular dendritic cells were not in contact with lymphocytes and their cytoplasmic processes were retracted (Fig. 10). Reticular cells were found in the lymph follicle and were abundant in its peripheral region. The border between the central region of the PALS and the lymph follicle was demarcated by reticular cells (Fig. 9). Gaps or discontinuities of the reticular cells, through which free cells could pass, were found.
Scanning Electron Microscopic Observations
Free cells in the central region of the PALS were easily washed away by perfusion and the basic structure of the PALS then became visible (Figs. 12, 13). However, free cells remained in the lymph follicle and in most of the peripheral regions of the PALS after perfusion (Fig. 12).Free cells still remained in the marginal zone bridging channel, but became sparse after perfusion (Fig. 16). In the central region of the PALS, two regions were distinguished: namely, a region which consisted of a large meshwork of stellate reticular cells (Fig. 13) and a region consisting of large spaces surrounded by flat reticular cells (Figs. 14, 15). These spaces surrounded the central artery situated in the central part of the PALS. The shapes of the stellate reticular cells within the large meshwork were similar to those of the red pulp and marginal zone, but the cells were larger and thicker. Processes of the stellate reticular cells attached themselves t o the outer surface of the central artery and supported it. The reticular cells which formed the large spaces were irregular andlor flat in shape. In the areas with the large spaces, the reticular cells were noted to have partly flat parts as well as irregular processes (Fig. 14). They were oriented in a line, like a wall or a fence, and completely surrounded the large space. These reticular cells were found a t the border between the central region of the PALS and the lymph follicle. The gaps formed by the irregular processes of the reticular cells were of various shapes and sizes, and were wide enough for lymphocytes to pass through. Some of the flat reticular cells were attached to and partially covered the wall of the central artery. There were also gaps between neighboring flat reticular cells, through which lymphocytes could pass. It was difficult to distinguish the lymphatic vessels detected by transmission electron microscopic examination, because spaces between the reticular cells often appeared to be vessels. However, vessellike structures, shown in Figures 17 and 18, were found in the PALS. Their walls had fenestrations or gaps which were wide enough for lymphocytes to pass through. In the peripheral region of the PALS (Fig. 15), there were free cells which had not been washed away by perfusion. Although the 3-dimensional fine structure of the peripheral region was not clear, flat reticular cells and reticular fibers were often observed among the free cells in the area where serous fluid had been washed away by perfusion. The peripheral region was continuous with the peripheral parts of the lymph follicle. Flat reticular cells were also found at the border between the PALS and the marginal zone, as we have already described (Sasou et al., 1980). A central artery ran through the middle part of the marginal zone bridging channel and entered the red pulp, then becoming a penicillar artery (Fig. 16).A few layers of flat reticular cells connected with those of the peripheral region and surrounded the central artery in a circumferential pattern. Spaces between the flat reticular cells formed a channel, as they connected with the large spaces and meshwork in the central region of the PALS and with the meshwork of the red pulp reticular cells. Furthermore, free cells found within the
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S. SASOU AND T.SUGAI
Figs. 2-5
19
PALS I N SPLEEN
Fig. 6. Low power electron micrograph showing the PALS after perfusion. In the central region (CR), the reticular cells are very few and large spaces are observed; lymphocytes and macrophages have become sparse because of perfusion. In the peripheral region (PR),
there are many reticular cells, among which lymphocytes, macrophages, and plasmacytes are observed to be more dense than in the central region (CR). A, central artery. x 2,400.
spaces formed a line in the marginal zone bridging channel from the red pulp t o the central region of the PALS. Free cells also passed through the gaps of the
flat reticular cells of the marginal zone bridging channel. DISCUSSION
Fig. 2. PALS after perfusion. Many free cells have been washed
away from part of the peripheral region of the PALS inside the rim (arrows) and it is difficult to distinguish the peripheral region of the PALS from the marginal zone or central region (CR). In the other part of the peripheral region (PR), many free cells still remain. A, central artery. H.E. stain. x 330. Fig. 3. Low magnification of the rat spleen treated by silver impregnation for reticular fibers. Many reticular fibers, forming a stratiform pattern, are observed in the peripheral region of the PALS. In the central region of the PALS, there are a few reticular fibers. Arrows show the lymphatic vessels. However, it is difficult to distinguish many of them from spaces between the reticular fibers. A, central artery; F, lymph follicle; MZ, marginal zone. x 130. Fig. 4. Reticular fibers of the PALS. Fine reticular fibers (arrows) form a structure like a wall or a fence. CR, central region. Reticulum stain. ~ 6 5 0 . Fig. 5. Marginal zone bridging channel, seen between the arrows. Reticular fibers show a parallel arrangement and form a channel-like structure. A, central artery. Reticulum stain. x 330.
The presence of deep lymphatics in the periarterial region of the white pulp of the spleen has been reported, but the lymphatics of the spleen appeared to vary in structure and/or in origin according to the different research reports that we examined (Snook, 1946; Fukuda, 1963; Janout and Weiss, 1972; Hokazono and Miyoshi, 1984).Janout and Weiss (1972) noted that the lymphatic vessels in the spleen lay in the connective tissue surrounding the central artery. The present study revealed that the lymphatics in the rat spleen were located in the large spaces surrounded by flat reticular cells (Figs. 1,3,6, 14, 13,although some true lymphatic vessels were found in the PALS near the hilus (Fig. 7). The lymphatic vessel structure in the PALS, as shown by scanning electron microscopic examination (Figs. 17, 18), had many fenestrations or gaps through which free cells could easily pass into the lymphatic vessel. A large meshwork of stellate reticular cells was noted in the central part of the PALS
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S. SASOU AND T. SUGAI
Figs. 7-10.
PALS I N SPLEEN
21
Fig. 11. Low power electron micrograph of the marginal zone bridging channel. Reticular cells arranged along the central artery (A) form a channel-like structure. Cells appear to be migrating through the channel. Many gaps or discontinuities (arrows) are seen in the
lines of reticular cells, through which cells appear to be penetrating from the red pulp to the marginal zone bridging channel or in the reverse direction. PA, penicillar artery; RP, red pulp. x 1,800.
through which the central artery ran. Free cells were easily washed away by perfusion; a strong lymph flow may exist, as reported by Koshikawa et al. (1984). This evidence shows that the deep lymphatics of the rat spleen may be characteristic in structure and different from the common thin-walled lymphatic capillaries of other animals, although the spleens of some
animals also have lymphatics similar to those of the rat (Janout and Weiss, 1972; Hokazono and Miyoshi, 1984). Many authors have described the deep lymphatic vessels as efferent channels (Snook, 1946; Fukuda, 1963; Janout and Weiss, 1972), which drain splenic fluid flowing within the spleen in a direction counter to the blood flow (Janout and Weiss, 1972). Koshikawa et al. (1984) thought that the PALS may act as a central station of the extravascular pathway, which is connected to both the deep lymphatic vessels and the marginal zone bridging channels. Their evidence is in agreement with our observations; namely, that the spaces and meshwork of the reticular cells in the central region of the PALS continue to the marginal zone bridging channel and then continue to the meshwork of the red pulp. Furthermore, we found that lymphocytes in some peripheral parts of the PALS, as well as in the central region, were washed away by perfusion (Fig. 2). This evidence suggests that the lymph of the rat spleen may be drained not only through the marginal zone bridging channel, but also through the peripheral region of the PALS, from the red pulp, or the marginal zone, and t h a t the peripheral region of the PALS may contain lymphocyte routes from the marginal zone to the deep lymphatic vessels.
Fig. 7. Low power electron micrograph showing lymphatic vessels (L) in the PALS. Some parts (arrows) of the outer side of the thin wall are not covered by cells. x 1,800. Fig. 8. High power electron micrograph of the wall of the lymphatic vessel shown in Figure 7. The endothelial cell is thin and has pinocytotic vesicles. An endothelial cell junction (arrow) is observed. x 13,500. Fig. 9. Electron micrograph of the border between the central region (CR) of the PALS and a lymph follicle (F). Reticular cells demarcate both areas. There are gaps (arrows) between the reticular cells. In the central region, free cells have decreased in number due t o perfusion. x 2,400. Fig. 10. High power electron micrograph of a lymph follicle. Spaces between cells have become wider after perfusion. Follicular dendritic cells have drawn in their cytoplasmic processes (arrows). x 7,200.
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S.SASOU AND T. SUGAI
Figs. 12-15.
PALS IN SPLEEN
23
Fig. 16. The marginal zone bridging channel (BC) runs through the marginal zone (MZ) to the red pulp (RP). The central artery runs through the central part of the marginal zone bridging channel and becomes a penicillar artery (PA) in the red pulp. Flat reticular cells
(arrows) of the marginal zone bridging channel surround the artery (A) and form channel-like spaces which communicate with the meshwork of the red pulp (RP). Migrating lymphocytes can be observed in the channels. MZ, marginal zone; S, venous sinus. x 680.
As described above, free cells passed through the spaces between reticular cells in the PALS and the marginal zone bridging channels around the central
artery (Figs. 6,11,15,16), suggesting t h a t this may be a route used by lymphocytes to migrate from the spleen to other places. Mitchell (1973) described the marginal zone bridging channels as a lymphocyte circulation route in the spleen, especially as a n exit pathway from the PALS to the red pulp. Other authors have reported that the PALS surrounding the terminal arterioles is one of the pathways of lymphocyte migration to the white pulp (Nieuwenhuis and Ford, 1976; van Ewijk and van der Kwast, 1980; Brelinska et al., 1984; van Ewijk and ~ i ~ ~19851, ~ although ~ ~ h B~cellsi also enter the PALS in the same way as T cells do (van Ewijk and van der Kwast, 1980; van Ewijk and Nieuwenhuis, 1985). According to Pellas and Weiss (1990), the marginal zone bridging channels may be a n entrance pathway for T and B lymphocytes from the red pulp to the PALS. In the present study, partially flat reticular cells, irregular in shape, were found at the border between the ~ymphocyte-depleted in the central region of the PALS and the lymph follicle, in which many free cells remained after perfusion (Figs. 9, 14). This finding suggests that reticular cells with characteristic structuresact as a barrier and prevent 1YmPhocYtes in the lymph follicle from entering the lymphatics of the PALS.
Fig. 12. General view of the rat spleen by scanning electron microscopy. Lymphocytes have been washed out of the PALS (PI by perfusion and the PALS is empty. However, follicular lymphocytes were not flushed out, and the follicle (F)retains its shape. In the marginal zone (MZ), a few lymphocytes still remain after perfusion. RP, red pulp; A, central artery. x 270. Fig. 13. A meshwork of stellate reticular cells, which is larger than those in the marginal zone and red pulp, can be observed in the central region (CR) of the PALS. Lymphocytes in the peripheral region (PR) have not been washed out by perfusion. MZ, marginal zone; A, central artery. x 300. Fig. 14. Large spaces of the PALS central region. Reticular cells (arrows) with a partially flat shape and irregular processes form a wall. The large gaps formed by the irregular processes of the reticular cells are observed. x 1,240.
Fig. 15. In the PALS central region (CR),flat reticular cells (arrows) form large spaces around the central artery (A). Numerous free cells are still observed in the space. Many free cells remain in the PALS peripheral region (PR). x 990.
~
,
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S. SASOU AND T. SUGAI
Fig. 17. Low magnification of the white pulp by scanning electron microscopy. Large vessels (L), suggesting lymphatic vessels, are observed in the PALS. A, central artery; PA, penicillar artery. x 270.
Fig. 18. High magnification of the vessels in Figure 17. Gaps or fenestrations are observed in the vessel wall (arrows). X 1,350.
LITERATURE CITED
electron microscopic features of spleen in the rat and human: a comparative study. Scanning Electron Microsc., 111:1063-1069. Sasou, S., R. Satodate, and A. Suzuki 1980 A scanning electron microscopic study of the perifollicular region of the rat spleen. J . Reticuloendothel. SOC.,27r461-469. Satodate, R., S. Ogasawara, S. Sasou, and S. Katsura 1971 Characteristic structure of the splenic white pulp of rats. J . Reticuloendothel. Sac., 10:248-433. Satodate, R., S. Sasou, S. Katsura, M. Yoshida, and M. Hirata 1977 Der Vergleichende histologicsche-morphometrische Untersuchung der Marginalzone and des Keimzentrums der Milzfollicle der Ratte nach Endotoxininjection. Exp. Pathol., 14r100-107. Satodate, R., H. Tanaka, S. Sasou, T. Sakuma, and H. Kaizuka 1986 Scanning electron microscopical studies of the arterial terminals in the red pulp of the rat spleen. Anat. Rec., 215r214-216. Snook, T. 1946 Deep lymphatics of the spleen. Anat. Rec., 94:43-55. Sprent, J. 1973 Circulating T and B lymphocytes of the mouse. I. Migratory properties. Cell. Immunol., 7:lO-39. Van Ewijk, W., and P. Nieuwenhuis 1985 Compartments, domains and migration pathways of lymphoid cells in the splenic pulp. Experientia, 41 :199-208. Van Ewijk, W., and T.H. van der Kwast 1980 Migration of B lymphocytes in lymphoid organs of lethally irradiated, thymocyte-reconstituted mice. Cell Tissue Res., 212:497-508. Veerman, A.J.P. 1974 On the interdigitating cells in the thymus: dependent area of the rat spleen: a relation between the mononuclear phagocytic system and T-lymphocytes. Cell tissue Res., 148:247-257, Veerman, A.J.P., and W. van Ewijk 1975 White pulp compartments in the spleen of rats and mice. A light and electron microscopic study of lymphoid and non-lymphoid cell types in T and B areas. Cell Tissue Res., 156:417-441. Waksman, B.H., B.G. Arnason, and B.D. Jankovic 1962 Role of the thymus in immune reactions in rats. 111. Changes in the lymphoid organs of thymectomized rats. J. Exp. Med., 116:187-206. Weiss, L. 1975 The structure of the normal spleen. Semin Hematol., 2t205-228.
Brelinska, R., C. Pilgrim, and I. Reisert 1984 Pathways of lymphocyte migration within the periarterial lymphoid sheath of rat spleen. Cell Tissue Res., 236:661-667. Fukuda, T. 1963 Deep lymphatics of the spleen. Tohoku J. Exp. Med., 79t281-292. Hokazono, K., and M. Miyoshi 1984 Scanning- and transmission electron-microscopic study of lymphatic vessels in the splenic white pulp of the macaque monkey. Cell Tissue Res., 237:l-6. Janout, V., and L. Weiss 1972 Deep splenic lymphatics in the marmot: an electron microscopic study. Anat. Rec., 172:197-220. Koshikawa, T., J. Asai, and S. Iijima 1984 Cellular and humoral dynamics in the periarterial lymphatic sheaths of rat spleens. Acta Pathol. Jpn., 34: 1301-13 11. Mitchell, J. 1973 Lymphocyte circulation in the spleen. Marginal zone bridging channels and their possible role in cell traffic. Immunology, 24:93-107. Muller-Hermelink, H.K. 1974 Characterization of the B-cell and Tcell regions of human lymphatic tissue through enzyme histochemical demonstration of ATPase and 5’-nucleotidase activities. Virchows Arch. [BI, 16t371-378. Nieuwenhuis, P., and W.L. Ford 1976 Comparative migration of Band T-lymphocytes in the rat spleen and lymph nodes. Cell. Immunol., 23t254-267. Pellas, T.C., and L. Weiss 1990 Deep splenic lymphatic vessels in the mouse: a route of splenic exit for recirculating lymphocytes. Am. J. Anat., 187:347-354. Sasou, S., T. Madarame, and R. Satodate 1982 Views of the endothelial surface of the marginal sinus in rat spleens using the scanning electron microscope. Virchows Arch. FBI, 4Ot117-120. Sasou, S., and R. Satodate 1979 A scanning electron microscopical study of the perifollicular region of the rat spleen. Recent Adv. Res., 19t43-53. Sasou, S., R. Satodate, and S. Katsura 1976 The marginal sinus in the perifollicular region of the rat spleen. Cell Tissue Res., 172:195203. Sasou, S., R. Satodate, T. Masuda, and K. Takayama 1986 Scanning
Periarterial Lymphoid Sheath in the Rat Spleen: A Light, Transmission, and Scanning Electron Microscopic Study SHUNICHI SASOU AND TAMOTSU SUGAI Division of Pathology, Central Clinical Laboratory, Zwate Medical University, School Medicine, Morioka, Japan
of
ABSTRACT
The periarterial lymphoid sheath (PALS) in the rat spleen was studied by light, transmission, and scanning electron microscopy. The PALS was divided into three regions: the central region, peripheral region, and marginal zone bridging channel. In the central region, lymphocytes were easily washed away by perfusion. Large spaces were found between flat reticular cells or in large meshworks of stellate reticular cells; these may be deep lymphatic vessels. True lymphatic vessels were found in the central region near the hilus. In the marginal zone bridging channel, flat reticular cells surrounded the central artery in a circumferential pattern and formed channel-like spaces between the flat reticular cells. These spaces were connected with the meshwork of the red pulp reticular cells and may be a route for lymphocytes between the deep lymphatic vessels and the red pulp. In the peripheral region of the PALS, it was usually difficult to wash away free cells by perfusion, and free cells were found among the reticular cells. In places in the peripheral region, however, free cells were washed away. I t is suggested that the lymph flow may start from the region surrounding the PALS, and that the peripheral region of the PALS may also be another route for lymphocyte migration.
The white pulp of the spleen is usually divided into two parts, namely, the periarterial lymphoid sheath (PALS), which contains T lymphocytes, and the lymph follicle, which contains B lymphocytes (Waksman et al., 1962; Weiss, 1965; Muller-Hermelink, 1974; Veerman, 1974; Veerman and van Ewijk, 1975). The lymphocytes in the PALS are continually recirculating (Sprent, 1973). Intravenously (IV) injected B lymphocytes enter the PALS through the marginal zone and reticulin sheaths surrounding the terminal arterioles. Small numbers of injected cells migrate into the efferent lymphatic vessels (van Ewijk and van der Kwast, 1980). Deep lymphatic vessels are found around the central arteries (Fukuda, 1963; Janout and Weiss, 1972; Koshikawa et al., 1984) and arising from the follicles (Fukada, 1963). The lymph flow direction of deep splenic lymphatics runs counter to the blood flow (Janout and Weiss, 1972). It has been reported that there is a strong lymph flow from the red pulp to the deep lymphatic vessels in the PALS, and lymphocytes in the PALS are easily depleted by perfusion of the spleen via the splenic artery (Koshikawa et al., 1984). However, little study has so far been carried out on the basic structure of the PALS. In previous studies, we examined the perifollicular region, the arteries, and the blood distribution in human and r a t spleens, and reported the significance of the marginal zone and termination of the splenic artery (Sasou et al., 1976, 1980, 1982, 1986; Sasou and Satodate, 1979; Satodate et al., 1971, 1977, 1986). Just a little evidence concerning the white pulp was reported in a previous paper (Sasou et al., 1986). The aim of the present study was to clarify the basic structure of 0 1992 WILEY-LISS, INC
the periarterial region of the white pulp in the rat spleen, using light microscopy and transmission and scanning electron microscopy. MATERIALS AND METHODS
Five untreated adult male rats of the Wistar strain, each weighing from 200-300 g, were used. Heparin (2,500 IU, IV) was given 15 minutes prior to sodium pentobarbital anesthesia. The thoracic aorta, hepatic artery, and mesenteric arteries were ligated. The spleens were then immediately perfused with Ringer’s solution, via the abdominal aorta, at a pressure of 150 cm H,O. During perfusion the portal vein was closed off with tweezers until i t became maximally dilated, after which i t was opened. This procedure was repeated until the spleen became pale in color. The spleens were fixed by perfusion with a 2.8% glutaraldehyde solution adjusted to pH 7.4 with a phosphate buffer. They were then removed and cut into several pieces. Some pieces were further fixed in 20% formalin for paraffin sectioning, some pieces were fixed in a 2.8% glutaraldehyde solution, adjusted to pH 7.4 with a phosphate buffer, for 2 hours, for transmission electron microscopic examination, and the remainder of the specimens were fixed in a 2.8% glutaraldehyde solution, adjusted to pH 7.4
Received September 11, 1989; accepted June 25, 1991. Address reprint requests to S. Sasou, M.D., Division of Pathology, Central Clinical Laboratory, Iwate Medical University, School of Medicine, 19-1Uchimaru, Morioka 020, Japan.
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S. SASOU AND T. SUGAI
Fig. 1. Low magnification of the rat spleen near the hilus after perfusion. Lymphocytes in the central region (CR) of the PALS around the central artery (A) are decreased in number. The lymphatic
with phosphate buffer, for 24 hours, for scanning electron microscopic examination. Paraffin sections were cut a t 4-6 pm and stained with hematoxylin and eosin, the periodic acid-Schiff technique, and silver impregnation for reticular fibers (Gomori’s method). For transmission electron microscopic examination, the sections were dehydrated in graded alcohols and embedded in Epon. Sections were cut with a n LKB ultramicrotome (LKB 2080) and were stained with uranyl acetate and lead citrate. The specimens were examined and photographed with a transmission electron microscope (Hitachi H-700). For scanning electron microscopic examination, the specimens were postfixed in a 1%osmium tetroxide solution for 16 hours, dehydrated in a graded alcohol series, and broken into small pieces in liquid nitrogen. The broken specimens were immersed in isoamyl acetate for 20 minutes, then dried in CO,, using the critical point method, coated with carbon in a vacuum evaporator, and coated with platinum or gold by the ion-sputtering method. They were then examined by scanning electron microscope (Hitachi HSM-2B) at 20 KeV. Light microscopically, the white pulp was divided into PALS and lymph follicle. The PALS was further divided into the central region, the peripheral region, and the marginal zone bridging channel. These areas were also observed by transmission electron and scanning electron microscopy.
vessels (L) are seen as empty spaces in the PALS. The lymph follicles (F) and peripheral region (arrows) of the PALS are filled with lymphocytes. MZ, marginal zone. H.E. stain. x 130.
RESULTS Light Microscopic Observations
In the central region of the PALS, the lymphocytes had been washed away by perfusion and large spaces surrounded by reticular cells appeared. Lymphatic vessels were found in the central region at the hilus (Fig. 1). The lymphatic vessel was often difficult to differentiate from the spaces surrounded by reticular cells. Lymphocytes tended to remain in the peripheral region of the PALS and the marginal zone bridging channel after perfusion (Figs. 1 , 5 ) . In some areas of the peripheral region, however, lymphocytes were depleted a s much as in the central region (Fig. 2). In the lymphocyte-depleted areas of the peripheral region of the PALS, lymphocytes were often found in gaps and discontinuities of the reticular cells. In many lymph follicles, little change was caused by perfusion (Fig. 1). Reticular fibers were scant in the central region of the PALS but abundant in the peripheral region and marginal zone bridging channel (Fig. 3). Long reticular fibers appeared as lines that ran parallel with the rim of the PALS a t the peripheral region end marginal zone bridging channel (Figs. 3, 5 ) . Some of the lines of reticular fibers consisted of a mesh of fine reticular fibers (Fig. 4). Several layers of reticular fibers parallel to the PALS showed a stratiform pattern in longitudinal sections of the PALS and a circumferential pattern
17
PALS IN SPLEEN
in cross section. Lines of reticular fibers in the peripheral region became continuous with lines of reticular fibers in the marginal zone bridging channel. Spaces between reticular fibers in the marginal bridging channel showed a channel-like structure (Fig. 5). Some layers of reticular fibers were found at the periphery of the lymph follicle. Very few reticular fibers were found in the central region of the lymph follicle. Transmission Electron Microscopic Observations
There were many reticular cells in the peripheral region and a few in the central region; large spaces appeared in the central region because of the depletion of free cells by perfusion (Fig. 6). In the peripheral region of the PALS, long and short thin reticular cells were arranged parallel with the rim of the PALS, and revealed a stratiform pattern. In places, they were connected with each other by their cytoplasmic processes. Stellate reticular cells were also found, especially in the central region of the PALS. The reticular cells enclosed reticular fibers (Fig. 6), but some reticular fibers were not enclosed by the reticular cells. Lymphocytes or macrophages were often found in discontinuities of the reticular cells (Fig. 6). The reticular cells connected with the pericytes of the central artery and arterioles. Almost all the free cells remaining in the PALS after perfusion were lymphocytes (Figs. 6, 7). A few macrophages, interdigitating dendritic cells, and plasma cells were also found. After perfusion, these free cells were very sparse in the central region of the PALS, but were dense among the reticular cells in the peripheral region (Fig. 6) and the marginal zone bridging channel (Fig. 11).Some red cells were found in the PALS. Some lymphatic vessels were found in the central region near the hilus (Fig. 7). Their walls consisted of three layers; that is, two layers of thin cells and a layer of reticular fiber-like substances between the cells, although some outer parts of the wall were not covered by these cells (Figs. 7, 8). It was often difficult to distinguish the wall of the lymphatic vessel from the long reticular cells enclosing the reticular fibers. In places, pinocytotic vesicles were found in the endothelium of the lymphatic vessels (Fig. 8). Free cells were rarely found in the vessels. In the marginal zone bridging channel (Fig. ll),the reticular cells were arranged circumferentially around the central artery and were continuous with the reticular cells of the peripheral region of the PALS. Spaces between the reticular cells formed a channel-like structure and free cells were found in these spaces. Discontinuities or gaps were found in the reticular cells, through which spaces of the marginal zone bridging channel were connected with the red pulp space (Fig. 11). Free cells were found in the gaps or discontinuities of the reticular cells. The lymph follicle was filled with numerous lymphocytes (Fig. 9).However, they were not tightly compacted after perfusion and some spaces were found between them. The follicular dendritic cells were not in contact with lymphocytes and their cytoplasmic processes were retracted (Fig. 10). Reticular cells were found in the lymph follicle and were abundant in its peripheral region. The border between the central region of the PALS and the lymph follicle was demarcated by reticular cells (Fig. 9). Gaps or discontinuities of the reticular cells, through which free cells could pass, were found.
Scanning Electron Microscopic Observations
Free cells in the central region of the PALS were easily washed away by perfusion and the basic structure of the PALS then became visible (Figs. 12, 13). However, free cells remained in the lymph follicle and in most of the peripheral regions of the PALS after perfusion (Fig. 12).Free cells still remained in the marginal zone bridging channel, but became sparse after perfusion (Fig. 16). In the central region of the PALS, two regions were distinguished: namely, a region which consisted of a large meshwork of stellate reticular cells (Fig. 13) and a region consisting of large spaces surrounded by flat reticular cells (Figs. 14, 15). These spaces surrounded the central artery situated in the central part of the PALS. The shapes of the stellate reticular cells within the large meshwork were similar to those of the red pulp and marginal zone, but the cells were larger and thicker. Processes of the stellate reticular cells attached themselves t o the outer surface of the central artery and supported it. The reticular cells which formed the large spaces were irregular andlor flat in shape. In the areas with the large spaces, the reticular cells were noted to have partly flat parts as well as irregular processes (Fig. 14). They were oriented in a line, like a wall or a fence, and completely surrounded the large space. These reticular cells were found a t the border between the central region of the PALS and the lymph follicle. The gaps formed by the irregular processes of the reticular cells were of various shapes and sizes, and were wide enough for lymphocytes to pass through. Some of the flat reticular cells were attached to and partially covered the wall of the central artery. There were also gaps between neighboring flat reticular cells, through which lymphocytes could pass. It was difficult to distinguish the lymphatic vessels detected by transmission electron microscopic examination, because spaces between the reticular cells often appeared to be vessels. However, vessellike structures, shown in Figures 17 and 18, were found in the PALS. Their walls had fenestrations or gaps which were wide enough for lymphocytes to pass through. In the peripheral region of the PALS (Fig. 15), there were free cells which had not been washed away by perfusion. Although the 3-dimensional fine structure of the peripheral region was not clear, flat reticular cells and reticular fibers were often observed among the free cells in the area where serous fluid had been washed away by perfusion. The peripheral region was continuous with the peripheral parts of the lymph follicle. Flat reticular cells were also found at the border between the PALS and the marginal zone, as we have already described (Sasou et al., 1980). A central artery ran through the middle part of the marginal zone bridging channel and entered the red pulp, then becoming a penicillar artery (Fig. 16).A few layers of flat reticular cells connected with those of the peripheral region and surrounded the central artery in a circumferential pattern. Spaces between the flat reticular cells formed a channel, as they connected with the large spaces and meshwork in the central region of the PALS and with the meshwork of the red pulp reticular cells. Furthermore, free cells found within the
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S. SASOU AND T.SUGAI
Figs. 2-5
19
PALS I N SPLEEN
Fig. 6. Low power electron micrograph showing the PALS after perfusion. In the central region (CR), the reticular cells are very few and large spaces are observed; lymphocytes and macrophages have become sparse because of perfusion. In the peripheral region (PR),
there are many reticular cells, among which lymphocytes, macrophages, and plasmacytes are observed to be more dense than in the central region (CR). A, central artery. x 2,400.
spaces formed a line in the marginal zone bridging channel from the red pulp t o the central region of the PALS. Free cells also passed through the gaps of the
flat reticular cells of the marginal zone bridging channel. DISCUSSION
Fig. 2. PALS after perfusion. Many free cells have been washed
away from part of the peripheral region of the PALS inside the rim (arrows) and it is difficult to distinguish the peripheral region of the PALS from the marginal zone or central region (CR). In the other part of the peripheral region (PR), many free cells still remain. A, central artery. H.E. stain. x 330. Fig. 3. Low magnification of the rat spleen treated by silver impregnation for reticular fibers. Many reticular fibers, forming a stratiform pattern, are observed in the peripheral region of the PALS. In the central region of the PALS, there are a few reticular fibers. Arrows show the lymphatic vessels. However, it is difficult to distinguish many of them from spaces between the reticular fibers. A, central artery; F, lymph follicle; MZ, marginal zone. x 130. Fig. 4. Reticular fibers of the PALS. Fine reticular fibers (arrows) form a structure like a wall or a fence. CR, central region. Reticulum stain. ~ 6 5 0 . Fig. 5. Marginal zone bridging channel, seen between the arrows. Reticular fibers show a parallel arrangement and form a channel-like structure. A, central artery. Reticulum stain. x 330.
The presence of deep lymphatics in the periarterial region of the white pulp of the spleen has been reported, but the lymphatics of the spleen appeared to vary in structure and/or in origin according to the different research reports that we examined (Snook, 1946; Fukuda, 1963; Janout and Weiss, 1972; Hokazono and Miyoshi, 1984).Janout and Weiss (1972) noted that the lymphatic vessels in the spleen lay in the connective tissue surrounding the central artery. The present study revealed that the lymphatics in the rat spleen were located in the large spaces surrounded by flat reticular cells (Figs. 1,3,6, 14, 13,although some true lymphatic vessels were found in the PALS near the hilus (Fig. 7). The lymphatic vessel structure in the PALS, as shown by scanning electron microscopic examination (Figs. 17, 18), had many fenestrations or gaps through which free cells could easily pass into the lymphatic vessel. A large meshwork of stellate reticular cells was noted in the central part of the PALS
20
S. SASOU AND T. SUGAI
Figs. 7-10.
PALS I N SPLEEN
21
Fig. 11. Low power electron micrograph of the marginal zone bridging channel. Reticular cells arranged along the central artery (A) form a channel-like structure. Cells appear to be migrating through the channel. Many gaps or discontinuities (arrows) are seen in the
lines of reticular cells, through which cells appear to be penetrating from the red pulp to the marginal zone bridging channel or in the reverse direction. PA, penicillar artery; RP, red pulp. x 1,800.
through which the central artery ran. Free cells were easily washed away by perfusion; a strong lymph flow may exist, as reported by Koshikawa et al. (1984). This evidence shows that the deep lymphatics of the rat spleen may be characteristic in structure and different from the common thin-walled lymphatic capillaries of other animals, although the spleens of some
animals also have lymphatics similar to those of the rat (Janout and Weiss, 1972; Hokazono and Miyoshi, 1984). Many authors have described the deep lymphatic vessels as efferent channels (Snook, 1946; Fukuda, 1963; Janout and Weiss, 1972), which drain splenic fluid flowing within the spleen in a direction counter to the blood flow (Janout and Weiss, 1972). Koshikawa et al. (1984) thought that the PALS may act as a central station of the extravascular pathway, which is connected to both the deep lymphatic vessels and the marginal zone bridging channels. Their evidence is in agreement with our observations; namely, that the spaces and meshwork of the reticular cells in the central region of the PALS continue to the marginal zone bridging channel and then continue to the meshwork of the red pulp. Furthermore, we found that lymphocytes in some peripheral parts of the PALS, as well as in the central region, were washed away by perfusion (Fig. 2). This evidence suggests that the lymph of the rat spleen may be drained not only through the marginal zone bridging channel, but also through the peripheral region of the PALS, from the red pulp, or the marginal zone, and t h a t the peripheral region of the PALS may contain lymphocyte routes from the marginal zone to the deep lymphatic vessels.
Fig. 7. Low power electron micrograph showing lymphatic vessels (L) in the PALS. Some parts (arrows) of the outer side of the thin wall are not covered by cells. x 1,800. Fig. 8. High power electron micrograph of the wall of the lymphatic vessel shown in Figure 7. The endothelial cell is thin and has pinocytotic vesicles. An endothelial cell junction (arrow) is observed. x 13,500. Fig. 9. Electron micrograph of the border between the central region (CR) of the PALS and a lymph follicle (F). Reticular cells demarcate both areas. There are gaps (arrows) between the reticular cells. In the central region, free cells have decreased in number due t o perfusion. x 2,400. Fig. 10. High power electron micrograph of a lymph follicle. Spaces between cells have become wider after perfusion. Follicular dendritic cells have drawn in their cytoplasmic processes (arrows). x 7,200.
22
S.SASOU AND T. SUGAI
Figs. 12-15.
PALS IN SPLEEN
23
Fig. 16. The marginal zone bridging channel (BC) runs through the marginal zone (MZ) to the red pulp (RP). The central artery runs through the central part of the marginal zone bridging channel and becomes a penicillar artery (PA) in the red pulp. Flat reticular cells
(arrows) of the marginal zone bridging channel surround the artery (A) and form channel-like spaces which communicate with the meshwork of the red pulp (RP). Migrating lymphocytes can be observed in the channels. MZ, marginal zone; S, venous sinus. x 680.
As described above, free cells passed through the spaces between reticular cells in the PALS and the marginal zone bridging channels around the central
artery (Figs. 6,11,15,16), suggesting t h a t this may be a route used by lymphocytes to migrate from the spleen to other places. Mitchell (1973) described the marginal zone bridging channels as a lymphocyte circulation route in the spleen, especially as a n exit pathway from the PALS to the red pulp. Other authors have reported that the PALS surrounding the terminal arterioles is one of the pathways of lymphocyte migration to the white pulp (Nieuwenhuis and Ford, 1976; van Ewijk and van der Kwast, 1980; Brelinska et al., 1984; van Ewijk and ~ i ~ ~19851, ~ although ~ ~ h B~cellsi also enter the PALS in the same way as T cells do (van Ewijk and van der Kwast, 1980; van Ewijk and Nieuwenhuis, 1985). According to Pellas and Weiss (1990), the marginal zone bridging channels may be a n entrance pathway for T and B lymphocytes from the red pulp to the PALS. In the present study, partially flat reticular cells, irregular in shape, were found at the border between the ~ymphocyte-depleted in the central region of the PALS and the lymph follicle, in which many free cells remained after perfusion (Figs. 9, 14). This finding suggests that reticular cells with characteristic structuresact as a barrier and prevent 1YmPhocYtes in the lymph follicle from entering the lymphatics of the PALS.
Fig. 12. General view of the rat spleen by scanning electron microscopy. Lymphocytes have been washed out of the PALS (PI by perfusion and the PALS is empty. However, follicular lymphocytes were not flushed out, and the follicle (F)retains its shape. In the marginal zone (MZ), a few lymphocytes still remain after perfusion. RP, red pulp; A, central artery. x 270. Fig. 13. A meshwork of stellate reticular cells, which is larger than those in the marginal zone and red pulp, can be observed in the central region (CR) of the PALS. Lymphocytes in the peripheral region (PR) have not been washed out by perfusion. MZ, marginal zone; A, central artery. x 300. Fig. 14. Large spaces of the PALS central region. Reticular cells (arrows) with a partially flat shape and irregular processes form a wall. The large gaps formed by the irregular processes of the reticular cells are observed. x 1,240.
Fig. 15. In the PALS central region (CR),flat reticular cells (arrows) form large spaces around the central artery (A). Numerous free cells are still observed in the space. Many free cells remain in the PALS peripheral region (PR). x 990.
~
,
24
S. SASOU AND T. SUGAI
Fig. 17. Low magnification of the white pulp by scanning electron microscopy. Large vessels (L), suggesting lymphatic vessels, are observed in the PALS. A, central artery; PA, penicillar artery. x 270.
Fig. 18. High magnification of the vessels in Figure 17. Gaps or fenestrations are observed in the vessel wall (arrows). X 1,350.
LITERATURE CITED
electron microscopic features of spleen in the rat and human: a comparative study. Scanning Electron Microsc., 111:1063-1069. Sasou, S., R. Satodate, and A. Suzuki 1980 A scanning electron microscopic study of the perifollicular region of the rat spleen. J . Reticuloendothel. SOC.,27r461-469. Satodate, R., S. Ogasawara, S. Sasou, and S. Katsura 1971 Characteristic structure of the splenic white pulp of rats. J . Reticuloendothel. Sac., 10:248-433. Satodate, R., S. Sasou, S. Katsura, M. Yoshida, and M. Hirata 1977 Der Vergleichende histologicsche-morphometrische Untersuchung der Marginalzone and des Keimzentrums der Milzfollicle der Ratte nach Endotoxininjection. Exp. Pathol., 14r100-107. Satodate, R., H. Tanaka, S. Sasou, T. Sakuma, and H. Kaizuka 1986 Scanning electron microscopical studies of the arterial terminals in the red pulp of the rat spleen. Anat. Rec., 215r214-216. Snook, T. 1946 Deep lymphatics of the spleen. Anat. Rec., 94:43-55. Sprent, J. 1973 Circulating T and B lymphocytes of the mouse. I. Migratory properties. Cell. Immunol., 7:lO-39. Van Ewijk, W., and P. Nieuwenhuis 1985 Compartments, domains and migration pathways of lymphoid cells in the splenic pulp. Experientia, 41 :199-208. Van Ewijk, W., and T.H. van der Kwast 1980 Migration of B lymphocytes in lymphoid organs of lethally irradiated, thymocyte-reconstituted mice. Cell Tissue Res., 212:497-508. Veerman, A.J.P. 1974 On the interdigitating cells in the thymus: dependent area of the rat spleen: a relation between the mononuclear phagocytic system and T-lymphocytes. Cell tissue Res., 148:247-257, Veerman, A.J.P., and W. van Ewijk 1975 White pulp compartments in the spleen of rats and mice. A light and electron microscopic study of lymphoid and non-lymphoid cell types in T and B areas. Cell Tissue Res., 156:417-441. Waksman, B.H., B.G. Arnason, and B.D. Jankovic 1962 Role of the thymus in immune reactions in rats. 111. Changes in the lymphoid organs of thymectomized rats. J. Exp. Med., 116:187-206. Weiss, L. 1975 The structure of the normal spleen. Semin Hematol., 2t205-228.
Brelinska, R., C. Pilgrim, and I. Reisert 1984 Pathways of lymphocyte migration within the periarterial lymphoid sheath of rat spleen. Cell Tissue Res., 236:661-667. Fukuda, T. 1963 Deep lymphatics of the spleen. Tohoku J. Exp. Med., 79t281-292. Hokazono, K., and M. Miyoshi 1984 Scanning- and transmission electron-microscopic study of lymphatic vessels in the splenic white pulp of the macaque monkey. Cell Tissue Res., 237:l-6. Janout, V., and L. Weiss 1972 Deep splenic lymphatics in the marmot: an electron microscopic study. Anat. Rec., 172:197-220. Koshikawa, T., J. Asai, and S. Iijima 1984 Cellular and humoral dynamics in the periarterial lymphatic sheaths of rat spleens. Acta Pathol. Jpn., 34: 1301-13 11. Mitchell, J. 1973 Lymphocyte circulation in the spleen. Marginal zone bridging channels and their possible role in cell traffic. Immunology, 24:93-107. Muller-Hermelink, H.K. 1974 Characterization of the B-cell and Tcell regions of human lymphatic tissue through enzyme histochemical demonstration of ATPase and 5’-nucleotidase activities. Virchows Arch. [BI, 16t371-378. Nieuwenhuis, P., and W.L. Ford 1976 Comparative migration of Band T-lymphocytes in the rat spleen and lymph nodes. Cell. Immunol., 23t254-267. Pellas, T.C., and L. Weiss 1990 Deep splenic lymphatic vessels in the mouse: a route of splenic exit for recirculating lymphocytes. Am. J. Anat., 187:347-354. Sasou, S., T. Madarame, and R. Satodate 1982 Views of the endothelial surface of the marginal sinus in rat spleens using the scanning electron microscope. Virchows Arch. FBI, 4Ot117-120. Sasou, S., and R. Satodate 1979 A scanning electron microscopical study of the perifollicular region of the rat spleen. Recent Adv. Res., 19t43-53. Sasou, S., R. Satodate, and S. Katsura 1976 The marginal sinus in the perifollicular region of the rat spleen. Cell Tissue Res., 172:195203. Sasou, S., R. Satodate, T. Masuda, and K. Takayama 1986 Scanning
