Microcirculation of intestinal lymphoid follicles in rat Peyer's patches

GASTROENTEROLOGY

1981;81:481-91

Microcirculation of Intestinal Lymphoid Follicles in Rat Peyer’s Patches DEEPAK

K. BHALLA,

TAKURO

MURAKAMI,

and

ROBERT L. OWEN Cell Biology and Aging Section, Veterans Administration Medical Center, and the Department of Medicine, University of California, San Francisco, California

Peyer’s patch microvasculature carries recirculating lymphoid cells into lymphoid follicles and supports vigorous metabolic demands of crypts and replicating germinal centers. Scanning electron microscopy of methyl methacrylate casts of blood vessels in rat intestinal Peyer’s patches revealed that ascending arterioles pierce follicles and feed a subepithelial capillary network beneath the follicle surface. These capillaries interconnect with baskets of capillaries beneath adjacent crypts and jlow into postcapillary venules in thymus-dependent areas around the follicle perimeters. This pattern is a modification of the fountain pattern in villi and reflects the secondary development of germinal centers of B lymphocytes between central ascending arterioles and peripheral postcapillary venules. Sections of glycol methacrylate’-embedded follicles injected with India ink revealed multiple fine capillaries in germinal centers not detected by previous techniques. Capillary flow from follicle apex to adjacent crypts provides a route for feedback control of replenishment of the specialized antigen-trapping epithelium covering follicles, and it is an opportunity for absorbed materials and cellular products beneath the epithelium to modify migration of lymphocytes out of postcapillary venules into follicles. Peyer’s patches have been characterized morphologically and functionally as secondary lymphoid organs initiating immune reactions against orally administered antigens. Like other secondary lymphoid Received December 23,198O. Accepted April 8, 1981. Address requests for reprints to: Robert L. Owen, M.D.. Cell Biology and Aging Section (151E). Veterans Administration Medical Center, San Francisco, California 94121. Dr. Murakami is affiliated with the Department of Anatomy, Okayama University Medical School, Okayama, Japan. This work was supported by the Veterans Administration, National Institutes of Health Grant AM 21969, and a grant from the Japanese Ministry of Education.

organs, Peyer’s patches are located in the path of continuous lymphocyte traffic. Isotopically labeled circulating T and B lymphocytes have been shown to migrate through the postcapillary venules into Peyer’s patches, where they segregate into peripheral T-cell areas surrounding germinal centers composed primarily of B cells and macrophages (l-5).It is apparent that the follicle-associated blood vessels play a central role in the migration and distribution of lymphocytes to the Peyer’s patches, and that an elaborate system of blood vessels is needed to cope with the heavy lymphocyte traffic in this region and to satisfy metabolic demands of replicating cells in germinal centers. Blau (6)and Abe and Ito (7)investigated the vascular architecture of Peyer’s patches by injecting India ink into the blood circulation. These studies were interpreted as showing a reticular microcirculation in Peyer’s patches resembling the loop pattern described in thymus (8)where the capillaries arise from the arterioles at the corticomedullary junction, ascend up the cortex, divide, curve back, and then join postcapillary venules in the medulla. Determination of vascular interconnections with adjacent villous vessels and spatial relationships of arterioles, capillaries, and venules were limited by difficulties of three-dimensional reconstruction of models from partially traceable vessels in thick sections. Using experimental combinations with methyl methacrylate resin, Murakami (9) developed a plastic-injection replica technique to demonstrate the three-dimensional architecture of microcirculation by scanning electron microscopy. This technique permits more complete reproduction of vascular interconnections than is possible with India ink injections. Using this plastic-casting technique with commercially available materials, we have examined the microvascular architecture of rat Peyer’s patches by scanning electron microscopy. Our injection replicas were correlated with the vascular pattern traced by

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light microscopy in methacrylate sections of Peyer’s patches injected with India ink to assess the relationships of blood vessels to B- and T-cell areas and to structures within the lymphoid follicles.

Materials and Methods Dissecting and Scanning Electron Microscopy intact tissue. Two nonfasted, 2-mo-old male Sprague-Dawley rats (Holtzman Co., Madison, Wis.) weighing 250 g were anesthetized by intraperitoneal administration of sodium pentobarbital (Nembutal sodium 50 mg/ml, Abbott Laboratories, North Chicago, Ill.) 0.25 ml/rat. Peyer’s patches were excised and fixed while still distended with fecal material, or inverted and rinsed with cold saline and then fixed for 72 h by immersion in 2.7% glutaraldehyde and 0.8% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3. Tissues were washed in phosphate buffer, dehydrated through a graded series of alcohols, and dried in a Bomar SPC-SOO/EX critical-point drying apparatus by using carbon dioxide. Tissues were mounted by colloidal graphite (Ted Pella, Inc., Tustin, Calif.) on aluminum studs, examined and photographed with an Olympus dissection microscope. Peyer’s patches were then sputter coated with 200 A of gold in a vacuum evaporator (Hummer II, Technics Inc., Alexandria, Va.) and observed in a Cambridge Stereoscan 150 scanning electron microscope (Cambridge Instrument Co., Monsey, N.Y.) using an acceleration voltage of 20 kV with a lanthanum hexaboride electron source. Vascular casts. Vascular casts of Peyer’s patches were prepared by the method described by Ohtani and Fujita (10). Two 3-mo-old Sprague-Dawley rats, weighing about 400 g, were fasted overnight and then anesthetized by intraperitoneal administration of sodium pentobarbital, 0.40 ml/rat, and exsanguinated by decapitation. Using large syringes, the animals were irrigated with about 60 ml of Ringer’s solution through the thoracic aorta until the effluent was clear, then manually injected at a rate of 10 ml/ min with about 40 ml of commerically available methacrylate-casting medium with 2% MA catalyst (Mercox 2R, Oken-Shoji, Tokyo, Japan). The methacrylate-injected abdominal organs were removed en bloc, immersed for 3 h in a warm-water bath (SO’C), macerated overnight with warm 10% NaOH solution (6O’C) and washed for about 12 h in running tap water. The vascular casts thus obtained were dissected with forceps and scissors and the arteries and veins of intestinal lymphoid patches and surrounding villi were isolated. The trimmed specimens were dried in air, sputter coated with a 400-A layer of gold in a vacuum evaporator, and photographed by scanning electron microscopy using an acceleration voltage of 10 kV. The specimens were then cut with razor blades or microdissected with sharpened needles under a dissecting light microscope in order to expose internal vascular arrangements of the patches. After each dissection, specimens were again sputter coated with 200 A of gold for the scanning electron microscopic observations.

Light Microscopy Two 5-mo-old male Sprague-Dawley rats weighing about 400 g were anesthetized as described above and injected with 10% colloidal carbon suspension, (India ink, Pelikan, Gunther Wagner, Hanover, Germany) through the left ventricle of the heart. Peyer’s patches were removed, pinned flat and fixed with a 10% solution of formalin containing 2.52% NaOH and 2.26% sodium phosphate, monobasic. The tissues were dehydrated through a graded series of alcohols and embedded in glycol methacrylate (Sorvall Embedding Medium, DuPont Biomedical Division, Newton, Conn.). Sections were cut at a thickness of 10 pm, stained with hematoxylin and eosin, and photographed with a Zeiss photomicroscope (Carl Zeiss, New York, N.Y.)

Results Dissecting Microscopy In intact specimens, one or more artery-andvein pairs could be seen beneath the serosal surface, running from the mesenteric margin toward the bulging white follicles making up each Peyer’s patch. Inversion of intestinal segments prevented the curling of cut edges over the Peyer’s patches that otherwise occurs during critical-point drying. The tongue- and ridge-shaped villi were generally in parallel with their long dimensions running circumferentially around the intestine. Inversion exposed surfaces of the individual follicles within each Peyer’s patch by splaying villi apart. Scanning Electron Microscopy The intestinal lymphoid follicles of the rats were round, with elevated centers below the level of the villus tips. By the inversion of intact specimens, the narrow mouths of the crypts were displayed in a ring separating the follicle surface from surrounding villi (Figure 1). In the en bloc cast of abdominal visceral vasculature, intestinal loops overlay one another, and it was not practical to determine whether specific Peyer’s patches were from jejunum or ileum. Although patches varied in composition from four to more than 11 follicles, the vascular architecture of the component follicles was similar at all levels of the small intestine examined. The commercially available Mercox was effective in casting the intestinal blood vascular beds in Peyer’s patch follicles. The fine capillaries and their connecting arteries and the veins of the mucous layers and muscular sheets were reproduced by forceful injection through the thoracic aorta, though some undesired resin leakage was observed. Resin injection into the fasted animals produced good casts

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Figure 1. Scanning electron micrograph of a single lymphoid follicle from a rat Peyer’s patch. The follicle is separated tongue-shaped and ridge-shaped villi by a ring of crypt mouths (arrows).

with no fecal contamination. The Mercox casts were appropriately hard and brittle, easily withstanding the electron beam, and yet permitted microdissection to study the detailed connections of intestinal capillaries with their parent arteries and veins in the lymphoid follicles. Distention of vessels by forceful injection of resin effaced any recognizable impressions of endothelial cells or transmigrating lymphocytes. Diameters of vessels varied depending on perfusion pressure, but relative proportions were consistent from follicle to follicle. Arterioles were identified by their straight courses, oblique angles of branches, and by their fine capillaries within the muscular wall of larger interfollicular arterioles. Veins ran more irregular courses with anastomoses at wider angles. Interfollicular veins were of larger diameter than arteries. When classification of particular vessels was in doubt, they were traced back to larger, identifiable vessels. Postcapillary venules were differentiated from collecting venules by their greater relative di-

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ameters, by spatial relationships to identifiable landmarks (villi, crypts, lumen, serosa), and by comparisons with similarly located vessels in India inkinjected sections where vessel walls could be examined. Measurement of diameters was not used to discriminate vessel types due to variations in size because of differing pressures of distention and perspectives due to tilt of specimens away from the electron source. Each Peyer’s patch is supplied by branches of the superior mesenteric artery entering along the mesenteric margin of the intestine. Upon reaching an aggregate of follicles that makes up a patch, the artery divides and gives off branches that enter the tissue between adjacent follicles and run close to the serosal surface. These interfollicular arteries divide as they progress, but they do not form anastomoses (Figure 2). The arteries towards the far side of the Peyer’s patch may enter follicles directly instead of ringing their peripheries. Subdivisions of interfollicular arteries enter the follicles and become seg-

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Figure 2. Scanning electron micrograph of the injection replica of a rat Peyer’s patch and adjacent intestine. A large vein (V) and artery (A) originate below at the mesenteric margin and divide into large branches running between the follicles. At least 11 round follicles (F) can be detected in this micrograph. Small arteriolar and venous branches enter around the periphery of follicles. To the left and right of the main follicular vessels, smaller arteries and veins supply the adjacent mucosa. Small circumferential and longitudinal vessels (arrowheads), which supplied the external muscle layers, overlie the main vessels.

regated into two sets of arterioles: a horizontal set running parallel to the serosal surface and a vertical set penetrating the follicles. The parallel arterioles progress along the base of the follicles and occasionally anastomose with other arterioles, but they rarely anastomose with venules. The vertical arterioles penetrate the follicle and undergo repeated divisions. In addition to these two sets of arterioles, a single, prominent vessel, which we call the central ascending arteriole, is given off either from a horizontal arteriole or from the interfollicular artery. It traverses the germinal center or adjacent T-cell areas and ascends through the follicle: at its distal end it gives off capillaries that form a continuous reticulum of interconnecting vessels immediately below the surface epithelium. The distribution of the

capillary network around the central ascending arteriole may be described as a “fountain pattern.” In the surface view, the capillaries appear to diverge from the apex in all directions (Figure 3). Some of these capillaries anastomose with the vertical set of arterioles while others empty into subepithelial collecting venules. Some of their branches also join with superficial capillaries from adjacent villi to surround intervening crypts distributed along the periphery of follicles. These capillaries form rings around the upper portion of crypts and connect with a capillary net that forms a basket beneath the base of the crypts (Figure 4). The crypt baskets are also supplied by vertical and horizontal arterioles, and the basket bases were characterized by remarkably rich or dense capillary networks. Figure 5 illustrates

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the capillary baskets of crypts and the connections between follicle and villus capillaries in a lateral view of a partially dissected cast. In the interior of the follicle, vessels exposed after dissection run into the germinal center and appear to end blindly. Some of these casts were broken during dissection while others with tapered ends constitute partially replicated vessels. An intact central ascending arteriole is shown in Figure 6. Most of the vessels of the follicle have been removed by dissection. Smaller follicles have only one central ascending arteriole, but two or three ascending arterioles may be seen in larger follicles (Figure 7). All ascending arterioles remain relatively unbranched as they pass through the follicles, but they divide repeatedly at the follicle apex. The venules form a highly branched network of vessels in T-cell areas, peripheral to the germinal centers in their distribution (Figure 7). Collecting venules often originate near the follicle apex. They

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join, forming postcapillary venules that progress toward basal parts of the follicle, ultimately joining the larger venules in the interfollicular area either individually or in the form of a few, relatively large, often interconnected vessels (Figures 6 and 7). The interfollicular venules run in close association with their arterial counterparts and form venous rings around the follicles. The larger follicle venules are evenly distributed along the follicle periphery and join the venous ring at several places around the follicles. Light Microscopy Blood vessels in methacrylate-embedded sections of Peyer’s patches were identified by the presence of India ink in the vascular lumen (Figure 6). Veins

corresponding

associated follicles.

with Smaller

to the venous them vessels

were

rings

localized

corresponding

and lateral

arteries to

to those

the de-

Figure 3. Scanning micrograph of the luminal surface of the vascular cast of a single lymphoid follicle (F). The central ascending arteriole (arrows) can be seen rising from the substance of the follicle to the apex from which a network of capillaries run to the periphery where they join the capillary baskets around adjacent crypts (C). The capillary framework of adjacent villi overlie and largely obscure interfollicular veins (arrowheads). Compare with Figure 1 showing an intact follicle and villi.

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Figure 4. Luminal capillary (vv) and capillary

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surface after microdissection of the junction of a portion of a follicle capillary net (F) and adjacent, richly supplied baskets (C), which lay beneath crypts. Villi have been microdissected away leaving only portions of villus venule arteriole (va) at the follicle-crypt junction. Collecting venules (V) descend into the substance of the mucosa, but some branches anastomose with crypt baskets (arrowheads).

tected by the plastic-casting technique were also localized in suitable sections. These vessels included horizontal and vertical arterioles, the central ascending arteriole traversing the germinal center, capillaries running immediately below the follicle epithelium, and the vessels surrounding the crypts. In addition, within germinal centers we detected multiple transfollicular capillaries (Figure 8) only partially preserved in our injection replicas, where they appeared to terminate bluntly (Figure 5). Postcapillary venules were identified by “high” endothelial cells bulging into the lumen creating a serrated periphery around such venules, located at the lower outer edges of germinal centers. The vascular architecture of a Peyer’s patch is schematically represented in Figure 9. Shown in this figure are the closely associated interfollicular arteries and veins, horizontal and vertical sets of arterioles, crypt baskets, the central ascending arteriole,

and its distal branches forming a fountain. Fine vessels of the musculature are also shown at the base, below the horizontal arterioles. These vessels, because of their superficial distribution and fragile nature, were often disrupted during the cast-preparative procedures, especially over the protruding follicles. Vessels serving the circular and longitudinal muscle layers join at right angles. In Figure 2 the rectilinear pattern of these muscle vessels is preserved at the edges of the Peyer’s patch.

Discussion Microvasculature of lymphoid organs is of interest because of its role in antigen transport and lymphocyte migration and recirculation necessary for the propagation of immune responses. Transmural passage of lymphocytes from the blood circulation to the lymphoid follicles takes place through

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the postcapillary venules, which have irregular walls formed by high endothelial cells that facilitate lodging of circulating lymphocytes as they migrate across the endothelial walls (1,11-18). In Peyer’s patches, the presence of such postcapillary venules in the interfollicular regions is well known. However, the detailed arrangements of the finer vessels and their interactions with larger vessels are not so well understood. Blau (6) and Abe and Ito (7) have used the India ink-injection technique to study microvasculature of Peyer’s patches. Vascular arrangement in these studies was reconstructed and inferred from examination of serial sections of portions of India ink-filled vessels. The three-dimensional vascular interconnections could not be fully determined by these studies. A variety of injection media, including Microfil, Vultex, silicone rubber, and Micropaque, had been used for constructing three-dimensional casts of vascular systems for light microscopy (6,14-16). The

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major drawbacks of these media were their tendency to shrink during preparation and their poor penetration properties. With the development of the plastic-casting technique (9), it became possible to follow the microvascular pattern of Peyer’s patches with more accuracy and with high resolution. Even with this new medium, partial reproduction of finer vessels was occasionally observed. Thus, capillaries passing among densely packed lymphocytes of the follicles appeared to end bluntly in our vascular replicas, but were seen to cross the follicles when injected with low-viscosity suspensions of India ink. The incomplete casting is believed to result partly from closure of vascular lumen by muscular contractions and partly from viscosity changes in the casting medium during injection. The methacrylatecasting technique has been used successfully in recent years to demonstrate the arrangement of vessels in a variety of tissues including bone marrow (17), spleen (18), gastrointestinal tract (19,20), ovary

Figure 5. Transverse section of the microvascular cast of a follicle (F) and surrounding villi (Vi). Arteries and veins decussate along the serosal surface below. The capillary network forms a shell over the top of the rounded follicle. Portions of the central ascending arteriole (aa) and collecting venules (arrowheads) are seen. Large interfollicular veins (v) and arteries (A) lie to each side of the follicle. The conjunction of capillary flow from vilii and follicle occurs at the intervening crypt basket (C).

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(21) liver (22), and pancreas (10). Occasional leakage of the casting medium through the capillary walls has been attributed to increased capillary permeability (2l), although it may also result from damage to capillaries induced by injection pressure. In Peyer’s patches, we observed a pattern of blood vessels that seems to be a modification of the fountain pattern first described in intestinal villi of dogs by Mall (23). We did not find the loop patterns that others have reconstructed from thick sections of lymph nodes, thymus, or Peyer’s patch. The fountain pattern consists of a distinct central arteriole ascending through the germinal center and reaching the follicle apex, where capillaries are given off in all directions. The relatively unbranched central ascending arteriole ensures an adequate supply of blood to the apical region of the follicle, which is occupied by a large number of M cells and is actively involved in transporting antigen from intestinal lumen to the underlying lymphoid tissue (24). In villi

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countercurrent exchange of oxygen between the central ascending arteriole and parallel venules is presumed to produce low oxygen tensions at the villous apex (25). Because of the spatial separation of arterioles and venules in Peyer’s patch follicles, this countercurrent exchange may not occur, allowing higher apical oxygen concentration than in villi. This remains to be determined. Although a large number of capillaries appear to form a network immediately below the epithelium, a substantial number of capillaries traverse germinal centers. Our results contrast with studies suggesting that blood vessels in Peyer’s patches and lymph nodes are displaced by and absent from germinal centers (6,26,27). We may have been successful in detecting fine germinal center capillaries because we embedded tissue for light microscopy in glycol methacrylate, which preserves tissue better and prevents the diffusion of India ink that occurs within paraffin sections. Displacement of venules away from central

Figure 6. Lateral wall of a lymphoid follicle after microdissection. A central ascending arteriole (aa) rises from an interfollicular artery (A) somewhat obscured by leakage from intimal capillaries in the arterial wall. Above, the ascending arteriole flows into the remaining portion of the capillary net at the follicle surface, which descends, forming a curtain in front of surrounding villi. The path of blood flow is marked by arrows through the capillary net into postcapillary venules (P) and into the interfollicular veins (v).

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Figure 7. Dissected view of a lymphoid follicle from the serosal surface. The follicle (F1) is ringed by interfollicular veins (V) and arteries (A). To the right, the space between the major vessels is filled with bottoms of crypt capillary baskets. Dissected portions of three central ascending arterioles (arrowheads) flow toward the apex at the center of this oval follicle. Multiple collecting venules (C) around the periphery of the follicle are collected into postcapillary venules (P), which flow into the interfollicular veins. To the left is an undissected follicle (FZ).

ascending arterioles may reflect secondary development of germinal centers after follicle formation. Although there are differences in vascular patterns between Peyer’s patches and lymph nodes, the two lymphoid organs exhibit certain similarities. Long, slender, unbranched arterial vessels of lymph nodes (13,27,28) are comparable to the central ascending arterioles of the Peyer’s patch follicles. In the rat, arteriovenous connections have been morphologically demonstrated in lymph nodes. We also observed them in rat Peyer’s patches. Such connections have not been definitely identified in lymphoid organs in other species. In rat intestinal villi, such connections are believed to be present (29), but they could not be demonstrated by the plastic-casting technique (19). In both Peyer’s patches and lymph nodes the venules are larger than the arterioles, do not penetrate the germinal centers, and are

located around the follicle periphery. In these respects they are similar to the intestinal venules that extend along the sides of the villi (19), although venules encircle the bases of Peyer’s patch follicles but do not encircle the bases of villi. Migration of lymphocytes out of postcapillary venules could be affected by luminal molecules or lymphoid cell products absorbed by capillaries as they run beneath the dome epithelium. The distribution of postcapillary venules around the follicle periphery appears to ensure uniform migration of lymphocytes over a large area around the follicle. The branched network of vessels provides an efficient supply of oxygen and nutrients to the crypts engaged in active cell proliferation, especially the crypt base where capillaries are richest. Although both columnar and goblet cells migrate from crypts to adjacent villi, the migration over the follicle sur-

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Figure 8. India ink-injected Peyer’s patch follicle partly extends below villi on the left. Large lightly staining lymphoblasts fill the germinal center (GC) surrounded by a rim of darker staining lymphocytes in thymus-dependent areas (TDA). Capillaries are seen beneath the surface of the follicle apex, beneath the epithelium of adjacent villi, and within the germinal center (arrowheads). Postcapillary venules lie to each side of the follicle (arrows) and a partially filled large interfollicular vein (V) is seen to the right next to a lymphatic (L) with clear space. (X 120).

of Figure 9. Diagrammatic recapitulation the vascular distribution in a typical rat lymphoid follicle and a portion of an adjacent villus. Arteries are white and capillaries are stippled, becoming darker as they flow into the venous collecting system. In both the villus and follicle, central ascending arterioles rise to the apex and flow into capillary networks descending over the surface and coming together in the baskets of capillaries surrounding intervening crypts. Compare with the cast in Figure 5 and the histologic structures in Figure 8.

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face is restricted mainly to columnar cells. The direct route of the capillaries from the follicle apex to the crypt plexus provides a pathway for movement of humoral factors from follicle to the crypts, modulating migration of cells from proliferation zones in the crypts to the region of desquamation at the follicle apex.

References 1. Gowans

JL, Knight EJ. The route of re-circulation of lymphocytes in the rat. Proc R Sot Lond (Biol) 1964;159-257-82. 2. Parrott DMV, deSousa M. Thymus dependent and thymus-independent populations: origin, migratory patterns and lifespan. Clin Exp Immunol 1971;8:663-84. 3. Parrott DMV, Ferguson A. Selective migration of lymphocytes within the mouse small intestine. Immunology 1973; 26:571-88. 4. Howard JC, Hunt SV, Gowans

JL. Identification of marrowderived and thymus-derived small lymphocytes in the lymphoid tissue and thoracic duct lymph of normal rats. J Exp Med 1972;135:200-19. lym5. Waksman BH. The homing pattern of thymus-derived phocytes in calf and neonatal mouse Peyer’s patches. J Immuno1 1973;111(3):878-84. in the 6. Blau JN. A comparative study of the microcirculation guinea-pig thymus, lymph nodes and Peyer’s patches. Clin Exp Immunol 1977;27:340-7. 7. Abe K, Ito I. A qualitative and quantitative morphologic study of Peyer’s patches of the mouse. Arch Histol Jpn 1977;16(5): 407-20. bar8. Raviola E, Karnovsky MJ. Evidence for a blood-thymus rier using electron-opaque tracers. J Exp Med 1972; 136:46698. 9. Murakami T. application of the scanning electron microscope to the study of the fine distribution of the blood vessels. Arch Histol Jpn 1971;32(5):445-54. of the pancreas with spe10. Ohtani 0, Fujita T. Microcirculation cial reference to periductular circulation. A scanning electron microscope study of vascular casts. Biomedical Research 1980;1:130-46. I, McGregor DD. Migration of lymphocytes 11. Goldschneider and thymocytes in the rat. I. The route of migration from blood to spleen and lymph nodes. J Exp Med 1968;127:155-68. 12. Schoefl GI. The migration of lymphocytes across the vascular endothelium in lymphoid tissue, a reexamination. J Exp Med 1972;136:568-588. 13. Cho Y, De Bruyn PPH. The endothelial structure of the postcapillary venules of the lymph node and the passage of lymphocytes across the venule wall. J Ultrastruct Res 1979;69:1321.

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LF, Noer RJ. The vascular pattern of the intestinal villi in various laboratory animals and man. Anat Ret 1952;114:85-101. 15. Miller DS, Rahman MA, Ranner R, et al. The vascular architecture of the different forms of small intestinal villi in the rat (Rattus norvegicus). Stand J Gastroenterol 1969;4:477-82. 16. Herman PG, Yamamoto I, Mellins HZ. Blood microcirculation in the lymph node during the primary immune response. J Exp Med 1972;136(4):697-714. 17. Irino S, Ono T, Watanabe K et al. SEM studies on microvascular architecture, sinus wall, and transmural passage of blood cells in the bone marrow by a new method of injection replica and non-coated specimens. In: Johari 0, ed. Scanning Electron Microscopy/l975 (part I). Chicago: IIT Research Institute, 1975:267-73. 18. Irino S, Murakami T, Fujita T. Open circulation in the human spleen. Dissection scanning electron microscopy of conductive-stained tissue and observations of resin vascular casts. Arch Histol Jpn 1977;40(4):297-304. 19. Ohashi Y, Kita S, Murakami T. Microcirculation of the rat small intestine as studied by the injection replica scanning electron microscope method. Arch Histol Jpn 1978;39(4):27182. 20. Nopanitaya W, Aghajanian JG, Gray LD. An improved plastic mixture for corrosion casting of the gastrointestinal microvascular system. In: Johari 0, Becker RP, eds. Scanning electron microscopy/l979/111. AMF O’Hare: SEM, Inc., 1979:7515. 21. Kardon RH, Kessel RG. SEM studies on vascular casts of the rat ovary. In: Johari 0, Becker RP, eds. Scanning electron microscopy/1979/111. AMF O’Hare: SEM Inc. 1979743-50. organization of 22. Kardon RH, Kessel RG. Three-dimensional the hepatic microcirculation in the rodent as observed by scanning electron microscopy of corrosion casts. Gastroenterology 1980:79:72-81. 23. Mall FP. Die Blut und Lymphwege in Dunndarm des Hundes. Abhandlungen des Mathematische-Physische Klasse des Saechsische Akademie der Wissenschaft 1887;14:153-200. peroxidase by 24. Owen RL. Sequential uptake of horseradish lymphoid follicle epithelium of Peyer’s patches in the normal unobstructed mouse intestine: an ultrastructural study. Gastroenterology 1977;72:440-51. 25. Lundgren 0. The circulation of the small bowel mucosa. Gut 1974;15:1605-13. 26. Mohiuddin A. Blood and lymph vessels in the jejunal villi of the white rat. Anat Ret 1966;156:83-90. of organized lymphoid tissues. 27. Herman PG. Microcirculation Monogr Allergy 1980;16:126-42. in 28. Herman PG, Ohba S, Mellins HZ. Blood microcirculation the lymph node. Radiology 1969;92:1073-86. 29. Nylander G, Olerud S. The vascular pattern of an isolated jejunal loop; a microangiographic study in the rat. Acta Chir Stand 1961;121:39-46.