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The intermediate zone (IZ) of nonperfused and perfused spleens in three species of primitive mammals (shrew, mole, platypus) was studied morphologically. The IZ is a tissue zone consisting of plexiform vessels, probably venous capillaries, and is located transitionally between the white and red pulp. The IZ is separated from the white pulp by the arterial net (AN), in which the white pulp arteries terminate. Development of the IZ differs between the three species examined being distinctive in the platypus and shrew. The IZ is thin in the mole spleen. A closed type of arteriovenous (A-V) anastomosis was demonstrated in or around the IZ in the two Insectivora species examined. In the shrew spleen, peripheral arterial branches running within the IZ anastomose with the AN around the follicle. The AN anastomoses eventually with venous plexiform vessels of the IZ around the nonfollicular area of the white pulp to form a closed system. In the mole spleen, A-V anastomoses were noted between white pulp arteries (follicular and AN) and veins of the red pulp, either by direct communication or through fenestrated IZ vessels compatible with the plexiform vessels of the shrew spleen. A-V anastomosis in the IZ is probable, but not confirmed, in the platypus spleen, as analysis was limited to a nonperfused specimen. Well-developed ellipsoids were noted around arterial terminals of the IZ in the shrew spleen. Ellipsoids were also noted around all arterial terminals of the mole spleen directed to the red pulp. Most ellipsoids of the mole spleen appeared located within the IZ. No ellipsoids were present around arterial terminals of the IZ in the platypus spleen. Closed circulation was noted in terminals of the pulp artery in spleens of all three species. All pulp arteries of the mole spleen are postellipsoid segments of white pulp (AN and follicle) arteries. No ellipsoids were found around terminals of the pulp artery (penicillar artery) in shrew and platypus spleens. The IZ is probably homologous to the perilymphatic sinusoid (vein) of the lungfish spleen and may be regarded as part of the red pulp. The IZ may be representative of primitive mammalian spleens that have closed circulation. The marginal zone (MZ) of common mammalian spleens is probably a modified IZ by differentiation (remodelling) of the intrasplenic vein. In this process, withdrawal of venous vessels from the IZ occurred, leaving a lymphoreticular zone with open circulation (MZ). The marginal sinus reported in some mammalian spleens is probably a modified AN formed during this process. Possible morphological alterations of the spleen in vertebrate phylogeny are discussed.

The marginal zone in the cat spleen consisted of a characteristic mixture of lymphocytes and other blood cells located mainly between the several layers of circumferential reticulum around white pulp. A region of fine-meshed reticulum between white pulp and red pulp, as present in some species, was absent from the cat spleen. Arterial capillaries to the marginal zone were few. Some were continuations of white pulp capillaries, whereas others were red pulp capillaries that likely were continuations of axial capillaries of periarterial macrophage sheaths (PAMS) (ellipsoids). Blood cells deposited in the marginal zone could reach red pulp by passing through the numerous openings in each layer of circumferential reticulum. Lymphocytes appeared to migrate across the marginal zone both toward and away from white pulp. Macrophages lying on the circumferential reticulum of the marginal zone phagocytized cells but did not ingest Thorotrast, although it coated their surfaces. Because of the scarcity of arterial endings and the lack of a macrophage-charged reticular meshwork, the marginal zone in cat spleen is not a major site of blood clearance and phagocytosis. These functions are better served in PAMS and red pulp.

Confusion regarding microcirculatory pathways in normal human spleen has arisen due to extrapolation from pathological material and from other mammalian spleens, not to mention difficulties in tracing intricate three-dimensional routes from the study of thin sections or cut surfaces of tissue. We examined microcirculatory pathways in normal human spleens freshly obtained from organ transplant donors. A modified corrosion casting procedure was used to obtain an open view of vessels and their connections. Our results demonstrate: 1) "arteriolar-capillary bundles" within lymphatic nodules and extensive branching of arterioles in the marginal zone (MZ); 2) the marginal sinus around lymphatic nodules; 3) the peri-marginal cavernous sinus (PMCS) outside the MZ or immediately adjacent to the nodule itself; the PMCS receives flow via ellipsoid sheaths and MZ, or directly from arterial capillaries, and drains into venous sinuses; 4) fast pathways for flow into venous sinuses via ellipsoid sheaths; 5) arterial capillary terminations in the reticular meshwork of the red pulp or MZ ("open" circulation); direct connections to venous sinuses also occur ("closed" circulation), although rarely; and 6) numerous open-ended venous sinuses in the MZ, allowing a large proportion of the splenic inflow to bypass the red cell filtration sites in the reticular meshwork and at venous sinus walls.

Periarterial macrophage sheaths (PAMS), a term we introduce to replace "ellipsoids," surround arterial capillaries in the red pulp of the spleen and are major sites for clearance of blood-borne particles. PAMS and their arterial capillaries in cat spleens in various states of congestion and contraction were studied by transmission electron microscopy. Thorotrast, a colloidal suspension of thorium dioxide, was injected to label macrophages. A PAMS consisted of a fine meshwork of reticular cells and reticular fibers which held macrophages and formed a cylindrical sheath around an arterial capillary lying in its longitudinal axis. Some PAMS were spongy due to loosening of cell associations by plasma infiltration; others were tightly compressed. Blood cells were both free in the interstices of the PAMS and attached to macrophages. Reticular cells formed a closely applied but incomplete layer adventitial to the arterial capillary and extended branches which contributed to the meshwork. Small villous processes on the major branches of reticular cells approached each other, sometimes forming intercellular junctions, and fit into complementary indentations in the surfaces of macrophages and endothelial cells. Thin filaments within reticular cells filled the villous processes and formed a border beneath the plasmalemma; intermediate filaments ran through the centers of the branches. Reticular fibers lay between reticular cells. Basement membrane fabricated of the same material as reticular fibers lay between the endothelium and reticular cells. Macrophages contained Thorotrast and abundant debris of phagocytized cells and were joined by extensive interdigitation of micropseudopodia. Endothelial cells were long rods which lay parallel and were joined along their bases by interdigitating lateral processes. Intercellular junctions were present at some points, but at others lateral processes were everted to form open interendothelial slits through which blood cells could pass. Endothelial cells possessed great numbers of randomly oriented intermediate filaments and small patches of thin filaments scattered along the basal plasmalemma and in lateral processes. Thin filaments may function to attach cells to one another and to the basement membrane and may assist in closing interendothelial slits. We believe that the endothelium responds to changes in arterial blood pressure and blood flow. It stretches to allow dilatation and recoils, probably due to the intermediate filaments, squeezing blood cells through interendothelial slits.

Rabbit spleens have been examined after perfusion fixation with and without prior washing with various fluids. The platelets were stored in the splenic sinuses and in the cord spaces as single platelets, or in loosely packed aggregates which appeared to be anchored to the endothelium by one or a few platelets. After washing prior to fixation most of the platelets disaggregated and regained their normal shape. Some platelets adhered to morphologically normal endothelium even after prolonged perfusion. Occasionally, platelets were observed inside splenic endothelial cells. Others were closely associated with macrophages, many of which also contained engulfed platelets. There was no morphological evidence of a particular platelet population being retained in the spleen after washing. In the sinuses special granule-rich cytoplasmic structures were observed. They were interposed between ordinary endothelial cells and contained a large number of small lysosome-like granules. Nuclei were never observed in these structures, probably because they consisted of pseudopod-like protrusions. Their origin and function are discussed. They may represent actively phagocytizing elements.

The migration of blood cells across the wall of splenic sinusoids has been studied by means of serial sections in rats inoculated with murine erythroblastosis virus (MEV). The sinusoidal walls of the normal spleen have no permanent openings. In the normal spleen, mature red blood cells and blood cells in the latest stages of maturation can be seen in diapedesis. The predominant form of transmural migration is intercellular. In MEV-inoculated animals, in which there is a vigorous erythroblastic proliferation, large numbers of erythroblasts are in transmural passage. Serial sections show that these cells migrate through the cytoplasm of the endothelial lining cells. On the basis of these observations, it is suggested that the transcellular mode of migration may be related to the delivery of newly formed red blood cells into the circulation while the intercellular mode may be part of the mechanism by which the spleen controls the removal of red cells from the circulation.

The present study is concerned with the ultrastructure of the spleen in the natterjack, Bufo calamita (Anura, Bufonidae), with special emphasis on the structure and function of the non-lymphoid elements occurring in the red and white pulp. The organ consists of two clearly distinguishable areas, the white and the red pulp, separated by a prominent marginal zone. Thus, the pattern of lymphocytic arrangement in the spleen of Bufo calamita corresponds to a follicular model and is similar to that reported in the primitive anurans of the Pipidae family, such as Xenopus laevis. The white pulp presents a reticular network consisting of two different cell types and free cells, such as lymphocytes, plasma cells, and macrophages. The red pulp is formed by cell cords, where reticular cells and fibers, macrophages and lymphocytes occur, and blood sinuses which sometimes contain developing erythroid elements. Colloidal carbon particles injected via the lymph sac are trapped exclusively by free macrophages in the red pulp which then move through the marginal zone to the white pulp. Giant, ramified, non-phagocytic cells appear in both white and red pulp. They have been functionally related with the trapping of antigen-antibody complexes on their surface, and a possible dendritic significance is discussed on the basis of their morphologic characteristics.

CBA/N mice carry an X-linked immune-deficiency gene, leading to a defect in the ability to form antibodies against T-independent type 2 antigens. By using immunohistochemistry, the organization of the spleen of the immune-deficient male (xid) CBA/N F1 and the normal female F1 were compared. Staining with antilymphocyte markers showed that the total number of cells in the various T- and B-cell areas was smaller in the xid mouse, resulting in very small white pulp compartments. Fewer B cells were seen in the marginal zone. When the spleens of the F1 mice were examined for macrophage markers, the rings of marginal-zone macrophages and the ring of marginal metallophilic macrophages were much thinner in the xid mouse. In particular, the marginal-zone macrophages are thought to play a role in the response against thymus-independent type 2 antigens, and their small numbers in the xid mouse are suggestive of a role for the microenvironment in the defects in these mice.

The development of splenules derived from slices of freshly removed autologous spleen implanted subcutaneously or intraperitoneally was followed by light and electron microscopy from day 2 to day 70. Within 48 hr after transplantation, a rough space filled with blood, unlined by endothelium, formed just under the surface of the splenic fragment. The tissue central to this vascular space was disrupted and necrotic. In the outer portion of the vascular space, fibroblasts appeared and created locules which developed into a highly vascular, hematopoietic red pulp. From the inner portion, blood percolated into the central necrotic tissue. At 1 week the splenule was divisible into concentric structures. The capsule was outermost. A shell of vascularized, highly hematopoietic red pulp lay within the capsule, having replaced the vascular space. Central to the red pulp lay a band of fibroblasts and macrophages. Next was a layer of fibroblasts in a matrix of degenerating cells, and, at the center, a necrotic core. As fibroblasts and macrophages moved centrad, the red pulp moved with them, expanding and replacing the necrotic tissue. The splenule differed in character from the original spleen. Splenular red pulp, especially near the surface, was unusually hematopoietic. The circumferential reticulum of white pulp was reduced or absent, and the boundary between red and white pulp was sometimes indistinct. Some white pulp was subcapsular, and the capsule and surrounding connective tissue were infiltrated by lymphocytes. The necrotic core of the splenule was typically surrounded by a zone containing large blood vessels, connective tissue, and adipocytes.

The capacity of the equine spleen to store and rapidly release as much as half the circulating blood volume after adrenergic stimulation depends upon the size of the spleen, its muscular capsule, and the distinctive structure of its red pulp. The unit, or lobule, of red pulp is a cylinder of pulp spaces organized in a reticular meshwork, supplied by a peripheral ring of arterial capillaries, and drained by a central venule. Reticular cells, which make up the meshwork of the pulp, contain an extraordinarily large complement of microfilaments and intermediate filaments and are richly innervated by nerves containing both dense and lucent core vesicles typical of adrenergic nerves. The wall of the pulp venule contains large apertures. The capacious red pulp would thus appear capable both of large-scale blood storage and, by the contractile adrenergic innervated reticulum and open venous vasculature, of rapid expression of stored blood into the circulation. Antigen-presenting cells are present not only in B and T cell zones in white pulp but in the periarterial macrophage sheath of red pulp as well. The periarterial macrophage sheath is one of the first sites of antigen capture, and the presence of these cells confers on it an immunological role.

The vascular and stromal arrangements of the red pulp in congested and contracted dog spleens were studied by transmission electron microscopy. Each dog had been injected intravenously with Thorotrast to label actively endocytizing cells. Only macrophages ingested Thorotrast. The proximal portion of each arterial capillary was surrounded by a "periarterial macrophage sheath" (PAMS), a term we introduce to replace the term "ellipsoid". PAMS were composed of a fine meshwork of reticular cells and reticular fibers which held tightly-packed macrophages and interspersed blood cells. These macrophages, as well as those in the reticular meshwork of red pulp, contained Thorotrast, cell debris, and deposits of hemosiderin. The arterial capillary at the center of each PAMS was formed by parallel, rod-shaped endothelial cells and discontinuous layers of basement membrane and reticular-cell cytoplasm. PAMS were tapered at their distal ends; the terminal portion of the arterial capillary continued beyond the PAMS to end in the reticular meshwork of red pulp. Endothelial cells in the terminal arterial capillaries were separated by gaps through which blood cells passed into the spaces of the reticular meshwork of red pulp. The reticular meshwork was formed by reticular cells which appeared to be specialized for contraction. These cells were filled with thin filaments and possessed plasmalemmal dense bodies as found in smooth muscle cells. Furthermore, the reticular meshwork was innervated by unmyelinated adrenergic axons which probably were derived from nerves that followed arterioles. Axons were enclosed in surface invaginations of cells which were similar to reticular cells in shape and cytologic detail and which we called "axon-bearing reticular cells". Axon-bearing reticular cells were inserted between the branches of the reticular cells that formed the meshwork. Venous sinuses formed an anastomosing system of vessels draining into pulp veins which then joined trabecular veins. Sinuses were formed by parallel, rod-shaped endothelial cells encircled by strands of basement membrane and reticular-cell branches. Endothelial cells lay closely side by side except where interendothelial slits were opened by blood cells passing into the lumen or by pseudopodia of macrophages which lay outside the sinus. Cell traffic across the sinus wall was greatest in areas where blood cells were mixed with plasma. Congested spleens stored concentrated red cells in both sinuses and the reticular meshwork; contracted spleens were emptied of blood. The reticular meshwork may contract to assist trabecular and capsular smooth muscle in expelling stored red cells and effecting hemoconcentration.