The hepatic sinusoids are preferentially supplied with portal venous blood and equipped with fenestrated endothelial cells that are distinct from capillary endothelial cells. We previously observed in rats that sinusoidal capillarization proceeded concurrently with arterial blood supply during hepatocarcinogenesis. This study aimed to clarify the inducing role of arterialization in sinusoidal capillarization by investigating phenotypical, morphological and functional alterations to sinusoidal endothelial cells (SECs) in arterialized rat livers induced by portal branch ligation. At one week, after massive hepatic necrosis following ligation, the livers were restored to their normal architecture without causing post-necrotic fibrosis. At 12-21 weeks, they exhibited a normal histology except for mild pericellular fibrosis which developed along sinusoids or between adjacent hepatocytes. SECs expressed factor VIII-related antigen and showed a decrease in the number of fenestrae and porosity, still lacking any basement membrane but further retaining the functional capacity for carmine dye uptake. Stellate cells, while occasionally associated with large amounts of collagen bundles, contained many lipid droplets and expressed no alpha-smooth muscle actin, indicating a quiescent property. Kupffer cells were commonly found within the sinusoids. The present results indicate that arterialization of the liver induces a partial (but not complete) transition of SECs into capillary-type endothelial cells, suggesting that arterialization might be one of the factors which induce sinusoidal capillarization in the development of hepatocellular carcinoma.

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The liver has a remarkable proliferative capacity after a partial hepatectomy. Previous studies have indicated that Kupffer cells have the potential to exert both stimulatory and inhibitory influences on hepatocyte proliferation. To elucidate the role of Kupffer cells in liver regeneration, mice were selectively depleted of Kupffer cells by injection of liposome-encapsulated dichloromethylene diphosphonate (lipo-MDP) at day 3 after a two-thirds hepatectomy. Results showed that liver regeneration was delayed after Kupffer cell-depletion. In control mice, hepatocyte growth factor (HGF) mRNA expressions were enhanced during liver regeneration and expressions of HGF were localized in fat-storing cells (Ito cells). In Kupffer cell-depleted mice, the number of HGF-expressing cells decreased in the regenerating liver, and expressions of HGF and its receptor (c-met) as well as other growth factors/cytokines were less prominent than in control mice. In contrast, expressions of TNF-α, another potent cytokine involved in liver regeneration, did not differ between Kupffer cell-depleted and control mice during the regeneration. Administration of TNF-α antibody did not reduce the expression of HGF or liver regeneration. These findings imply that Kupffer cells play a stimulatory role in liver regeneration by enhancing HGF expression via TNF-α-non-mediated mechanisms.

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The mechanisms for the formation of autophagic vacuoles were investigated using GH4C1 cells, a rat pituitary tumor cell line, whose induction increases intracellular levels of lysosomal proteinases and their mRNA by treatment with a combination of hormones (17β-estradiol, insulin and EGF). By ordinary electron microscopy, autophagic vacuoles containing various undigested structures with or without limiting membranes were abundant in the hormone-induced cells. These vacuoles, also containing numerous small vesicles, appeared to be derived from multivesicular bodies. In fact, there were also numerous C-shaped multivesicular bodies which enclosed cytoplasmic portions, suggesting that these unique structures are involved in the production of the autophagic vacuoles. Moreover, the cytoplasmic portions enlapped by the C-shaped multivesicular bodies were high in electron density and contained filamentous structures. By the cryothin-section immunogold method, the C-shaped multivesicular bodies in some cases contained lysosomal marker proteins such as cathepsins B and H, and lgp 120. Using an anti-actin monoclonal antibody, immunogold particles clearly labeled the cytoplasmic portions enclosed by the C-shaped multivesicular bodies. Pulse-chase experiments with horse radish peroxidase, a fluid-phase endocytic marker, revealed that the incidence of the C-shaped multivesicular bodies labeled with horse radish peroxidase peaked at 30 min after the beginning of chase incubation, whereas no C-shaped multivesicular body with horse radish peroxidase was detected in the cells by cytochalasin D treatment. These results suggest that the C-shaped multivesicular bodies occur in a transitional process from endosomes to lysosomes by the action of actin filaments, and that this morphological change may be essential for the production of autophagic vacuoles in the hormone-induced GH4C1 cells.

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The present immunocytochemical study examined the localization of Trk B, a high affinity neurotrophin receptor, in the neural elements of the periodontal ligament of the rat incisor. In light microscopy, the immunoreactivity was demonstrated in dendritic profiles in the alveolar half of the periodontal ligament. Their location and morphological features indicated that they were periodontal Ruffini endings. Occasional rounded cells associated with periodontal Ruffini endings, which had immunonegative kidney-shaped nuclei, were immunoreactive; these were judged to be terminal Schwann cells. Immunoelectron microscopy revealed the heterogeneous localization of Trk B among individual Ruffini endings. Some terminal Schwann cells contained immunoreactive products for Trk B in the cytoplasm, while others did not. Similarly, a part of the Schwann sheaths covering the axon terminals showed Trk B immunoreactivity. Most axon terminals associated with periodontal Ruffini endings were immunopositive for Trk B, though a few of them were immuno-negative. The ordinary Schwann cells did not contain Trk B immunoreactive products. These findings imply that Trk B is required for the maintenance of periodontal Ruffini endings. The different expression pattern of Trk B suggests that neuronal and glial elements comprising individual periodontal Ruffini endings are subject to heterogeneous conditions with regard to the requirement of Trk B.

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Light microscopic immunolocalization of taurine, a sulfur-containing free amino acid, was investigated in the developing retina of a lefteye flounder, Paralichthys olivaceus, which exhibits metamorphic changes with rod cell addition for 3-5 weeks after hatching. This immunocytochemical study of the developing retina revealed: 1) From 3 to 13 days after hatching, intense immunostaining was shifted from the surroundings of neural cells to the neural somata and processes in the inner retina. 2) Intense immunoreactivity appeared also in the outer and inner segments and basal processes (pedicles) of cone cells within 6 days or 13 days after hatching. 3) Lack of immunoreactivity was found in the outer segment of rod cells from their appearance during metamorphosis. These findings are discussed with the possible functional roles of taurine in the fish retina: 1) involvement in cell differentiation and/or development; 2) protection of the outer segments against light stimuli; and 3) regulation of neural transmission.

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The high pressure freezing/freeze substitution technique is known to yield a deep vitreous freezing of tissues. Combination of this technique with Lowicryl K4M embedding allows us histochemical studies of dynamic cellular processes with improved structural preservation. The disadvantage of Lowicryl K4M embedding is its poor electron density in electron microscopy. To address this problem, we examined the effects of KMnO₄ oxidation applied to Lowicryl K4M embedded rat gastric glands processed by high pressure freezing. The KMnO₄ oxidation-uranyl acetate-lead citrate sequence succeeded not only in contrast enhancement of cellular components, but also in differential staining of the zymogen granules of rat gastric chief cells. This technique could be applied to semi-thin sections of Lowicryl K4M embedded rat gastric glands. The KMnO₄ oxidation-toluidine blue staining provided sufficient contrast with regard to the zymogen granules. Various experiments used in this study verified that the KMnO₄ oxidation plays an essential role in the differential staining of the zymogen granules. Combined use of the KMnO₄ oxidation with phospholipase A₂-immunostaining demonstrated that gold labeling was localized to the zymogen granules without the loss of immunolabeling. Energy dispersive X-ray microanalysis revealed some manganese depositions on the zymogen granules. It is highly anticipated that the KMnO₄ oxidation will become a useful tool for histochemical investigations combined with cryofixation/freeze substitution and low temperature embedding techniques.

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Facilitated-diffusion glucose transporter GLUT1 is abundant in the blood-nerve barrier. To observe the relationship between glucose transfer across the barrier and the molecular architecture of the barrier, we examined the localization of GLUT1 and tight junction proteins, occludin and zonula occludens-1 (ZO-1), by immunofluorescence microscopy and immunogold electron microscopy in the rat sciatic nerve. GLUT1 was enriched at the whole aspects of the plasma membranes of the cells of the barrier: perineurial cells, and endothelial cells of the blood vessels in the endoneurium. These GLUT1-positive cells were also positive for occludin and ZO-1, both of which were localized at tight junctions. ZO-1 additionally was present in the GLUT1-negative cells not serving as the blood-nerve barrier. These observations suggest that occludin in the tight junctions and GLUT1 at the plasma membranes in the cells of the barrier may constitute a mechanism for the selective transfer of glucose across the barrier while preventing the non-specific flow of blood constituents.

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The epithelial basement membrane of intestinal villi is perforated with numerous small pores, through which free cells in the lamina propria communicate with the enterocytes. This study was a comparative analysis of the pores in the basement membrane by SEM after removal of the gut epithelium with OsO₄ maceration. The porosity as represented by the area fraction of the pores varied along the baso-apical axis of villi in patterns specific for each animal species examined: consistent scantiness along the entire length of villi in mice, acute elevation in the second and third distal one-sixths of villi in rats, and gradual augmentation toward the villus tips in guinea pigs. Size distribution analyses of the pores indicated their heterogeneous enlargement in the regions of elevated porosity. Concomitant observation of lamina propria macrophages by histochelnical labelings and by conventional TEM showed that the cells specifically clustered beneath the hyperporous basement membrane, with their thick processes penetrating it. The spatially-regulated patterns of perforation of the epithelial basement membrane indicate phase-specific interventions of lamina propria macrophages in the maturation or aging of enterocytes, which steadily proliferate in crypts and exfoliate at the villus tips.

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The expression of heat shock protein 25 (Hsp 25) was investigated in the rat temporomandibular joint by immunocytochemistry combined with confocal and electron microscopy. Immunostaining with an antibody to Hsp 25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp 25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp 25-positive cells with cytoplasmic processes: one extended thick and long processes towards the articular cavity, and the other prejected horizontally slender processes which covered the synovial membrane. Under the electron microscope, the immunoreactive synovial lining cells were characterized by a well-developed rough endoplasmic reticulum and secretory granules, suggesting that they can be categorized as fibroblastic type B cells. The covering by the cytoplasmic extensions was confirmed by immuno-electron microscopic observations. This cytoplasmic covering presumably performs a barrier function and expedites the effective secretion/resorption of synovial fluids. Since it has been proposed that Hsp 25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp 25 was also observed in the chondrocytes of the maturative and hypertrophic cell layers as well as in the cells of the articular disk. A suggestion was made that Hsp 25 might be involved in the inhibition of apoptosis of those cells.

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