Contents

1. Phenotypical and morphological alterations to rat sinusoidal endothelial cells in
   arterialized livers after portal branch ligation (YAMASAKI, M.)
   
2. The role of Kupffer cells in liver regeneration (TAKEISHI, T.)
   
3. The induction of autophagic vacuoles and the unique endocytic compartments, C-shaped multivesicular bodies, in GH4C1 cells after treatment, with 17b-estradiol, insulin and EGF (WAGURI, T.)
   
4. Heterogeneous localizations of Trk B among individual periodontal Rufinni endings in the rat incisor (ATSUMI, Y.)
   
5. Immunocytochemical localization of taurine in the developing retina of the lefteye flounder Paralichthys olivaceus (OMURA, Y.)
   
6. Reappraisal of potassium permanganate oxidation applied to Lowicryl K4M' embedded tissues processed by high pressure freezing/freeze substitution, with special reference to differential staining of the zymogen granules of rat gastric; chief cells (SAWAGUCHI, A.)
   
7. Immunolocalization of tight junction proteins, occludin and ZO-1, and glucose transporter GLUT1 in the cells of the blood-nerve barrier (TSERENTSOODOL, N.)
   
8. Porosity of the epithelial basement membrane as an indicator of macrophage- enterocyte interaction in the intestinal mucosa (TAKAHASHI-IWANAGA, H.)
   
9. Immunocytochemical demonstration of heat shock protein 25 in the rat temporo-mandibular joint (NOZAWA-INOUE, K.)
   

Summary

Title
Phenotypical and Morphological Alterations to Rat Sinusoidal Endothelial Cells in Arterialized Livers after Portal Branch Ligation

Author
Masato YAMASAKI, Kazuo IKEDA, Kazuki NAKATANI, Takatsugu YAMAMOTO, Yukiko KAWAI, Kazuhiro HIROHASHI, Hiroaki KINOSHITA and Kenji KANEDA

Address
Departments of Anatomy and Surgery, Osaka City University Medical School, Osaka; and Department of Anatomy, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

Summary
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 func. tional alterations to sinusoidal endothelial cells (SECs) in arterialized rat livers induced by portal branch ligation. At one week, after massive hepatic necrosis fol. lowing 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.

Prof. Kenji KANEDA, M. D,
Department of Anatomy
Osaka City University Medical School 1-4-3 Asahimachi, Abeno-ku, Osaka
545-8585 Japan
Tel: +81-6-6645-3705
Fax: +81-6-6646-3603
E-mail: kkaneda@med.osaka-cu.ac.jp

Title
The Role of Kupffer Cells in Liver Regeneration

Author
Toshiyuki TAKEISHI, Kenichiro HIRANO, Takashi KOBAYASHI, Go HASEGAWA, Katsuyoshi HATAKEYAMA and Makoto NAITO

Address
Second Department of Pathology, and First Department of Surgery, Niigata University School of Medicine, Nijgata, Japan

Summary
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 Kuplrer 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-a, another potent cytokine involved in liver regeneration, did not differ between Kupffer cell depleted and control mice during the regeneration. Administration of TNF-a 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-a non-mediated mechanisms.

Prof. Makoto NAITO
Second Department of Pathology
Niigata University School of Medicine Asahimachi-dori 1, Niigata
951-8510 Japan
Tel: +8l-25-227-2102
Fax: +8l-25-227-0761
E-mail: 2byori@med.niigata-u.ac.jp

Title
The Induction of Autophagic Vacuoles and the Unique Endocytic Compartments, C-Shaped Multivesicular Bodies, in GH4C1 Cells after Treatment with 17b-Estradiol, Insulin and EGF

Author
Satoshi WAGURI, Mari KOHMURA, Takahiro GOTOW, Tsuyoshi WATANABE, Yoshiyuki
OHSAWA, Eiki KOMINAMI and Yasuo UCHIYAMA

Address
Department of Cell Biology and Neuroscience (Al), Osaka University Graduate School of Medicine, Suita, Osaka and Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan

Summary
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 17b-estradiol, insulin and EGFI. 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 Cshaped 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.

Dr. Satoshi WAGURI
Department of Cell Biology and Neuroscience (Al) Osaka University Graduate School of Medicine
2-2 Yamadaoka, Suita, Osaka
565-0871 Japan
Tel: +81-6-6879-3124 or 3121
Fax: +81-6-6879-3129
E-mail: waguri@anat1.med.osaka-u.ac.jp

Title
Heterogeneous Localizations of Trk B among Individual Periodontal Ruffini Endings in the Rat Incisor

Author
Yukako ATSUMI, Saehiko HAYASHI, Kuniko NAKAKURA-0HSHIMA, Takeyasu MAEDA, Kojiro KURISU and Satoshi WAKISAKA

Address
Departments of Oral Anatomy and Developmental Biology and Oral and Maxillofacial Surgery II, Osaka University Faculty of Dentistry, Osaka; and Departments of Oral Anatomy and Pedodontics, Faculty of Dentistry, Niigata University, Niigata, Japan

Summary
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 immunonegative. The ordinary Schwann cells did not contain Trk B immunoreactive products. These findings imply that TrkB 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.

Dr. Satoshi WAKISAKA
Department of Oral Anatomy and Developmental Biology
Osaka University Faculty of Dentistry 1-8, Yamadaoka, Suita,Osaka
565-0871 Japan
Tel: +81-6-6879-2872
Fax: +81-6-6879-2875
E-mail: wakisaka@dent.osaka-u.ac.jp

Title
Immunocytochemical Localization of Taurine in the Developing Retina of the Lefteye Flounder Paralichthys olivaceus

Author
Yuri OMURA and Rie YOSHIMURA

Address
Laboratory of Animal Information Biology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

Summary
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.

Dr. Yuri OMURA
Laboratory of Animal Information Biology Graduate School of Bioagricultural Sciences
Nagoya University Chikusa, Nagoya
464-8601 Japan
Tel: +81-52-789-4082
Fax: +81-52-789-4083
E-mail: yuromura@agr.nagoya-u.ac.jp

Title
Reappraisal of Potassium Permanganate Oxidation Applied to Lowicryl K4M Embedded Tissues Processed by High Pressure Freezing/Freeze Substitution, with Special Reference to Differential Staining of the Zymogen Granules of Rat Gastric Chief cell

Author
Akira SAWAGUCHI, Soyuki IDE, Jun-ichi KAWANO,Ryoko NAGAIKE, Tsutomu OINUMA, Hiromasa TOJO, Mitsuhiro OKAMOTO and Tatsuo SUGANUMA

Address
Department of Anatomy, Miyazaki Medical College, Miyazaki; and Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Osaka, Japan

Summary
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 efrects 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 difrerential 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.

Prof. Tatsuo SUGANUMA
Department of Anatomy
Miyazaki Medical College 5200 Kihara, Kiyotake-cho
Miyazaki, 889-1692 Japan
Tel: +81-985-85-1784
Fax: +81-985-85-8406
E-mail: suganumat@post1.miyazaki-med.ac.jp 

Title
Immunolocalization of Tight Junction Proteins, Occludin and ZO-1, and Glucose Transporter GLUT1 in the Cells of the Blood-Nerve Barrier

Author
Nomingerel TSERENTSOODOL, Bo-Chul SHIN, Haruko KOYAMA, Takeshi SUZUKI and Kuniaki TAKATA

Address
Laboratory of Molecular and Cellular Morphology, Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan

Summary
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.

Prof. Kuniaki TAKATA, Ph.D.
Laboratory of Molecular and Cellular Morphology Department of Cell Biology Institute for Molecular and Cellular Regulation
Gunma University, Maebashi
371-8512 Japan
Tel: +81-27-220-8840
Fax: +81-27-220-8844
E-mail: takata@akagi.sb.gunma-u.ac.jp

Title
Porosity of the Epithelial Basement Membrane as an Indicator of Macrophage-Enterocyte Interaction in the Intestinal Mucosa

Author
Hiromi TAKAHASHI-IWANAGA, Toshihiko IWANAGA and Haruhiko ISAYAMA

Address
Department of Anatomy, Hokkaido University Graduate School of Medicine; and Laboratory of Anatomy, Hokkaido University Graduate School of Veterinary Medicine, Sapporo, Japan

Summary
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 histochemical 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.

Dr. Hiromi TAKAHASHI-IWANAGA
Department of Anatomy
Hokkaido University
Graduate School of Medicine
Kita-15, Nishi-7, Sapporo
060-8638 Japan
Tel: +81-11-706-5895
Fax: +81-11-706-7151
E-mail: hiromiva@med.hokudai.ac.jp

Title
Immunocytochemical Demonstration of Heat Shock Protein 25 in the Rat Temporomandibular Joint

Author
Kayoko NOZAWA-INOUE, Hayato OHSHIMA, Yoshiro KAWANO, Hitoshi YAMAMOTO, Ritsuo TAKAGI and Takeyasu MAEDA

Address
Department of Oral Anatomy and 2nd Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Niigata University, Niigata, Japan

Summary
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 Hsp25 was able to demonstrate various cellular elements in the synovial membrane of the joint. Intense immunoreaction for Hsp25 was recognized in certain cells comprising the synovial lining layer. Confocal microscopic observation revealed two characteristic profiles of the Hsp25-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 immunoelectron 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 Hsp25 is associated with an estrogen receptor, the immunopositive synovial lining cells were considered estrogen-target cells. Immunoreactivity for Hsp25 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 Hsp25 might be involved in the inhibition of apoptosis of those cells.

Dr. Kayoko NOZAWA-INOUE
Department of Oral Anatomy
Faculty of Dentistry
Niigata University
2-5274 Gakkocho-dori, Niigata
951-8514 Japan
Tel: +81-25-227-2817
Fax: +81-25-223-6499
E-mail : nozawa@dent.niigata-u.ac.jp