The process of the arrangement of D-periodic collagen fibrils and their growth in maturing tail tendon of mice were studied with the association of type VI collagen, from fetal day 10 to 10 weeks after birth. In tail tendons, the amount of collagen fibers gradually increased along with the diameters of D-periodic collagen fibrils during. maturation. Type VI collagens first appeared on fetal day 10, when D-periodic collagen fibrils were not recognizable. Type VI collagens were observed around the fibroblastic cells in early stages of development, but were among thick collagen fibrils in the adult tendon. While the periodic distances of type VI collagen fibrils were over 100nm at fetal days, they were packed to 80-90nm after birth. The periodic bands were stained well with ruthenium red in adult but not in young tendons, indicating the close association of proteoglycans or glycosaminoglycans (PGs/GAGs) with maturing type VI collagens. Since type VI collagen in native form is known to associate with D-periodic collagen fibrils via PGs/GAGs, ruthenium red-stainability on the surface of D-periodic collagen fibrils was also examined; results showed that ruthenium red-stainable elements were D-periodically associated.
When the surface morphology of D-periodic collagen fibrils in adult animals was examined by atomic force microscopy, a large depth of the groove between elevated and depressed surfaces became prominent when the fibril surface was digested with hyaluronidase. Thus, it is possible to observe topologically the association of PGs/GAGs and probably that of type VI collagens with D-periodic collagen fibrils.

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Negative charged sites in the normal rabbit articular cartilage were investigated using cationic colloidal iron methods. In light microscopy of the cartilage stained with the colloidal iron at pH 1.5, a distinct Prussian blue reaction was observed in the pericellular matrix, and a weak blue reaction in the territorial and interterritorial matrices. At pH 7.0, a diffuse Prussian blue reaction was observed in the pericellular and interterritorial matrices.
Digestion with chondroitinase ABC, hyaluronidase and keratanase could not erase the Prussian blue reaction. However, the sections digested with collagenase followed by chondroitinase ABC showed significant elimination of the Prussian blue reaction.
Electron microscopy of ultrathin sections stained with the colloidal iron at pH 1.5 revealed that the cationic colloid particles were deposited abundantly in the pericellular matrix and dotted along collagen fibrils in the territorial and interterritorial matrices.
The present results suggest that negatively charged sites in the articular cartilage derive mostly from chondroitin sulfate, whose proteoglycans firmly bind to collagen fibrils. Such an ultrastructure may maintain the electrostatic microenvironment in the collagen plexus, holding much water in the cartilage matrix, and also producing biomechanical properties such as tensile strength and elasticity of the cartilage.

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The electrocyte of the electric organ of the electric eel, Electrophorus electricus, L was investigated by light and electron microscopy as well as immunoelectron microscopy, in order to clarify the fine structures and distribution of cytoskeleton filaments and their relations to proteins, especially desmin and actin. Cytoskeleton-enriched fractions of the electrocytes were analysed with SDS-PAGE. It was verified that a meshwork of filaments was distributed in the electrocytes, more abundantly in the anterior than in the posterior part of the cell, and that this could be associated with membrane invaginations. Desmin and actin were the components of this meshwork, suggesting that desmin intermediate filaments and actin filaments might play a role in the maintenance of the morphology of electrocytes and, as an intracellular filamentous meshwork, they may contribute to the organization of the components of membranes and papillae formation on the anterior face of the electrocytes.

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The present study was conducted to examine the response of amoeboid microglial cells in the postnatal rat brain to colchicine administration. One-day-old postnatal rats were given intraperitoneal injections of colchicine and sacrificed at 7, 14 and 21 days of age. In rats killed at 7 days age, the number of OX-42, OX-18 and ED1 positive amoeboid microglial cells was considerably reduced when compared with the control rats. At 14 and 21 days, the number of cells immunoreactive with the above antibodies was comparable to that of the control rats. The intensity of the immunoreaction with the various antibodies was also comparable in colchicine injected and control rats. When rhodamine isothiocyanate (RhIC) was administered, amoeboid microglial cells emitted a bright fluorescence in control rats as well as in colchicine-injected rats, although in the latter, the number of RhIC labelled cells was considerably reduced. With the antibody bromodeoxyuridine a large number of stained cells were observed in the control rats. On the other hand, occasional labelled cells were recognized in colchicine-injected rats. Apoptotic amoeboid microglial cells were observed in 4-day-old colchicine-injected rats. At the electron microscopic level, amoeboid microglial cells in colchicine-injected rats killed at 7 days of age showed a large number of phagosomes in their cytoplasm compared with the corresponding control rats. At 14 and 21 days, in colchicine-injected and control rats, amoeboid microglial cells did not display any noticeable defferences. It is concluded from the present study that colchicine suppresses the number of amoeboid microglial cells, and that this may be attributed to the antimitotic effect of the drug as well as apoptosis induced by it; the phagocytic activity, however, was not affected. The cells returned to their normal population and morphological features once the drug was discontinued, indicating the reversible nature of the drug effect.

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Effects of bFGF, TGFα and TGFβ on cultured mesenchymal cells of rabbit phalli were investigated. A remarkable cell proliferation reaching confluency was only obtained from the media containing bFGF by the first week of culture. By subsequent 4 weeks culture using such confluent cells in the media containing bFGF, TGFα or TGFβ, respectively, cells cultured in the media containing bFGF showed an active transformation to endothelial precursor cells determined by positive immunoreactions for factor VIII-related antigen at 2 weeks. By electron microscopy, such vasof ormative cells appeared in contact with each other, occasionally forming a solid cell cord. The use of Matrigel's basement membrane matrix (Matrigel) in each culture bed promoted the capillary ingrowth, implying that certain extracellular matrix (ECM) components included in Matrigel may accelerate the formation of neocapillaries. Enhancement in the production of ECM components and transformation to smooth muscle cells determined by positive immunoreactions for actin were also seen in cells cultured in the media containing TGFα and TGFβ, respectively. The present results indicate that the above growth factors play crucial roles in the differentiation of the rabbit cavernous body.

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Argininosuccinate synthetase (ASS) and nitric oxide synthase (NOS) comprise part of the cyclic metabolic pathway to produce nitric oxide (NO). ASS is one of the arginine synthesis enzymes which synthesizes argininosuccinate from aspartate and citrulline, and NOS forms NO and citrulline from arginine. This study examines the localization of ASS and NOS in the cat hypothalamus using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and immunohistochemistry against ASS and NOS.
NADPH-d positive and/or ASS-immunoreactive neurons were localized in the following areas: the anterior hypothalamic area, the anterior hypothalamic nucleus, the supraoptic nucleus, the suprachiasmatic nucleus, the periventricular complex, the paraventricular nucleus, the parvocellular nucleus, the lateral hypothalamic area, the dorsomedial hypothalamic nucleus, the dorsal hypothalamic area, the posterior hypothalamic area, and the supramammillary nucleus. NOS and ASS double-labeled neurons were found in the anterior hypothalamic area, the supraoptic nucleus, the central part of the paraventricular nucleus of the hypothalamus, the lateral hypothalamic area, ventral part of the parvocellular hypothalamic nucleus, the posterior hypothalamic area, and the supramammillary nucleus. Double-labeled neurons in the hypothalamus comprised 20.7-32.0% of ASS-immunoreactive neurons and 10.2-26.3% of NOS-immunoreactive neurons. The results suggest the existence of the ‘NO cycle’ in situ and the physiological importance of NO and argininosuccinate in several regions of the cat hypothalamus.

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Duct-associated lymphoid tissue (DALT) of the main excretory duct in the monkey parotid gland was first demonstrated by light microscopy and by transmission and scanning electron microscopy. The DALT included a follicular area, a parafollicular area and a specialized overlying epithelium with distinct fine-structural elements. There was usually a solitary lymphoid follicle located in the subepithelial area near the orifice of the parotid duct. The lymphoid follicles typically had a distinct germinal center. Numerous immune cells often infiltrated into the epithelium overlying the lymphoid follicle. The superficial epithelial cells of the DALT were larger and flatter than the ordinary duct epithelial cells, and had short irregular microvilli on their luminal surface. They were also in close contact with immune cells such as dendritic cells and lymphocytes. Goblet cells were rare in this area. In addition, bacteria, seen at the duct orifice, were sometimes taken up by the flattened epithelial cells near the orifice. Latex microspheres administrated as particulate antigens at the duct orifice were selectively taken up by the flattened epithelial tells and also by the intraepithelial dendritic cells of the DALT.
These morphological findings suggest that the epithelial cells of the DALT in parotid glands take up antigens from the duct lumen and transport them to adjacent immune cells, and that the DALT in parotid glands may serve as one of the inductive sites in the common mucosal immune system.

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Pathways of peritoneal fluids to the pleural cavity in the rat were investigated by light microscopy and transmission electron microscopy (TEM). Intraperitoneally injected India ink was demonstrated to enter the subperitoneal lymphatics through lymphatic stomata, and to drain through the subpleural collecting lymphatics, into the parasternal, paravertebral and mediastinal lymphatic trunks as well as the thoracic duct. Five to 10min after the intraperitoneal injection of India ink, the parasternal lymphatic trunk was ligated at the third intercostal space. Thirty minutes, 1h, or 2h after the ligation of either the right or the left trunk, India ink was macroscopically recognized only around the ligated trunk. When the right and left trunks were simultaneously ligated, India ink leaked around both trunks. Five hours after the ligation of both trunks, a massive amount of ink was located in the interstitium of the anterior thoracic wall. TEM revealed carbon particles passing through gaps of the lymphatic endothelial cells into the interstitial space, and partly reaching the medothelial surface lining the anterior thoracic wall. Results show that obstruction or narrowing of the lymphatic trunks draining the diaphragmatic lymph causes a hydrothorax, indicating that this is at least one mechanism causing this during continuous ambulatory peritoneal dialysis and diseases with ascites.

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The pineal organ possesses highly fenestrated capillaries, and is devoid of the so-called blood-brain barrier. The present study indicated that the pineal epithelium of the teleost fish, ayu Plecoglossus altivelis, possesses an unusually thick and convoluted basement membrane (2.2-2.4μm in width) which is visible even under the light microscope. This pineal basement membrane was observed by scanning and transmission electron microscopy and its detailed composition and relationships with the fenestrated capillaries and the perivascular space were investigated. As the basement membrane was composed of three to eight layers of basal laminae interspersed with laminae lucidae, we termed it the “multilayered basement membrane”. In consideration of our previous demonstration that macromolecules such as HRP are trapped by the basement membrane, it is suggested that this multilayered basement membrane may prevent foreign substances from reaching the pineal epithelium.

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