We assessed changes in neuropeptide Y (NPY), vasoactive intestinal peptide (VIP) and Choline acetyltransferase (ChAT) immunoreactivities when neonatal rat chromaffin cells were cultured in a medium containing nerve growth factor (NGF) or the synthetic glucocorticoid dexamethasone (DEX), examining whether their expression was correlated with the morphological changes induced by NGF and DEX. All of the chromaffin cells in culture were tyrosine hydroxylase (TH)-immunopositive regardless of whether they extended processes. TH-immunoreactive materials of NGF-treated chromaffin cells were distributed in all the cytoplasmic processes, even at the tips of growth cones. The percentage of NPY-positive chromaffin cells did not change markedly when treated with NGF or DEX throughout the 14 days in culture. The proportion of VIP-positive chromaffin cells increased gradually in the NGF-treated group and that of ChAT-positive cells in the group was similar to VIP. The morphological alterations induced by NGF were not correlated with the changes in proportions of NPY-, VIP- or ChAT-positive chromaffin cells. The percentages of VIP- or ChAT-immunopositive chromaffin cells in the NGF-treated group showed much greater increases than those in the DEX-treated group. These findings suggest that NGF might modulate the phenotypic changes of neuropeptides and amines in rat chromaffin cells.

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This study demonstrates that many neurons in the somatosensory cortex, cingulate cortex, retrosplenial cortex and hippocampal subiculum of the mouse brain are covered by sulfated proteoglycans which are intensely negative-charged and stained with cationic iron colloid, while being digested with hyaluronidase. Neurons with similar perineuronal proteoglycans are also recognized in the extrapyramidal system (superior colliculus, red nucleus, reticular formation, vestibular nuclei and cerebellar nuclei), in the secondary auditory system (cochlear nuclei, nucleus of trapezoid body, inferior colliculus and nucleus of lateral lemniscus), in the vestibulo-ocular reflex system (vestibular nuclei and extraocular motor nuclei), and in the pupillary reflex system. The neurons with perineuronal sulfated proteoglycans in the cerebral cortices and hippocampal subiculum are usually labeled with the lectin Vicia villosa agglutinin, though those in the cerebellar, vestibular and cochlear nuclei may not be reactive to this lectin. Double staining of the retrosplenial cortex, hippocampal subiculum and cerebellar nuclei with Golgi's silver nitrate and cationic iron colloid indicates that the perineuronal sulfated proteoglycans are identical with the Golgi's reticular coating or glial nets.

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Many neurons in the human somatosensory cortex (Area 7 of Brodmann) possess an intensely negative-charged surface coat consisting of perineuronal sulfated proteoglycans which were stained with cationic iron colloid. This surface coat was stained doubly with cationic iron colloid and Golgi's silver nitrate. The result indicates that the perineuronal sulfated proteoglycans are identical with Golgi's reticular coating, whose existence and nature have previously been controversial. The result also suggests that Golgi's silver nitrate stains the core proteins of proteoglycans.

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The choroid plexus in rats exhibited ultra. structural changes following a non-penetrative blast. The immunophenotypic features of epiplexus cells associated with the choroid plexus epithelium were also altered. In rats killed at 1 and 7 days after the blast, the intercellular spaces between the epithelial cells were greatly widened, coupled with the massive eruption and possible extrusion of the apical cytoplasm into the ventricular lumen. Associated with these changes was the passage of some monocytes/lymphocytes across the epithelium. The incidence of such a migratory phenomenon was more frequent in rats killed 7 days after the blast. In rats killed 14 days after the blast, the ultrastructural changes of the epithelial cells became less pronounced. At 21 and 28 days after the blast, the ultrastructure of the choroid plexus was comparable to that of normal specimens. The immunoreactivity of epiplexus cells in terms of their cell number and staining intensity with the monoclonal antibodies OX-42, OX-18, OX-6 and ED1 was noticeably augmented at 7 and 14 days after the blast; this, however subsided at 21 and 28 days. It is concluded that the choroid plexus is extremely sensitive to a blast wave as manifested by its structural alterations and the vigorous expression of CR3 receptors and MHC antigens by the epiplexus cells. It is suggested that a possible immune response might have been triggered in the cerebrospinal fluid ventricular system following the blast.

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To examine localization of cysteine and aspartic proteinases, and ubiquitin in rat and human urinary bladders, immunocytochemistry was applied to the tissues. In semi-thin sections, immunoreactivity for cathepsins B and D was densely localized throughout epithelial layers of rats and humans, while that for cathepsins H and L was mainly localized in rat superficial and human intermediate cells. Immunoreactivity for cathepsin C was relatively high in rat and human epithelia, especially in humans. Immunoreactivity for ubiquitin was detected in rat and human epithelial cells. By electron microscopy, vesicular or heterogeneously dense lysosomes labeled with immunogold particles indicating cathepsin B were seen in rat and human epithelial cells; particularly, they often appeared near fusiform vesicles in rat superficial cells and in human intermediate and superficial cells. By double immunostaining, lysosomes with or without vesicular structures were co-labeled with immunogold particles showing both cathepsin B and ubiquitin. The results suggest that cathepsins B, C, H, and L, and cathepsin D are involved in the lysosomal system of rat and human bladder epithelia. Moreover, considering that ubiquitin is a cofactor in the soluble ATP-dependent proteolysis, the results may also indicate that epithelial cells actively form autophagolysosomes.

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The prohormone convertases PC1/PC3 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. To determine the cellular and subcellular distribution of PC1/PC3 and PC2 in the rat pancreas, we generated their polyclonal antisera in rabbits, using as immunogens two synthetic peptide antigens corresponding to amino acids 442-459 (ST-28) of PC1/PC3 and 613-629 (ST-29) of PC2 and two bacterially expressed antigens covering amino acids 145-414 (KN-1) of PC1/PC3 and 385-637 (KN-2) of PC2. Western blot analysis revealed the presence of PC1/PC3 (87 and 68kDa) and PC2 (75 and 70kDa) in rat pancreatic islets, indicating that the antisera are specific for the corresponding antigens. Immunocytochemical staining of serial sections demonstrated that the antibody against PC1/PC3 immunostained only insulin-producing cells, whereas the PC2 antibody stained insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-producing cells. Double-immunolabeling of the prohormone convertases and pancreatic hormones with gold particles of different sizes revealed that insulin-positive secretory granules were also immunolabeled with PC1/PC3 and PC2 antibodies, whereas glucagon-, somatostatin-, or pancreatic polypeptide-positive granules were labeled only with the PC2 antibody. This differential localization of PC1/PC3 and PC2 provides a further problem on the substrate-specificity of these enzymes in the processing of pancreatic prohormones.

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The distribution of lymphatics in human palatine tonsils was studied by enzyme-histochemistry for 5′-nucleotidase (5′-Nase) and scanning electron microscopy (SEM) of lymphatic corrosion casts. The palatine tonsils were found to possess lymphatics in the parafollicular area (i. e., interfollicular, interfolliculo-septal, and folliculo-septal area), in the connective tissue septa, and in the capsules, but not in the subepithelial area between the follicles and the follicle-associated epithelia or within the follicles. The tubular lymphatics originated some 200-300μm below the epithelium and formed a three-dimensional network in the parafollicular area. Some lymphatics around the lower part of the follicle were flat, wide, and irregular in shape, and thus appeared to be lymphatic sinuses, referred to as perifollicular lymphatic sinuses. The lymphatics in the parafollicular area drained into the septal lymphatics, which ran rather straight in the connective tissue septa. The septal lymphatics finally gathered into the broader capsular lymphatics. Most of the septal and capsular lymphatics were endowed with valves. Our results indicate that lymphocytes and fluid from the follicles and the subepithelial region enter the perifollicular lymphatic sinuses and/or the interfollicular lymphatics, pass through the interfolliculo- and folliculoseptal lymphatics, and finally enter the septal and capsular lymphatics to leave the tonsil.

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Meissner corpuscles in the palatine mucosae of ddY-mice of various ages were studied histochemically and electron microscopically. As the age of the animals advanced, regressive Meissner corpuscles with lessened or depleted activity of non-specific cholinesterase (ChE) increased in number. Immunohistochemistry for Na⁺/K⁺-ATPase showed that the complex ramification of axon terminals in Meissner corpuscles attained at maturation gradually changed to a sparse distribution as the animals aged. Only a few putative rudiments of Meissner corpuscles, which were completely devoid of nerve terminals, were encountered. Electron microscopy showed that the regressive Meissner corpuscles contained atrophic axons and lamellae in a disordered arrangement, although the severity of the atrophy differed depending on the degree of the regression. Furthermore, the Meissner corpuscles were found to progressively accumulate fibrous long-spacing fibers having a periodicity of 160nm and unique spherical corpuscles 150-300nm in diameter in their intercellular spaces.
From these results we concluded that: 1) the atrophy of the axon and lamellae proceeds almost simultaneously during the age-related regression of Meissner corpuscles; and 2) not only the atrophy of the axon and the lamellar cell but microenvironmental changes such as the accumulation of intercellular substances is also involved in the regression of these corpuscles.

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Distributions of growth-associated protein-43 (GAP-43) in the periodontal ligament and dental pulp of adult rats were studied by light and electron microscopy. The mature periodontal ligament and dental pulp contained numerous GAP-43-positive neural elements, comprising periodontal Ruffini endings and thin nerve fibers, but expression patterns differed among the kinds of nerves. In the periodontal ligament of rat molars, immunoelectron microscopy revealed that GAP-43 like immunoreactivity in the Ruffini ending, an essential mechanoreceptor, was confined to the Schwann sheaths around the axon terminals and was not in the axon terminals themselves, unlike free endings that revealed axonal GAP-43. However, the lamellar Schwann cells associated with the cutaneous receptors did not exhibit any GAP-43 like immunoreactivity though they were intensely reactive for low affinity nerve growth factor receptor (p75-NGFR), a marker for lamellar Schwann cells in mechanoreceptors. The characteristically uniform expression of GAP-43 in the Schwann lamellae that surround the Ruffini mechanoreceptors of rat molar ligament suggests that Schwann cells are involved in the GAP-43 mediated plasticity of these receptors. On the other hand, the pulpal nerves were filled with the reaction products in their axonal spaces, suggesting the potential for neuronal plasticity during normal function and after tooth injury.

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