Extracorporeal shock wave lithotripsy (ESWL) is generally considered as an effective cure for cholelithiasis. The present study is to explore whether ESWL does damage to hepatocytes. After the liver area was exposed to a given period of extracorporeal shock wave (ESW), the livers of 14 rabbits were taken out and samples for electron microscopy were prepared. The Na⁺-K⁺- ATPase activity in hepatocytes and the extent of damage by ESW was quantified by the cytochemical method using cerium (Ce) as the capture and by the electron probe X-ray microanalysis. It was observed that the Na⁺-K⁺-ATPase reaction products containing Ce were located on the sinusoidal, lateral and bile canalicular membrane, and were most concentrated on the latter one. The peak and background ratio (P/B) of Ce on the canalicular membrane in the control samples was 1.18±0.36; and in the samples exposed to ESW for 15, 30, 45 min, was 0.88±0.26, 0.86±0.22 and 0.48±0.31 respectively. The differences between the control and ESW-treated samples were statistically significant (p<0.01). However, the P/B of Ce in the samples of 60^th day after 30 min ESW exposure was 1.04±0.26. No difference existed as compared with the control (P>0.05). In addition, the Ce content of the reaction products on the canalicular membrane decreased to about 1/3 by ouabain treatment, indicating that rich Na⁺-K⁺- ATPase activity exists. These findings indicate that the ESWL performed on cholelithiasis may exert certain harmful effects on adjacent hepatocytes. Fortunately, the effects disappear within two months.

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To study the expression and role of adhesion molecules in normal salivary glands and their tumors, we have studied simultaneously the expression of E-cadherin, and associated cytoplasmic proteins, α-catenin and β-catenin. Paraffin sections were evaluated using the streptavidin biotin peroxidase complex (SBPC) method for E-cadherin, and the SBPC method with tyramide signal amplification (TCA) for α- and β-catenin.
Normal submandibular (n=10), parotid (n=4) and oral minor glands (n=4) showed cell membrane staining for E-cadherin and a positive reaction in intercalated and striated ducts for α - and β -catenin. Pleomorphic adenoma (n = 17) showed cell membrane staining for E-cadherin and α-catenin in luminal and non-luminal cells, but were negative for β-catenin. E-cadherin was also focally or irregularly positive in modified myoepithelial cells. Warthin's tumors (n = 4) expressed Ecadherin only and were negative for a- and β-catenins. Papillary adenocarcinoma (n = 4) showed predominant staining for E-cadherin, with no expression in the other tumor cells. Adenoid cystic carcinoma (n = 4) showed only cell membrane positivity for E-cadherin, with an absence of α- and β-catenins in the luminal cells. Non-luminal tumor cells of adenoid cystic carcinoma were negative for E-cadherin and positive for α- and β-catenins. The results were concluded as follows, 1) there was uniform cell membrane staining for E-cadherin in normal and benign tumor epithelium, but α- and β-catenins showed different expression and heterogeneous localization, 2) loss of E-cadherin reactivity was seen in malignant lesions, 3) there was no correlation between the distribution of Ecadherin, α-catenin and β-catenin in benign or malignant tumors of salivary gland origins. The results show variability and heterogeneous expression for E-cadherin, α- and β-catenins in salivary gland tumors.

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The existence and distribution of mammosomatotrophs (MS cells) containing growth hormone (GH) and prolactin (PRL) were detailed in swine adenohypophysis. MS cells were positively identified and were first recognized in the adenohypophysis of fetuses with 33.0 cm in body length, when GH cells existed earlier and PRL cells occurred. The proportion of MS cells transiently increased in the glands of three month-old females and were densely populated in the fore ventral (FV) and the hind ventral (HV) region except for the zona tuberalis (ZT). The distribution was similar to GH cells and PRL cells. MS cells were divided into three subtypes by the intracellular intensity of both GH and PRL stainings. Type I MS cells which were strongly stained with anti-GH antiserum, constituted 100% of MS cells in fetuses and the proportion decreased with age in females. Type II MS cells which were evenly stained with both anti-GH and PRL antisera were detected in all animals except for fetuses, and were proportioned moderately. Type III MS cells which contained more PRL than GH existed predominately in three and six month-old females, castrated males and aged males. The findings indicated that an amount of GH and PRL within MS cells was changeable in response to physiological conditions. The results suggest that GH cells may transform into PRL cells, with the MS cell acting as an intermediate cell in swine adenohypophysis, and that the GH and PRL in MS cells are synthesized synchronously or asynchronously according to the secretory activities.

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To examine the relationship between induction of phenobarbital (PB) -inducible cytochrome P-450 (P-450) 2B1/2 and the PB-induced proliferation of endoplasmic reticulum (ER) in hepatocytes, we measured the amount of P-450 2B1/2, amount of total P-450 isoforms, and area of ER in the cytoplasm of perivenular, midzonal and periportal hepatocytes from adult male rats injected with 80 mg/kg of PB once a day for 3 days by quantitative immunohistochemistry, microphotometry and morphometry, respectively. Then, the density of P-450 2B1/2 or total P-450 molecules in the ER membrane (number of molecules per μm² of ER) was calculated by dividing P-450 2B1/2 or total P-450 content by the ER area followed by multiplying Avogadro's number. P-450 2B1/2 content in hepatocyte cytoplasm increased markedly in perivenular (15 times), midzonal (20 times) and periportal hepatocytes (10 times) after PB injection. The area of ER increased all hepatocytes (1.7-1.9 times) after PB injection. Thus the density of P-450 2B1/2 in the ER membrane increased markedly in hepatocytes in the three zones, whereas that of total P-450 increased slightly. The proportion of P-450 2B1/2 density to total P-450 density in the ER membrane increased markedly in perivenular hepatocytes and moderately in midzonal and periportal hepatocytes after PB injection. Thus P-450 2B1/2 molecules in the ER membrane increased in perivenular hepatocytes with reduction in molecules of other P-450 isoforms after PB injection (substitutional increase), whereas the value increased in midzonal and periportal hepatocytes without reduction in other P-450 isoforms (additive increase).

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Proliferative and aberrant cellular activities of human tissues were evaluated using a novel double staining technique for histone H3 (with in situ hybridization) and immunohistochemistry for p53 protein. Simultaneous analyses were run on tissue sections of papillomas, squamous cell carcinomas (SCC) and normal non-neoplastic stratified squamous epithelia from human oral specimens using formalin-fixed, paraffin-embedded material. Sections were first FITC-labeled histone H3 DNA probe and then treated with alkaline phosphatase-conjugated anti-FITC rabbit antibody. Following reaction with anti-p53 protein mouse monoclonal antibody using the labeled streptavidin-biotin (LSAB) method, sections were stained with 3-amino-9-ethylcarbazole-HCI to detect p53 protein, and with 5-bromo-4-chloro-3-indoxy phosphate/nitro blue tetrazolium to detect histone H3 mRNA. Several methods differing only in the order of detection procedures were employed and subsequently compared. Using these methods, histone H3 mRNA and p53 protein were detected both within the cytoplasm and the nucleus, respectively. Histone H3 mRNA and p53 protein were expressed in larger cell populations in the following order: SCC, papilloma, and nonneoplastic stratified squamous epithelia. The double staining as employed here proved effective for simultaneous evaluation of cell proliferative activity as well as the overexpression of aberrant gene on the same tissue sections.

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The localization of tyrosine hydroxylase (TH) -immunoreactive (IR) (TH-IR) neurons, which are transiently observed in the anterior olfactory nucleus (AON) of the mouse, was studied. A double-labelling method was used with antibodies against TH, L-3, 4-dihydroxyphenylalanine (DOPA), phenyletha-nolamine-N-methyltransferase (PNMT), γ-aminobutyric acid (GABA), calretinin (CR) and calbindin-D28k (CB). When the total number of TH-IR neurons observed in all regions of the AON obtained from 10- to 14- day-old mice was set at 100%, 30% of TH-IR neurons were observed in the lateral (AOL), 25% in the dorsal (AOD), 20% in the ventral (AOV), 15% in the medial (AOM) and 5% in both the posterior (AOP) and external (AOE) regions. Approximately 40% of the TH-IR neurons in the AOL to the AOV regions also exhibited GABA immunoreactivity. This corresponds to 20% of the total number of TH-IR neurons observed in all regions of the AON, and also corresponds to 12% of the total number of GABA-IR neurons observed in all regions of the AON. However, TH-IR neurons did not exhibit concomitant PNMT, CR, and CB immunoreactivities. These findings suggest that the TH-IR neurons in the AON of the mouse, a proportion of which is also GABA-IR, may function as a source of neuromodulators for the postnatal growth of GABA-IR neurons.

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A distribution of abnormal catecholaminergic and serotonergic fibers was in the spinal cord of 12-month-old zitter mutant rats which are characterized by abnormal metabolism of superoxides studied using antisera for tyrosine hydroxylase (TH) and serotonin. Similar to our previous report [13], abnormal serotonergic fibers characterized by swollen varicosites were observed mainly in the anterior and lateral columns of the cervical and lumber segments of zitter rats. On the other hand, abnormal TH-immunoreactive fibers with swollen varicosities were located in the anterior and lateral funiculus of lumbar and sacral segments of these mutant rats. The present results suggest that the degeneration patterns differ between catecholaminergic and serotonergic fibers in aged-zitter rats.

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To elucidate the effects of synthetic salmon calcitonin (sCT) on the cells in the rat pituitary gland, we histopathologically and immunohistochemically examined the early changes, after 4 or 13 weeks treatment with sCT 120 IU/kg. Focal proliferative lesions of the anterior pituitary glands were found in 100% of the cells after treatment for 13 weeks with sCT. Histologically, the cells which make up the focal proliferative lesions were classified into the following groups, i. e., 1) enlarged basophilic focus, 2) vacuolated cell focus and 3) chromophobe cell focus. The majority of nuclei in those focal proliferative lesions were positively stained by BrdU. Furthermore, those focal proliferative lesions had positive staining for only the alpha-subunit and failed to show Pit-1 protein. Thus, sCT-induced pituitary tumors are considered to be endocrinologically inactive, pure alpha-subunit-producing tumors. Therefore, these tumors were thought to be potentially useful models of pure alpha-subunit-producing pituitary tumors in humans.

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The reduction of nitroblue tetrazolium (NBT) produces diformazan which shows localization of superoxide anion (O₂^-) in polymorphonuclear leukocytes (PMNs). The detection of O₂^- in PMN is performed exclusively by using a microscopic NBT test, while an ultracytochemical method in NBT has seldom been employed. The present study compared various tetrazolium salts, including nitroblue tetrazolium (NBT), tetranitroblue tetrazolium (TNBT) and 2- (2′-benzothiazolyl) -5-styryl-3- (4′-phthalhydrazidyl) tetrazolium chloride (BSPT), by detecting O₂^- using a histochemical and an ultracytochemical method. Peripheral rabbit leukocytes which ingested opsonized zymosan were prepared for detection of O₂^-. The reacted leukocytes in each tetrazolium salt contained a reaction medium which was examined by light and electron microscopy. Each reaction of the tetrazolium salts took place in the phagosomal membrane. The color of formazan in the TNBT was black rather than dark purplish-blue, and darker than that of NBT. On the other hand, the reaction color of BSPT was purplish-red. The reaction formazans of NBT and TNBT were coarse and electron-dense by ultracytochemistry. Some of these diformazans were not associated with the membrane, while the formazan of BSPT demonstrated fine deposits and was electron-dense. These reaction products corresponded well with the membrane. These results suggest that BSPT could be employed for detection of O₂^- not only in the histochemical method but also in ultracytochemistry and can be used as a new method.

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Primate spermatogenesis is characterized by yet unidentified mechanisms to regulate its rate-limiting process, spermatogonial proliferation. In the previous study using a seasonal breeder, Japanese monkey (Macaca fuscata), we demonstrated that the spermatogonial proliferation is suppressed through cytoplasmic liberation of PGP 9.5 when the spermatogenetic activity declines in the nonbreeding season. By comparing the morphology of the seminiferous tubules between the breeding and nonbreeding seasons, the present study demonstrates that in the nonbreeding season a basal lamina of seminiferous epithelium, with which spermatogonia directly keep contact, enlarges its extent at levels of lamina densa and lamina fibroreticularis, and is frequently invaginated. The invagination is composed of only the three laminae, not of cell components. The invaginations expand contact area between the spermatogonia and the basal lamina, which would interrupt spermatogonial proliferation. An ultrastructural distribution of laminin suggests that the invagination during the nonbreeding season is due to rearrangement of laminin molecules inside the basal lamina. The present results indicate that the alteration of the basal lamina of the seminiferous epithelium would mediate effects of the hormonal regulators and enable the fully restorable, transient suppression of spermatogenesis in primate.

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The localization of glutathione S-transferase P (GST-P) in postnatal rat liver was examined by an immunohistochemical technique. Dexamethasone (DEX) was injected intraperitoneally into postnatal rats at a dose of 2 μg/g body weight daily for 4 days. GST-P was detected only in the cytoplasm of epithelial cells of the interlobular bile ducts in the livers of 8-day-old rats that did not receive DEX-administration. In contrast, dispersed-positive hepatocytes were observed in the liver lobules of rats that were administered DEX from 4 days after birth. In these rats, GST-P was found in the cytoplasm of those hepatocytes and epithelial cells of the interlobular bile ducts. GST-P was observed only in the cytoplasm of epithelial cells of the interlobular bile ducts in the livers of 13-day-old rats that did not receive DEX-administration and those that began DEX-administration at 9 days. The immunoreactivity was stronger compared to the 8-day-old rats. These results indicate that DEX promotes the appearance of hepatocytes that contain the GST-P protein in early postnatal rat liver.

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Paraquat has previously been shown to reduce the viability of rat C6 glioma cells, suggesting that this drug may be toxic to normal glial cells. However, there is no direct evidence for the cytotoxic effect of paraquat on glial cell in vivo. To investigate the toxic effect of paraquat, the damage to hippocampal neurons and astrocytes in paraquat-poisoned rat brain was immunohistochemically examined using microtubule associate protein-2 (MAP-2) and glial fibrillary acidic protein (GFAP) as a histological marker. Paraquat poisoning reduced the number of astrocytes in rat hippocampus in a concentration-dependent manner. In addition, paraquat caused the damage to hippocampal neurons at relatively lower doses (25 and 50mg/kg), and this neuronal damage was unexpectedly less pronounced at a higher dose (100mg/kg). These findings indicate that paraquat is toxic to astrocytes in vivo, and furthermore suggests that the toxic effect of paraquat on glial cells may be different from that on neuronal cells in the rat brain.

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