The effects of barnidipine hydrochloride, a new calcium antagonist, on the development of atherosclerosis were evaluated in cholesterol-fed rabbits. Two groups of twelve rabbits received l or 3mg/kg of barnidipine hydrochloride orally daily for 10 weeks. Another 12 rabbits received 12mg/kg/day of nifedipine, 6mg/kg twice a day, for 10 weeks as a positive control. These animals were fed 1% cholesterol diet, 100g daily during the treatment period. Twelve cholesterol-fed animals served as a cholesterol control group.
Over the 10-week period, serum cholesterol levels were significantly elevated in all groups, but there were no difference among these groups. After 10-week treatment, the aortas were removed and opened longitudinally. Sclerotic areas on the intimal surface were determined by planimetry. The ratios of sclerotic foci were significantly suppressed in the groups of barnidipine hydrochloride 3mg/kg and nifedipine 12mg/kg. Cholesterol contents in the arterial wall of aortic arch and abdominal portion in animals given barnidipine hydrochloride were significantly decreased in a dose dependent manner. The heart was cut into three transverse sections, and subepicardial and intramyocardial coronary arteries were examined microscopically. These examinations revealed no difference between the groups given bar-nidipine hydrochloride or nifedipine and cholesterol control group in the incidence and degree of sclerotic changes.
From these data, it is concluded that barnidipine hydrochloride suppresses the development of atherosclerosis and the cholesterol contents in aortic wall without reduction of serum cholesterol levels in cholesterol-fed rabbits. Also, histopathological examination revealed no effects on the cardiac coronary arteries as in other calcium antagonists such as lanthanum, diltiazem, and flunarizine.

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The effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), on bleomycin-induced diffuse alveolar damage (DAD) were investigated in male Syrian golden hamsters. Six-week-old animals were treated with a single intratracheal administration of bleomycin (BLM; 5mg/5ml/kg) or an equivalent volume of saline. Starting 1 day after the treatment, animals were given 1% BHA or 1% BHT in the diet or basal diet alone for 31 days. The survival rate of the BLM/BHA group (55%) was significantly higher than that of the BLM alone (15%) or BLM/BHT (15%) groups (p<0.01). Histopathologically, the lungs of hamsters in the BLM alone group, which were found dead 1 week after the treatment, showed severe pulmonary edema with hemorrhage. These were clearly attenuated by the BHA treatment, which in addition, somewhat accelerated fibrosing and regenerative changes. Moreover, surviving animals in the BLM/BHA group showed a tendency for reduction of emphysematous changes as compared to those receiving BLM alone and consequently to retain the normal alveolar architecture. On the other hand, the BHT administration did not exert any apparent inhibitory effects on early alveolar damage. Our results thus clearly indicate that BHA exerts a protective effect on BLM-induced DAD in hamsters although BHT did not inhibit the lung injury under the present experimental conditions. Possible mechanisms for the differences in influence between BHA and BHT on BLM-induced DAD are discussed.

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This study was conducted to establish a novel model of airway mucous cell hyperplasia and mucous hypersecretion suitable for both morphological and physico-biochemical examinations, using a xenotransplanted in vivo culture system. Tracheal epithelial cells of rabbits were cultured in de-epith-elialized rat tracheal grafts transplanted to nude mice. At four weeks, human neutrophil elastase (300μg/graft) or saline was injected into the graft lumina. Three weeks later, intratracheal mucus was collected from the retrieved tracheas, and the grafts were examined histologically.
The percentage of mucous cells in the elastase-treated tracheas was significantly increased compared to the control level (25.5±0.8% vs. 12.9±0.9%, p<0.001). Histochemically, mucous cells containing acidic glycoprotein were more abundant in the elastase-treated tracheas. The volume of mucus did not differ in the untreated and treated tracheas. The acidity of the mucus in the control and the elastasetreated tracheas was pH 7.7±0.2 and 6.3±0.1, respectively (p<0.001). Amounts of protein, N-acetyl-neuraminic acid, and fucose were all significantly higher in the elastase-treated tracheas.
These results indicate that elastase induced mucous cell hyperplasia and the hypersecretion of acidic mucus. This culture model will be useful for studying the role played by elastase in the development of this pathological condition.

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The effect of ethinylestradiol (EE), a synthetic estrogen which is tumorigenic to the mammary gland, pituitary gland, and liver, on the cell proliferation in 5 organs was investigated in male rats. Cell proliferation was estimated by bromodeoxyuridine (BrdU) immunohistochemistry after 72 hr continuous administration of BrdU through an osmotic minipump prior to sacrifice. Two experimental protocols were applied. In one, EE was administered to rats at l ppm in feed for 3, 7, 14, 28, and 91 days. The BrdU labeling indices (LI) in the pars distalis of the pituitary gland were significantly increased in the EE-treated rats compared to the control rats throughout the experimental period. The LI in the Harderian gland, a non-target organ of EE tumorigenesis, were also higher in the EE-treated rats on day 14 and thereafter. Other non-target organs, including the adrenal cortex and medulla, kidneys, and pancreas, showed variable or consistently lower LI in the EE-treated rats. In the other protocol, EE wa administered to rats at 0.1, 0.5, and 2 ppm in feed for 3, 7, and 14 days, and the serum prolactin (PRL) concentration and LI in the pars distalis were compared. EE treatment increased the serum PRL concentration, the LI in the pars distalis, and the numerical density (No. of cells/mm²) of BrdU-labeled PRL cells in a dose-dependent manner with complete parallelism among them. The increase in LI was observed within 14 days after the start of EE treatment, and the profiles of cell proliferation were unique to each organ, including tumorigenic target and non-target organs.

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Morphologic changes occurring in the endocrine organs and tissues of rats in single doss multiple doses, or primed estradiol study of a psychotropic drug were examined light and electro microscopically, or immunohistochemically. When a single dose of a psychotropic drug was given to female rats, prolactin mRNA contents in the pituitary gland was increased immediately after dosing of the drug. Ultrastructurally, fewer and smaller secretory granules of lactotrophs in the pituitary anterior lobe were observed 2 hours after dosing, and morphologic activation of rough endoplasmic reticulum and Golgi apparatus followed after its initial changes. In light microscopic examination, however, sligl changes in the pituitary anterior lobe and mammary glands were observed only 24 hours after dosing. When multiple doses of the drug were given to female and male rats, the pituitary changes and mammar development were seen light and electron microscopically. The pituitary changes were evidenced by immunohistochemistry using anti-BrdU or anti-PCNA monoclonal antibody, and AgNORs stain as indication of proliferating tissue. In the primed estradiol study, although microscopic changes in the pituitary gland and mammary glands due to hormonal effects could be advanced, there were no apparent differences between estradiol and estradiol/psychotropic drug treated animals. The findings in these studies revealed that the endocrine morphologic changes due to a psychotropic drug occurred soon after dosing, and electron microscopy and immunohistochemical techniques were useful for these investigations.

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Immunocytochemical localization of glutathione-reductase (GSSG-RD) in rat adrenal glands was studied in 1) normal untreated, 2) hypophysectomized, and 3) ACTH administered groups in order to clarify the biological significance of GSSG-RD in the adrenal cortical lipid metabolism including steroidogenesis. In the normal untreated group, GSSG-RD was immunohistochemically localized in all three zones of the adrenal cortex. In immunoelectron microscopic investigations, GSSG-RD was observed mainly in mitochondria. The hypophysectomized group showed atrophy of the zonae fasciculata and reticularis. GSSG-RD staining of the atrophic cortical zones was weakened. In immunoelectron microscopic investigations, GSSG-RD was observed not only in mitochondria but also in cytoplasmic matrix. On ACTH administration, the adrenal cortex became markedly hypertrophic, especially in the outer fasciculata. Immunohistochemically, GSSG-RD was mainly localized in the cells of those hypertrophic cortical zones. In immunoelectron microscopic investigations, GSSG-RD was observed not only in mitochondria but also in cytoplasmic matrix. These staining patterns were similar to those of glutathione-peroxidase (GSH-PO). GSH-PO reduces the lipid peroxides with concomitant oxidation of glutathione. Oxidized glutathione is reduced by GSSG-RD under the presence of NADPH. These two enzymes are known to be key enzymes of the lipid peroxides scavenging system. Based on our findings, it is strongly suggested that a very close relationship lies between the adrenal cortical GSSG-RD and steroidogenesis.

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Four kinds of polymer materials (polyurethane sheet, polyurethane beads, polysulfone powder, and polysulfone beads) were implanted into the subcutaneous tissue of F-344 male rats for 104 weeks. Subcutaneous tumors developed in 6 out of 20 rats with implanted polyurethane sheets. In the group with implanted polyurethane beads, 2 out of 20 rats also developed a subcutaneous tumor. All of these tumors were histologically diagnosed as malignant fibrous histiocytoma (MFH). No tumors were noted in the groups with implanted polysulfone powder or polysulfone beads.
For investigation of early cellular reactions to implanted materials during foreign body carcinogenesis, polyurethane sheets were implanted into the subcutaneous tissue of F-344 male rats, and the implanted sites were examined by light and electron microscope at 2, 4, 8, and 12 weeks after implantation. Uptake of bromodeoxyuridine (BrdU) or ³H-thymidine was also studied with enzyme histochemistry or electron microscopic autoradiography. By 8 weeks after implantation, focal proliferation of atypical cells was observed in the subcutis adjacent to the polymer sheet. These atypical cells showed a high labeling index for BrdU as well as ³H-thymidine.
These results indicate that atypical cell foci play the most important role as lesions in foreign body tumorigenesis.

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Regeneration of the heterotopically transplanted bladder (HTB) was histologically evaluated in a total of 30 rats sacrificed on days 3, 7, 14, 28, 42, and 56 after transplantation. The mucosa underwent severe necrosis and inflammatory change by day 3, but by 7 days regeneration occurred and was accompanied by simple hyperplasia and even papillary or nodular hyperplasia by 28 days. Regeneration gradually subsided, and by 56 days the mucosa and submucosa were nearly normal. 5-bromo-2'-deoxyuridine labeling indices correlated with the histologic changes observed in the regenerative process. The values reached a maximum by 28 days (1.9%), and then decreased. The generative process in the HTB differed from cyclophosphamide-induced cystitis of the homotopic (non-transplant-ed) bladder. It is concluded that considering the mucosal regenerative process of the HTB, 4 weeks after transplantation seems to be the proper period for instillation of test chemicals to be started.

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Comparison of immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) was studied in rat livers. Experiment 1: Rats were subjected to twothirds partial hepatectomy (PH). Three animals were killed at 1, 2, 3, 4, 7, and 14 days after PH. Experiment 2: Rats were given a single intraperitoneal injection of 500 mg/kg body weight of Dgalactosamine (D-gal). Three animals were sacrificed at 24 and 32 hours, 3, 7, and 14 days after administration. One hundred mg/kg body weight of bromodeoxyurine (BrdU) were administered in both experiments as a single intraperitoneal injection at I hour before sacrifice. Sequential changes in cellular proliferations were investigated by immunohistochemical staining of PCNA and BrdU using formlin-fixed and paraffin was embedded sections. In experiment 1, mitotic index of hepatocytes reached the highest values at 3 days after PH. Both the largest PCNA and BrdU labeling were found at 24 hours after PH. In addition, PCNA and BrdU labeling in bile ducts reached a peak 2 days after PH. In experiment 2, necrosis and vacuolar degeneration of hepatocytes were marked at 24-32 hours after D-gal administration, and proliferation of bile ducts and oval cells was noted at 3 days after administration. PCNA and BrdU labeling in the bile duct epithelial cells and hepatocytes had the highest values at 24 hours and 3 days after administration, respectively. A good correlation between PCNA and BrdU labeling was obtained not only in hepatocytes but in bile ducts in both experiments. These results strongly support that immunohistochemical staining of PCNA is a useful marker to examine cell proliferation even in tissues fixed by formalin and embedded in paraffin wax in the field of toxicologic pathology.

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A congenital basal cell tumor was found in a 21-day-old Sprague-Dawley rat. Macroscopically, a pinkish soft nodule about 10 mm in diameter was present on the mandibular skin region of the animal. The surface of the nodule was alopecic and ulcerated. Histologically, nuclei resembled those of the cells in the basal layer of the epidermis or the external root-sheath, but tumor cells were more closely packed and had a larger ratio of nucleus to cytoplasm. Mitotic figures were frequent. A large number of tumor cells formed various-sized epithelial nests, which appeared to coalesce into larger solid masses, suggesting a histogenetic derivation of the tumor from hair follicles. Immunohistochemistry revealed that most tumor cells were positive for cytokeratins. Ultrastructurally, the tumor cells had many desmosomes, hemidesmosomes, and tonofilaments.

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