[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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[in Japanese]

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Freeze-dried sections (14μm thick) of the whole layers of retinas were prepared from normal (C57BL strain, male) and retinal-degeneration (rd-mice, C 3 H strain, male) mice from neonate to 20 weeks of age. The samples of layers and sublayers were dissected out from the sections and weighed on a highly sensitive quartz fiber balance (sensitivity±150pg). A new microassay method for γ-aminobutric acid transaminase (GABA-T) was developed, based on an enzymatic amplification reaction and NAD cycling, to determine the distribution of GABA-T activities in normal and degenerated retinas. (1) The new microassay method is much more sensitive than the previous methods and applicable to single cell analysis. (2) In the normal samples containing evenly all retinal layers, GABA-T activity increased from neonate to 20 weeks of age. In contrast, the activity increased from neonate to 6 weeks of age and then decreased by 25% until 20 weeks of age in the similar samples from rd-mouse. (3) In the normal retinas from 6 weeks old mice, GABA-T activity had a peak in the ganglion cell layer samples and decreased in the outer retinal layer. However, the outer plexiform layer, inner nuclear layer, choroid and optic nerve samples contained substantial GABA-T activities. (4) In the rd-retinas from 6 weeks old mice, an activity peak was detected in the outer side of inner plexiform layer and a relatively high activity was present in the inner side of inner nuclear layer. The activity of GABA-T in ganglion cell layer was 70% of control. Outer retinal layers were absent. The decrease of GABA-T activity seems to be an advantage in maintaining the concentration of GABA in the degenerated mouse retinas.

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To clarify the charactaristics of adriamycin (ADR) -induced cardiotoxicity, we studied rat hearts morphologically in 3 experimental models : adult hearts, cultured heart cells, and fetal hearts. (1) Adult rats were intravenously injected with 0.2-3.0mg /kg /week of ADR for 1 to 8 weeks. By light microscopy, vacuolization of myocytes in the inner layer of the left ventricle and myocytolysis and fibrosis in middle layer were observed. Ultrastructural changes of electron microscopy consisted of fragmentation of myofibrils, disorganization of mitochondria and dilation of the sarcotubular system. These findigs were similar to those of human dilated cardiomyopathy. (2) New born rat cardiac cells were cultured, and 2.5 μg/ml of ADR was added to the medium on the day 4 th of culture for 24 hrs. On day 6 of culture, the number of cardiac muscle cells in the ADR group was markedly decreased as compared with non-muscle cells. Ultrastructurally, nucleolar segregation, margination of chromatin and /or cytoplasmic vacuolization were observed in the ADR group. These observations suggest that the ADR has selective cytotoxicity on cultured cardiac muscle cells. (3) Pregnant rats were intraperitoneally injected with 1.0-2.0mg/kg/day of ADR for 3 or 4 days. On the 17th day of gestation, the viable fetal hearts were arrested at the maximum diastolic state and were observed under a stereomicroscope. Twenty-three percent of 79 fetuses in the ADR group had malformation of the heart and great vessels, included persistent truncus arteriosus and ventricular septal defect. These results demonstrated that the ADR acts as a teratogen in fetal hearts as well as damaging adult hearts or cultured heart cells.

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In vivo smoking was investigated from the aspect of its effect on serum complement. Items of the investigation were complement activities before and after acute smoking in young adults, the complements in smokers, ex-smokers, and non-smokers among elderly subjects and in vitro influence of tobacco tar on serum complement activity. After 10 minutes of acute smoking, a significant decresase was observed in ACH50 (p<0.01), but no change was noted in CH50, C 3, or C 4. ACH50 in smokers, ex-smokers and non-smokers among elderly subjects showed low values as compared with those in young adults. Thus, a trend was seen that the values were further lower in aged smokers (p<0.01). In C 3 a, there was a inclination of showing high values in aged subjects as compared with young adults. When comparing C 3 a, the elderly subjects, who smoke showed higher values than those of ex- and non-smokers. As the in vitro results, tobacco tar lowered both CH50 and ACH50 in the serum. However, with a low tar concentration, there was a trend that was observed. As compared with CH50, there was a decrease in ACH50. The above results suggest that smoking activates the alternative pathway. Thus, in chronic elderly smokers, the repetition of the influence reflects as a decrease in serum ACH50.

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The pathogenesis of thrombocytopenia in the patient with liver cirrhosis was investigated. The effects of the conditioned medium from phytohemagglutinin-P stimulated human peripheral mononuclear cells (PHA-LCM) was studied before and after splenectomy by in vitro colony forming units-megakaryocyte (CFU-meg) originated colony forming technique. The mononuclear cells separated from human bone marrow were cultured in 0.9% methylcellulose gel containting 30% fetal calf serum, 1% bovine serum albumin, 2U of erythropoietin, and 20% PHA-LCM at 37°C fully humidified atmosphere containing 5% CO² in the air for 10 days. CFU-Meg originated colonies were identified by means of Avidin-Biotin peroxidase complex staining method using human antifactor VIII associated with rabbit IgG. More than four cells were scored as a CFU-MEG originated colony under a fluorescent mictoscope. Then 20% PHA-LCM obtained from 10⁶ mononuclear cells was added to each previous culture system. As a result, CFU-Meg in patients with liver cirrhosis were not damaged. However, the effects of PHA-LCM on CFU-Meg originated colony formation were significantly decreased in preoperative patients, as well as, splenectomized patients. These results suggest that in the pathogenesis of thrombocytopenia in patients with liver cirrhosis, the reduced production of megakaryocyte colony stimulating factor from peripheral blood lymphocytes may be participating as an important role.

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In MRL /lpr mice bearing an autosomal recessive gene, lpr, generalized lymphoproliferative disorder is associated with autoimmune disease resembling systemic lupus erythematosus (SLE) and rheumatoid arthritis which occurs spontaneously. It may start at ages as early as 2-3 months. The proliferating cells belong to unique T cells with unusual cell surface phenotypes (lpr cells). In the present study, we investigated the age-associated changes in the blood leukocytes and bone marrow cells in MRL /lpr mice. Thus we found that granulocytosis, particularly the increase in the number of neutrophils, and monocytosis developed in these mice at 2 months of age, and progressed thereafter. The granulocytosis in the blood was associated with the increase in the number of neutrophils in the bone marrow. Plasma cell proliferation was also observed in the bone marrow of these mice, but only in later life (6 months). The investigation of proliferating lpr cells revealed that these cells appeared first in the lymph node and spleen at approximately 2 months of age when the onset of granulocytosis occurred, suggesting that these two abnormalities are related. However, although the lpr cells were distributed thereafter into the thymus, lung and blood. These cells were never found in the bone marrow throughout the life span of MRL /lpr mice. Taken together, a possibility exists that the observed granulocytosis can be attributed to certain humoral factors produced by the lpr cells. From this viewpoint, a granulocyte colony-stimulating factor, G-CSF, was injected intraperitoneally into normal BALB/c and MRL/n mice. Although granulocytosis appeared in such mice to the extent found in MRL/lpr mice, the monocytosis, plasmacytosis and hypergammaglobulinemia, which are also characteristic clinical features of MRL/lpr mice, did not develop. Whether the effect of a single factor or the combined effects of multiple factors are responsible for the myeloid disorders in MRL /lpr mice is discussed.

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