Analysis of active oxygen metabolism in organ microcirculation using confocal laser microscopy

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We have established the experimental set up for intravital observation using confocal laser scanning imaging system. Our experiments were performed using fluorescence probes ; dichlorofluorescin diacetate (a fluorochrome sensitive to intracellular hydroperoxide formation), rhodamine 123 (a fluorochrome sensitive to the mitochondrial membrane potential, ) and propidium iodide (which labels the unclei of non viable cells).By using above mentioned set up ;1. It has been observed that leukocyte adhesion along postcapillary venules, the oxidative stress and the mitochondrial dysfunction occur in rat mesenteric microcirculation after ischemiareperfusion.2. Intralobular heterogeneity of the oxidative stress and the mitochondrial dysfunction were visualized in relation to their topographic distributions with the cell death during low-flow hypoxia in perfused liver. The oxidative stress and the resultant cell death during low-fow hypoxia are spatially restricted in the intermediate zone between … More the periportal and pericentral regions.3. Cold ischemia followed by reperfusion induced leukocyte adhesion to the sinusoidal wall and the damage of parenchymal cells. Superoxide dismutase attenuated these ischemiareperfusion-induced injuries.4. The metabolic changes in tumor cells after coculturing with Kupffer cells were visualized and analyzed. In cocultured hepatoma cells and colon cancer cells, Kupffer cells induced the oxidative stress and the mitochondrial dysfunction followed by cell death. Superoxide dismutase did not inhibit the mitochondrial dysfunction and cell death. L-NMMA,an inhibitor of nitric oxide production eliminated decrease in mitochondrial energization and the tumor cell injury induced by Kupffer cells. Kupffer cell-derived nitric oxide may present tumor cell cytotoxicity via inhibiting mitochondrial dysfunction.The metabolic changes in tumor cells were also determined ex vivo perfused liver. The oxidative stress and the mitochondrial dysfunction in tumor cells, which adhered to the liver sinusoid, were also obvious as well as those in vitro experiments. Less