Co-Investigator(Kenkyū-buntansha) |
HAMAMATSU Hisayoshi KEIO UNIVERSITY SCHOOL OF MEDICINE, INSTRUCTOR, 医学部, 助手 (00208597)
SUEMATSU Makoto KEIO UNIVERSITY SCHOOL OF MEDICINE, INSTRUCTOR, 医学部, 助手 (00206385)
KATO Shinzo KEIO UNIVERSITY SCHOOL OF MEDICINE, INSTRUCTOR, 医学部, 助手 (30177448)
柳沢 徹 慶應義塾大学, 医学部・消化器内科, 助手 (80191154)
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Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1990: ¥6,000,000 (Direct Cost: ¥6,000,000)
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Research Abstract |
Oxidative stress in the perfused rat liver was attempted to be analyzed by digital microfluorography using a fluorescent probe for hydroperoxide, dichlorofluorescin (DCFH). DCFH is known to be oxidized by hydorperoxide to dichlorofluorescein (DCF). Hepatic damage was also assessed by propidium iodide (PI), which is known to label the nuclei of non viable cells. Fluorescence of these probes was observed by fluorescence microscope equipped with silicon intensified target camera, and digitally processed by a image processor (ARGUS-200, Hamamatsu photonicus, Japan). Carbon tetrachloride induced oxidative stress was demonstrated in zone 3 by DCF fluorescence, followed by cell damage assessed by PI. These changes were prevented by SKF525A, an inhibitor of cytochrome P450, or promethazine, a radical scavanger. Low flow hypoxia increase DCF fluorescence in zone 2 first, and spread to zone 3. PI fluorescence was observed in zone 3 after increase in DCF fluorescence, suggesting midzonal oxidative stress preceed cell death. These changes were prevented by addition of allopurinol, a xanthine oxidase inhibitor, or PGE1. Rhodamine 123(Rh123), an indicator of mitochondrial energization, was used to assess lipololysaccharide induced liver damage. Addition of LPS to perfusate decreased Rh123 fluorescence, and the change was blocked by LNMA, and in hibitor of nitric oxide synthetase. Mitochondrial dysfunction in hepatocytes induced by LPS was observed only in the presence of Kupffer cell, suggesting that nitric oxide released from activated Kupffer cell mediates hepatic mitochondiral dysfunction in LPS induced liver damage.
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