Intravital Analysis of Oxidative Stress During Organ Circulatory Failure

本项目旨在建立研究微循环装置中氧自由基形成微形貌的方法。近年来，数字成像光子显微镜的发展使得分析组织损伤过程中氧自由基生成的时空变化成为可能。鲁米诺依赖光子成像显示血小板活化因子或内毒素诱导微血管损伤时粒细胞介导的氧化应激。我们的数据还表明，静脉内皮和粘附的粒细胞之间的界面可能是氧化依赖性细胞损伤的最关键部位。我们开发了另一种新的方法，利用数字成像显微荧光技术和氢过氧化物敏感的荧光染料，二氯荧光素二醋酸酯，来观察离体灌注大鼠肝脏细胞内氧化应激。在四氯化碳诱导肝损伤的实验模型中，该方法揭示了细胞色素p450依赖性氧化剂的形成主要发生在中心周围区域，在同一区域发生细胞死亡。目前的技术将为研究与外源性代谢或循环衰竭相关的器官微循环氧化应激的显微形貌提供有用的方法。

This project was aimed to establish the methodology to investigate microtopography of oxygen radical formation in microcirculatory units. Recent development of digital imaging photonic microscopy has made it possible to analyze spatial and temporal alterations of oxyradical generation during tissue injury. Luminol-dependent photonic imaging revealed granulocyte-mediated oxidative stress during microvascular damage induced by platelet-activating factor or endotoxin. Our data also demonstrated that the interface between venular indothelium and sticking granulocytes may be the most critical site of oxidant-dependent cellular damage. We have developed another new method to visualize intracellular oxidative stress in the isolated perfused rat liver using digital imaging microfluorography and a hydroperoxide-sensitive fluorescence dye, dichlorofluorescin diacetate. In the experimental model of carbon tetrachloride-induced hepatic injury, the method has revealed that cytochrome P450-dependent oxidant formation takes place predominantly in pericentral regions, preceding cell death at the same area. The current technique will provide a useful method to investigate the microtopograp hy of organ microcirculatory oxidative stress associated with xenobiotic metabolism or circulatory failure.