Intravital Analysis of Oxidative Stress During Organ Circulatory Failure

本项目的目的是建立一种方法学来研究微循环单位中氧自由基形成的微地形。数字成像光学显微镜的最新发展使得分析组织损伤过程中氧自由基产生的时空变化成为可能。鲁米诺依赖的光子成像显示，在血小板激活因子或内毒素诱导的微血管损伤过程中，粒细胞介导的氧化应激。我们的数据还表明，小静脉内皮细胞和黏附的粒细胞之间的界面可能是氧化剂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，使用数字成像显微荧光照相术和氢过氧化氢敏感的荧光染料二氯荧光素二乙酸酯来显示隔离灌流的大鼠肝脏细胞内的氧化应激。在四氯化碳诱导的肝损伤的实验模型中，该方法揭示了依赖细胞色素P450的氧化剂的形成主要发生在中心周围区域，在同一区域的细胞死亡之前。目前的技术将为研究器官微循环氧化应激与异物代谢或循环衰竭相关的微地形图提供有用的方法。

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.