In Vivo Visualization of Reactive Oxidants and Leukocyte-Endothelial Adherence Following Hemorrhagic Shock

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Abstract
The generation of oxygen radicals during leukocyte-endothelial cell interaction is considered to represent one of the fundamental steps of microvascular injury following ischemia and reperfusion. Indirect evidence also suggests that this relationship may be important following hemorrhagic shock. The purpose of this study was to characterize the temporal changes of reactive oxygen species (ROS) in the mesenteric microvascular endothelium, in vivo, as a consequence of hemorrhagic shock and reperfusion, and to correlate this ROS production to leukocyte adherence. Following a control period, blood was withdrawn to reduce the mean arterial pressure to 40 mmHg for 1 h in urethane-anesthetized rats. Mesenteric venules in a transilluminated segment of small intestine were examined to quantitate changes in ROS generation and leukocyte adherence. Sprague-Dawley rats were injected with dihydrorhodamine 123, a hydroperoxide-sensitive fluorescent probe that is trapped within viable cells as a nonfluorescent form and then converted to the mitochondrion-selective form rhodamine 123 by hydroperoxides. The fluorescent light emission from rhodamine 123 was recorded with digital microscopy and downloaded to a computerized image analysis program. Our results demonstrated an 80% increase in ROS generation beginning within 5 min into resuscitation and a 10-fold increase in leukocyte adherence that occurred at 10 min after resuscitation. Both ROS generation and leukocyte adherence were attenuated with pre-shock administration of platelet activating factor (PAF) antagonist, WEB 2086, and the CD11/CD18a antibody, anti-LFA-1β. Our findings suggest that ROS production in endothelial cells is increased during reperfusion following hemorrhagic shock and that the mechanism of expression is mediated in part by both PAF expression and subsequent leukocyte adherence.