Studies demonstrating that free radical scavengers prevent postischemic injury have provided evidence for a free radical mechanism of reperfusion injury. Using Electron Paramagnetic Resonance (EPR) techniques we have been able to demonstrate a burst of oxygen free radical generation during the early minutes of postischemic reperfusion. Endothelial cells have been shown to be important sources of this radical generation and leukocytes, in the presence of complement factors, have been shown to further amplify radical generation and injury. The nitric oxide radical, NO, is a potent vasodilator and it has been suggested that oxygen radicals decrease postischemic vascular reactivity by decreasing NO. EPR techniques have been developed enabling direct measurement and quantitation of NO in the heart and coronary effluent. This project has the following four specific aims. 1) To determine the cellular mechanisms of free radical generation in reoxygenated endothelial cells and leukocytes. EPR spin trapping techniques will be used to quantitate and characterize free radical generation, and trypan blue exclusion along with electron microscopy to assess cell injury. 2) To directly measure NO production in th normal and postischemic heart and determine the role of oxygen free radical generation in the pathogenesis of endothelial dysfunction. EPR measurements of NO- myoglobin formation in myocardial tissue will be performed. EPR and spectrophotometric measurements of NO in the coronary effluent will be performed via methemoglobin production or direct trapping by the Fe(II)- DETC complex, and correlated with measurements of vascular reactivity and contractile function. 3) To determine the effect of oxygen radicals, and NO on PMN and endothelial adhesion molecules. Fluorescence flow cytometry and functional adhesion assays will be performed in isolated cells and hearts to determine the effects of oxygen free radicals and NO on adhesion molecule expression. EPR techniques will be used to measure radical generation and immunohistochemistry to localize the observed effects in myocardial tissue. 4) To determine if free radicals directly or indirectly activate the complement pathway in the postischemic heart. Complement activation will be determined by measuring consumption of functionally active C3 and C5, radioimmunoassay for the generation of C3a and C5a, as well as by immunohistochemical studies of complement attack complex deposition. EPR techniques will be used to measure exogenous or endogenous free radical generation. Overall this project is designed to determine the basic cellular and molecular mechanisms of free radical mediated reperfusion injury as well as to provide insight regarding optimal strategies for preventing this injury.
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