This paper describes a histochemical study of superoxide generation in buffer-perfused, isolated rat hearts during the first 2 minutes of reperfusion after 60 minutes of warm ischemia. Superoxide radical production was demonstrated by a modification of Karnovsky's manganese/diaminobenzidine technique, in which superoxide oxidizes Mn++ to Mn ions, which in turn oxidize diaminobenzidine to form amber, osmiophilic polymers, observable by light or electron microscopy. Isolated hearts were rendered ischemic, reperfused with oxygen equilibrated buffer containing Mn++ and diaminobenzidine, fixed by perfusion with Trump's solution, and processed for light and electron microscopy. The method consistently demonstrated evidence of superoxide generation near the luminal surfaces of arterial, capillary, and venular endothelial cells during the first 2 minutes of reoxygenation after ischemia. The histochemical reaction was absent or markedly reduced in non-manganese-treated or nonischemic hearts, as well as in hearts perfused with calcium-free or oxygen-free buffers. The histochemical differences were statistically significant on quantitative morphometric analysis. These results provide direct, visual evidence of the existence and endothelial localization of a burst of superoxide radicals in intact, postischemic myocardium and suggest the pathophysiologic importance of calcium-dependent endothelial cell activation in the initiation of reperfusion injury.
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