Endothelial superoxide production in the isolated rat heart during early reperfusion after ischemia: A histochemical study

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 super-oxide 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.