Histochemical studies of myocardial injury caused by hydrogen peroxide after reperfusion using the cerium method

Free radicals have been implicated in myocardial reperfusion injury. Hydrogen peroxide (H 2 O 2 ) is a precursor of highly reactive oxygen intermediates. In this study, we investigated myocardial injury caused by endogenous H 2 O 2 during the early reperfusion period following brief ischemia with electron microscopy and the cerium method. This method involves formation of an electrondense precipitate when H 2 O 2 reacts with cerium chloride (CeCl 3 ). We used isolated, functioning hearts prepared according to the working heart model, which were reperfused with a solution containing 0.5mM CeCl 3 for 5 min after 10 min of ischemia. Some hearts were treated with 3-amino-1,2,4-triazole (ATZ) to inhibit catalase; others were treated with ATZ and superoxide dismutase (SOD), which dismutates the superoxide anion to hydrogen peroxide. In the control group (no drugs given) and the ATZ-treated group, the CeCl 3 −H 2 O 2 -dependent reaction products during the reperfusion period appeared in 12% and 28%, respectively, of the microvascular spaces. Treatment with SOD did not produce a decrease in electron-dense precipitates or a decrease in myocardial injury during ischemia-reperfusion. Moreover, in the ATZ group, moderately injured myocytes were seen (swelling of mitocondria, intermyofibrillar edema). Our results indicate that in myocytes, catalase plays an important role in the defense against H 2 O 2 and that the increase in H 2 O 2 is a cause of reperfusion injury. However, SOD does not protect against H 2 O 2 in the absence of catalase.