Cerium chloride as a histochemical marker of hydrogen peroxide in reperfused ischemic hearts

Abstract Hydrogen peroxide (H 2 O 2 ) has been implicated in cardiac damage due to ischemia and reperfusion. We adapted an electron microscopic, histochemical method for demonstrating H 2 O 2 produced by isolated cells to isolated, buffer-perfused rabbit hearts. The method involves formation of an electron-dense precipitate when H 2 O 2 reacts with cerium chloride (CeCl 3 ). We perfused hearts retrograde via the aorta with well-oxygenated bicarbonate-buffered solution, followed by one in which bicarbonate was replaced with imidazole (IPSS) to prevent precipitation of bicarbonate and CeCl 3 . Some hearts were made globally ischemic (30 min, 37°C), reperfused 5 min with well-oxygenated IPSS containing 1 m m CeCl 3 , then processed for electron microscopy. Others were perfused with IPSS containing catalase (300 U/ml) or albumin before ischemia and upon reperfusion, followed by CeCl 3 administration. Nonischemic control hearts perfused with IPSS (± catalase) were also studied. Electron micrographs were assessed visually and by computer for precipitate localization and amount. There was abundant precipitate on the luminal face of the coronary vascular endothelium in ischemic-reperfused, cerium-treated hearts, including those treated with albumin. There was significantly less in reperfused catalase-treated or nonischemic control hearts. X-ray microbeam analysis of the endothelial precipitate indicated the presence of Ce. This appears to be the first visual demonstration of a CeCl 3 -H 2 O 2 -dependent reaction product in intact isolated ischemic hearts. The data indicate that at the time of reperfusion some H 2 O 2 is accessible to the vascular space, and that its amount can be reduced by perfused catalase. Further modifications this technique may be useful for assessing the sites and pathways by which H 2 O 2 is generated by hearts or other buffer-perfused organs subjected to stresses such as ischemia or hypoxia.