Oxidative stress developed during open heart surgery induces apoptosis: Reduction of apoptotic cell death by ebselen, a glutathione peroxidase mimic

Apoptosis, a genetically controlled programmed cell death, has been found t o play a role in ischemic reperfusion injury in several animal species incl uding rats and rabbits. To examine whether this also is true for other anim als, a surgically relevant model was established using an isolated in situ swine heart. Hearts were subjected to 15 min of normothermic regional ische mia by left anterior descending artery (LAD) occlusion followed by 30 min o f normothermic cardioplegic arrest and 3 h of reperfusion. Oxygen free radi cals have been shown to be the inducers of apoptosis and because reperfusio n of ischemic myocardium is associated with the generation of free radicals , an additional group of hearts was preperfused with three different doses (5, 10, and 25 nM) ebselen, a glutathione peroxidase mimic, for 15 min befo re 15 min of LAD occlusion. Hearts were then subjected to 30 min of normoth ermic cardioplegic arrest followed by 3 h of reperfusion at normothermia. C ontrol experiments were performed by perfusing the hearts for 4 h at normot hermia. Two other groups of hearts were subjected to either 30 or 60 min of LAD occlusion followed by 30 min of cardioplegic arrest without subjecting them to reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis and DNA laddering. The in situ end-labeling (ISEL) technique was used to detect apoptotic cardiomyocyte nuclei while D NA laddering was evaluated by subjecting the DNA obtained from the cardiomy ocytes to 1.8% agarose gel electrophoresis followed by photographing under UV illumination. The apoptotic cells appeared only after 90 min of reperfus ion, as demonstrated by the intense fluorescence of the immunostained genom ic DNA when observed under fluorescence microscopy. None of the ischemic he arts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation showing increased ladders of DNA bands i n the same reperfused hearts. The presence of apoptotic cells and DNA fragm entation in the myocardium was abolished by preperfusing the hearts in the presence of 10 nM ebselen, which also moderated the oxidative stress develo ped in the heart. Apoptotic cells and DNA ladders were completely absent in the hearts subjected to either 30 or 60 min of LAD occlusion. The results demonstrate that reperfusion of the ischemic heart induces apoptosis, which can be reduced with ebselen by reducing the oxidative stress associated wi th ischemia/reperfusion.