Apoptotic cell death during ischemia/reperfusion and its attenuation by antioxidant therapy

Apoptosis is a form of programmed cell death that can be induced in suscept ible cells by a wide variety of normal physiological stimuli as well as by deleterious environmental conditions and cytotoxic agents. The common induc ers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ whi ch are also implicated in the pathogenesis of myocardial ischemic reperfusi on injury. To examine how free radicals are directly involved in apoptosis, rats were divided into three groups. The first group of rat hearts were pe rfused for 15 min with KHB buffer, the second group of rat hearts were perf used with superoxide dismutase plus catalase, and the hearts were subjected to 30 min of ischemia and 120 min of reperfusion. The third group of rat h earts, served as control which were subjected to 165 min of perfusion with KHB buffer (where n = 6 rats in each group). At the end of each experiment, hearts were saved (at - 70 degrees C) and analysed for apoptosis, DNA ladd ening and MDA production. During the reperfusion continuous cardiac pressur e measurements were recorded in real time with a data acquisition and analy sis system (CORDAT II, Triton Technologies). Direct measurements of heart r ate, developed pressure and the first derivative of the developed pressure were recorded before the intervention and during the reperfusion. Coronary flow was measured by timed collection of coronary effluent. The results of our study revealed apoptotic cells after 120 min of reperfusion as demonstr ated by the intense fluorescence of the immunostained digoxigeninlabeled ge nomic DNA when observed under fluroscence microscopy. Evaluation of DNA fra gmentation showed increased ladders of DNA bands in the same reperfused hea rts representing integer multiples of the internucleosomal DNA, about 180 b p. The presence of apoptotic cells and DNA fragmentation in the myocardium were abolished by preperfusing the hearts in the presence of SOD and catala se, which also reduced the oxidative stress as evidenced by the MDA product ion. In concert, myocardial function was significantly better when compared with the ischemic control. Taken together, these results clearly demonstra te that oxidative stress developed in the ischemic repel fused myocardium i nduces apoptosis and free radical scavengers can play a crucial role in apo ptotic cell death associated with ischemia/reperfusion. (C) 2000 Elsevier S cience Ireland Ltd. All lights reserved.