prior to the sham-operation. in myocardial infarct size. Sense or scrambled oligodeoxynucleotides did not abolish either Mn-SOD induction or tolerance to ischaemia/reperfusion. The simultaneous administration of the antibodies to TNF- (0.5?ml) and IL-1 (0.5?mg) prior to IP abolished the cardioprotection and the increase in Mn-SOD activity induced by IP. We conclude that the induction and activation of Mn-SOD, mediated by TNF- and IL-1 after IP, plays an important role in the acquisition of late-phase cardioprotection against ischaemia/reperfusion injury in rats. synthesis of proteins such as heat shock proteins (HSPs) (Marber synthesis of Mn-SOD 24?h later with marked protection against prolonged hypoxic insult (Yamashita has not been presented yet. Cardiac resistance to ischaemia/reperfusion injury is increased by exposure to such sublethal stress as a brief period of ischaemia, exercise, and whole-body hyperthermia in a biphasic manner (Kuzuya CD40LG a right parasternal sternomectomy. Silk thread (7-0 type) was passed around the LCA about 3C4?mm distal to the LCA origin, and an occlusive snare was placed around it. The animals were subjected to one of six different protocols with repetitive brief ischaemia, a sham operation or control (Figure 1). Preconditioned rats received four 3-min LCA occlusions, each separated by 10?min RAF709 of reperfusion. Sham operated rats were instrumented with a suture around the LCA in the same manner as the preconditioned rats. The chest wound was closed, and air was evacuated from the chest (on day 1). At 24?h after the final 10-min reperfusion, rats in these groups were again anaesthetized, RAF709 and the chest was reopened (on day 2). The right femoral artery was cannulated using polyethylene tubes for the continuous measurement of arterial blood pressure with a pressure transducer (TP-300T; Nihon Kohden, Tokyo, Japan). The heart rate, the incidence of arrhythmias, and ST-segment changes were monitored. Haemodynamic variables were also continuously recorded (model WT-645G recorder; Nihon Kohden, Tokyo, Japan). The arterial pressure was measured with a transducer the femoral artery cannula, and the LCA was ligated. After 20?min of coronary occlusion, the snare was released; reperfusion was indicated by a change in the colour of the ventricular surface. The surgical wounds were repaired 60?min after reperfusion, and the rats were returned to their cages to recover. Aseptic surgical techniques were used throughout. Benzylpenicillin (30,000?u?kg?1) RAF709 was injected intramuscularly as prophylaxis against infection (on day 1 and on day 2). Open in a separate window Figure 1 Experimental protocol. Preconditioned rats received four 3-min left coronary artery (LCA) occlusions, each separated by 10?min of reperfusion. At 24?h after the final 10-min reperfusion, the LCA was occluded for 20?min and followed by 48?h reperfusion. The animals were sacrificed 48?h after the restoration of cardiac perfusion. Antisense, sense, or scrambled oligodeoxynucleotides (ODN) was administered i.p. just after ischaemic preconditioning. TNF- (0.1C1?ml) and/or IL-1 (0.1C1?mg) antibodies were injected i.p. 20?min before the brief repetitive ischaemia. Rats in the control group received saline or TNF- antibody (0.5?ml) and IL-1 antibody (0.5?mg) 24?h before sustained ischaemia. Arrhythmias were monitored by ECG. Ventricular fibrillation (VF) was defined according to the criteria of the Lambeth Conventions (Walker the right femoral vein to estimate the area perfused by the occluded artery (ischaemic region). The left ventricle was then cut into six pieces perpendicular to the apex-base axis. These specimens were incubated with 1% triphenyltetrazolium chloride at 37C to stain the non-infarcted region. The ischaemic, infarcted, and non-ischaemic areas of tissue were separated with scissors and weighted. The area at risk and the infarct size were defined as the ratios of the mass of the ischaemic region to the left ventricular mass and the mass of the infarct region to that of the ischaemic region, respectively, and were expressed as percentages. Myocardial tissue sampling To obtain tissue samples for the measurement of Mn-SOD content and activity, rats were killed by an overdose of sodium pentobarbitone 24?h after preconditioning or sham-operation as described in Figure 1 (on day 2). The RAF709 myocardial tissue was rinsed in phosphate-buffered saline (PBS), and then blood in left and right coronary arteries was washed out with an adequate volume of PBS from ascending aorta retrogradely. Evans blue dye (2%) was introduced after reocclusion of LCA to estimate the area perfused by the occluded artery (LCA region), and the myocardium in the LCA region was cut out with scissors (Yamashita delivery of systemically injected oligodeoxyribonucleotides, we evaluated the time-course of their accumulation in the heart. In experiments with 5-FITC labeled ASODN to Mn-SOD, we found that significant labelling of these tissues occurred at these times following the intraperitoneal injection; in endothelial cells at 2C4?h, in vascular smooth muscle at 4?h, and in cardiac myocytes at 8?h (Yamashita test for multiple comparisons. A level of test for multiple comparisons. Open in a separate window Figure 3 Effects of oligodeoxynucleotides (ODN) on manganese superoxide dismutase (Mn-SOD) activity.