The involvement of cytokines in the second window of ischaemic preconditioning

Abstract

We utilized a rat model of myocardial infarction to investigate whether manganese superoxide dismutase (Mn-SOD), an intrinsic radical scavenger, and tumour necrosis factor- alpha (TNF-alpha) and/or interleukin-1beta (IL-1beta) are involved in the late phase of ischaemic preconditioning (IP). IP was induced in anaesthetized rats by four 3-min left coronary artery (LCA) occlusions, each separated by 10 min of reperfusion. Twenty-four hours after the repetitive brief ischaemia, the LCA was occluded for 20 min followed by reperfusion for 48 h. IP reduced the infarct size by approximately 46% as determined after 48 h of reperfusion. Antisense oligodeoxynucleotides to Mn-SOD inhibited the increases in Mn-SOD content and activity, and abolished the expected decrease 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-alpha (0.5 ml) and IL-1beta (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-alpha and IL-1beta after IP, plays an important role in the acquisition of late-phase cardioprotection against ischaemia/reperfusion injury in rats.

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-α (0.1–1 ml) and/or IL-1β (0.1–1 mg) antibodies were injected i.p. 20 min before the brief repetitive ischaemia. Rats in the control group received saline or TNF-α antibody (0.5 ml) and IL-1β antibody (0.5 mg) 24 h before sustained ischaemia.

Figure 2

Effects of oligodeoxynucleotides (ODN) on the size of myocardial infarct in rats. Infarct size was calculated as the ratio of the mass of the infarct region to that of the ischaemic region in hearts from rats exposed to treatments as described in Figure 1. Rats in the control ‘C', sham-operated, and preconditioning groups received saline i.p. 24 h before sustained ischaemia. ‘Sham' indicates sham-operated control group, which was instrumented with a suture around the left coronary artery in the same manner as ischaemic preconditioning group with 24 h recovery; ‘IP' indicates rats from ischaemic preconditioning group with 24 h recovery; ‘IP+Sense ODN' indicates 24 h-recovery rats receiving sense ODN just after ischaemic preconditioning treatment; ‘IP+Scrambled ODN' indicates 24 h-recovery rats receiving scrambled ODN just after ischaemic preconditioning treatment; ‘IP+Antisense ODN' indicates 24 h-recovery rats receiving antisense ODN just after ischaemic preconditioning treatment. The number of rats used was indicated in each column. The infarct size in the sham-operated control rats was not significantly different from that in the control rats. Data are expressed as the mean±s.e.mean. *Indicates a value of P<0.05 versus sham-operated control rats and #indicates n.s. versus sham-operated rats by ANOVA with the Bonferroni's post hoc test for multiple comparisons.

Figure 3

Effects of oligodeoxynucleotides (ODN) on manganese superoxide dismutase (Mn-SOD) activity and content in rat myocardium. Activity of Mn-SOD was determined by the nitroblue tetrazolium method and the content was determined by ELISA in cardiac tissue homogenates from rats 24 h after preconditioning or sham-operated as described in Figure 1. Groups are defined as in the legend to Figure 2. Data are expressed as the mean±s.e.mean of values from at least six rats. *Indicates a value of P<0.05 versus sham-operated control rats and #indicates n.s. versus sham-operated rats by ANOVA with the Bonferroni's post hoc test for multiple comparisons.

Figure 4

Effects of TNF-α and/or IL-1β antibodies on infarct size in rats. Infarct size was calculated as the ratio of the mass of the infarct region to that of the ischaemic region following reperfusion in rats exposed to treatments as described in Figure 1. Rats in the control group ‘C' received TNF-α (0.5 ml) and IL-1β (0.5 mg) antibodies i.p. 24 h before coronary occlusion. Rats in the sham-operated group received TNF-α (0.5 ml) and IL-1β (0.5 mg) antibodies i.p. prior to the sham-operation. ‘Sham' indicates sham-operated group, which was instrumented with a suture around the left coronary artery in the same manner as ischaemic preconditioning group with 24 h recovery; ‘IP' indicates rats from ischaemic preconditioning group; ‘IP+TNF-αAb' indicates rats receiving TNF-α antibody (0.5 ml) prior to ischaemic preconditioning treatment; ‘IP+IL-1βAb' indicates rats receiving IL-1β antibody (0.5 mg) prior to ischaemic preconditioning treatment; ‘IP+TNF-αAb+IL-1βAb' indicates rats receiving TNF-α(0.1, 0.5, 1 ml) and IL-1β (0.1, 0.5, 1 mg) antibodies prior to ischaemic preconditioning treatment. The number of rats used was indicated in each column. Data are expressed as the mean±s.e.mean. *Indicates a value of P<0.05 versus sham-operated rats and #indicates n.s. versus sham-operated rats by ANOVA with the Bonferroni's post hoc test for multiple comparisons.

Figure 5

Effects of TNF-α and/or IL-1β antibodies on manganese superoxide dismutase (Mn-SOD) activity in rat myocardium. Activity of Mn-SOD was determined by the nitroblue tetrazolium method in cardiac tissue homogenates from rats 24 h after preconditioning or sham-operated as described in Figure 1. Groups are defined as in the legend to Figure 4. ‘IP+TNF-αAb+IL-1βAb' indicates rats receiving TNF-α (0.5 ml) and IL-1β (0.5 mg) antibodies prior to ischaemic preconditioning treatment. Data are expressed as the mean±s.e.mean of values from at least five rats. *Indicates a value of P<0.05 versus sham-operated rats and #indicates n.s. versus sham-operated rats by ANOVA with the Bonferroni's post hoc test for multiple comparisons.