Limb remote-preconditioning protects against focal ischemia in rats and contradicts the dogma of therapeutic time windows for preconditioning

Abstract

Remote ischemic preconditioning is an emerging concept for stroke treatment, but its protection against focal stroke has not been established. We tested whether remote preconditioning, performed in the ipsilateral hind limb, protects against focal stroke and explored its protective parameters. Stroke was generated by a permanent occlusion of the left distal middle cerebral artery (MCA) combined with a 30 min occlusion of the bilateral common carotid arteries (CCA) in male rats. Limb preconditioning was generated by 5 or 15 min occlusion followed with the same period of reperfusion of the left hind femoral artery, and repeated for two or three cycles. Infarct was measured 2 days later. The results showed that rapid preconditioning with three cycles of 15 min performed immediately before stroke reduced infarct size from 47.7+/-7.6% of control ischemia to 9.8+/-8.6%; at two cycles of 15 min, infarct was reduced to 24.7+/-7.3%; at two cycles of 5 min, infarct was not reduced. Delayed preconditioning with three cycles of 15 min conducted 2 days before stroke also reduced infarct to 23.0+/-10.9%, but with two cycles of 15 min it offered no protection. The protective effects at these two therapeutic time windows of remote preconditioning are consistent with those of conventional preconditioning, in which the preconditioning ischemia is induced in the brain itself. Unexpectedly, intermediate preconditioning with three cycles of 15 min performed 12 h before stroke also reduced infarct to 24.7+/-4.7%, which contradicts the current dogma for therapeutic time windows for the conventional preconditioning that has no protection at this time point. In conclusion, remote preconditioning performed in one limb protected against ischemic damage after focal cerebral ischemia.

Fig. 1

Protocols for surgery. A. Control ischemia was induced by 30 minute occlusion of the bilateral CCA combined with permanent occlusion of the left MCA. Rats were killed 2 days later for infarct size measurement. B. Rapid limb preconditioning. Rapid preconditioning was conducted in the left limb by 2 cycles of 5 minute occlusion/reperfusion, or 2 or 3 cycles of 15 minute occlusion/reperfusion. Brain ischemia was induced immediately after preconditioning. Rats in the isoflurane-control group were subjected to sham surgery by exposing the femoral artery without occlusion, but with 90 minutes of isoflurane. C. Intermediate limb preconditioning. Limb preconditioning of 3 cycles of 15 minutes was induced 12 hours before brain ischemia. A corresponding isoflurane group was used as a control. D. Delayed limb preconditioning. Preconditioning with 2 or 3 cycles of 15 minutes, and isoflurane control were conducted 2 days before brain ischemia.

Fig. 2

Rapid limb-preconditioning reduced infarct size after stroke. Limb ischemia was induced immediately before brain ischemia. Rats were killed 2 days after stroke and rat brains were chopped into 5 slices. Infarcts were stained by TTC, and infarct cortex of each slice was measured and normalized to non-ischemic cortex as a percentage. Representive infarcts stained by TTC from each group are shown (A); arrows indicate islands of injury in the spared tissues in the spared tissues of ischemic brains treated with remote preconditioning. Note that isoflurane alone and preconditioning with 2 cycles of 5min did not reduce infarction compared with ischemic control. Bar graphs show the average infarct size of the 5 slices. Con-isc, control ischemia; Iso-isc, isoflurane of 90 minutes before ischemia onset; 2c-5 min, 2 cycles of 5 minutes occlusion/reperfusion of the femoral artery; 2c-15 min, 2 cycles of 15 minutes; 3c-15 min, 3 cycles of 15 minutes. * vs each of other groups, P<0.001.

Fig. 3

Delayed limb-preconditioning reduced infarct size. Limb preconditioning was induced 48 hours before brain ischemia. Representative infarcts stained by TTC are shown (A); arrows indicate islands of injury. For statistical results, infarct size of the control ischemia in Fig. 2 was represented in this figure for comparison with the control group of isoflurane. 48h iso-isc, 90 minutes of isoflurane was induced 48 hours before brain ischemia; 2c-15 min, 2 cycles of 15 minutes occlusion/reperfusion of the femoral artery; 3c-15 min, 3 cycles of 15 minutes. *, **, *** vs 2c-15 min (p=0.011), 48 h iso-isc (p=0.006), con isc (p<0.001), respectively.

Fig. 4

Intermediate limb-preconditioning also protects against ischemic injury. To test whether limb preconditioning performed 12 hours before brain ischemia has protection, the strongest condition, 3 cycles of 15 minutes, was used. Representative TTC stained-infarcts are shown (A); similarly, islands of injury (arrow) in the spared tissues were also observed in ischemic brains treated with preconditioning. In the bar graph (B), infarct size of control ischemia was presented again for comparison. 12 h-iso-isc, 90 minutes of isoflurane was induced 12 hours before brain ischemia; 3c-15 min, 3 cycles of 15 minutes occlusion/reperfusion of the femoral artery. * vs each of the other groups, P<0.001.