We found that both rapid and delayed remote postconditioning, which were conducted immediately or 3h after reperfusion in the ipsilateral hind limb, protected against focal cerebral ischemia in rats. It is plausible that remote postconditioning protects the ischemic brain through protein synthesis and afferent nerve activity, since inhibition of protein synthesis and nerve activity abolished its protection. However, the effect of rapid remote postconditioning on infarct size was lost at two months after stroke while it improved the neurological outcomes.
Currently, there are at least two types of ischemic postconditioning, conventional and remote postconditioning. The conventional postconditioning refers to a series of brief, repetitive mechanical occlusion/reperfusion of the occluded blood vessels after ischemia/reperfusion [31
]. We demonstrated in 2006 that conventional postconditioning, which was performed by 3 cycles of 30 sec occlusion/10 sec release of the bilateral CCA after reperfusion, robustly reduced infarct size in the same focal ischemia model used in this current study [31
]. This protective concept of ischemic postconditioning has been confirmed by a number of groups using in vivo global and focal ischemia models [6
], and in vitro ischemic models [19
In our current study, we further tested our hypothesis that remote postconditioning, which is conducted in the ipsilateral hind limb, protects against focal ischemia. This idea was derived from our most recent study showing that remote limb preconditioning reduces infarct size [23
]. In our previous study, remote preconditioning performed immediately, 12h and 48h before the onset of cerebral ischemia robustly reduced infarct size; remote preconditioning was conducted in the ipsilateral hind limb by 3 cycles of 15 min occlusion/15min release of the femoral artery [23
]. In addition, other previous studies have shown that remote postconditioning reduces heart injury after ischemia [1
]. We therefore speculated that limb ischemia could be extrapolated to remote postconditioning. Indeed, as expected, we have demonstrated that remote postconditioning conducted immediately after reperfusion markedly reduced infarct size. To our surprise, the therapeutic time windows of remote postconditioning can be as late as 3h after reperfusion, which provides a wide time window for clinical translation.
To further examine the reproducibility of the protective effect of rapid remote postconditioning, another individual in our laboratory independently repeated the experiment, and the result shows significant protection at 2d after stroke. Although it seems less protective in the latter experiment (24% reduction of infarction) compared with that in the former experiment (67% reduction in ), this difference may be caused by the experimental variability from different operators. Although in both experiments the CCA occlusion time was the same (30 min), the actual ischemic severity can be different due to the exact occlusion sites of the MCA; the more proximal the occlusion site, the worse the injury. The infarct size of the control ischemia is 58.4% for the confirming experiment, which is larger than the infarction of 45.8% in the former experiment (). Therefore, the protective effect of remote postconditioning may be dependent on ischemic severity.
The underlying protective mechanisms of remote postconditioning are unknown. Nevertheless, research from remote preconditioning against ischemia in other organs may shed light on our understanding of the protective mechanisms of remote postconditioning. In the heart, accumulating evidence suggests that neural pathways serve as a connection between the remote preconditioned organ and the heart. Wolfrum et al. reported that remote preconditioning with brief mesenteric artery occlusion/reperfusion reduced heart infarction by activating εPKC in rats [29
]. However, pretreatment with the ganglion blocker, hexamethonium, inhibited εPKC activation thus blocking remote preconditioning's protection [29
]. In addition, Schoemaker and colleagues showed that bradykinin released during remote preconditioning stimulates sensory nerves and offers protection, which is blocked by the ganglion blocker, hexamethonium [25
]. Moreover, inhibition of afferent nerves with capsaicin also abolishes remote preconditioning's protection against gastric ischemia, in which remote preconditioning was conducted in the heart or liver by two-5 min ischemic occlusions of the coronal or hepatic arteries [4
]. Accordingly, we have found that both hexamethonium and capsaicin abolished the protective effect of remote preconditioning against stroke (unpublished results). As expected, we currently demonstrated that capsaicin treatment reversed remote postconditioning's protection, suggesting that the afferent nerve pathways may sever a connection between the remote organ, limb, and the ischemic brain.
Moreover, we demonstrated that the protein synthesis inhibitor, cycloheximide, also robustly attenuated the protective effect of remote postconditioning. Cycloheximide is usually used to test the hypothesis that preconditioning protects against ischemic injury via protein synthesis [2
]. In those studies, preconditioning was carried out a few hours to days before ischemia onset [2
]. Thus, preconditioning may stimulate the organ to adapt to a future ischemic event, including protein synthesis. It is thus not surprising that a protein synthesis inhibitor blocks the protective effect of preconditioning. Nevertheless, remote postconditioning is performed immediately after stroke onset; it seems that there is no time for the brain to synthesize the new protein for neuroprotection. Therefore, the result that protein synthesis inhibition abolished remote postconditioning's protection is beyond of our expectation. Our result implies that protein synthesis may play critical roles in brain recovery after remote postconditioning.
Remote postconditioning no longer reduced infarct size at 2 months after stroke, but still improved neurological functions, suggesting its transient protection on infarct size with long-term protection on neurological function. Therefore, infarct size alone does not reflect the protective effect of a neuroprotectant. Such transient protection for infarct size is not new for neuroprotectants. A previous report has demonstrated that post-ischemic hypothermia (30 °C) attenuated hippocampal CA1 neuronal loss measured at 3 days but not at 2 months after transient global ischemia [5
]. Similar transient protection was reported for the protective effect of rapid ischemic preconditioning [18
]. Nevertheless, future studies need to address why remote postconditioning did not reduce infarction but still improved neurological functions at 2 months after stroke.
In conclusion, limb remote postconditioning reduced infarct size of focal ischemia in rats in a prolonged time window as late as 3h after reperfusion. It appears that remote postconditioning protects against ischemia via the nerve pathway and via modulating protein synthesis.