| Objective:The purpose of this study was to construct animal model of moderate hypothermic low flow (MHLF) in adult SD rats and to detect the phenotype of the model, also to investigate the effects of ischemic preconditioning (IP) on apoptosis of brain injury and the mitochondrial apoptosis pathway, furthermore, to explore the mechanism on cerebral protection of upstream PI3K/AKT signaling pathway following the MHLF procedure.Methods:A total of180adult male SD rats (8-10weeks old, weight:76-85g) were randomly divided into three groups:the surgery group, the sham-surgery group and the ischemic preconditioning (IP)-treated surgery group, then60rats in each group. The surgery group was subjected to body temperature reduction and bilateral common carotid artery (CCA) occlusion for120min at25±0.5℃, followed by artery reopening and rewarming. The sham-surgery group underwent same procedure, but CCAs were not occluded. The ischemic preconditioning (IP)-treated surgery group was pre-treated with four cycles of2min occlusion of bilateral CCAs and5min reperfusion, before120min of cerebral ischemia and reopen occlusion of bilateral CCAs. Another30adult male SD rats (8-10weeks old, weigh:76-85g) were randomly divided into three groups:the surgery group, the sham-surgery group and the ischemic preconditioning (IP)-treated surgery group, then10rats in each group. Regional cerebral blood flow (rCBF) was measured continuously in10rats per group using laser Doppler flowmetry and record the rCBF at the time point of before hypothermia, at the temperature of (25.0±0.5)℃, MHLF0～5min、MHLF10～15 min、MHLF30～35min, MHLF55～60min, MHLF65-70min、MHLF75～80min, MHLF85～90min、MHLF105～110min、MHLF115～120min and after MHLF0～5min, rewarming. The oher60rats for each group mentioned before were randomly redistributed into five subgroups:1hour (1h),6hours (6h),24hours (24h),72hours (72h) and7days (7d) after reperfusion,12rats in each subgroup. We investigated brain cell apoptosis levels and mitochondrial apoptosis signaling pathway components at various time points following reperfusion. In situ end-labeling of nuclear DNA fragmentation (TUNEL) was employed to determine the level of apoptosis. The mRNA expressions of cytochrome c and caspase-3were determined by RT-PCR, and the protein expressions of cytochrome c and caspase-3were determined by immunohistochemistry. Cytochrome c, caspase-3and caspase-9, also the upstream BCL-2, BAX and AKT were detected using western blot.Results:The rCBF was decreased about84%during bilateral common carotid artery occlusion in the P-treated surgery group and the surgery group. The changes trends of rCBF and internal environment was consistent with the changes trends of cerebral perfusion level and internal environment on clinic. The IP-treated surgery group experienced decreased mortality after24h of cerebral I/R, and displayed decreases in apoptotic cell numbers and apoptotic protein expression levels after the procedure at the time point of6h,24h,72h and7d after reperfusion compared with the surgery group (P<0.05or P<0.01). IP inhibited cytochrome c release, caspase-3and caspase-9activation and mitochondrial apoptosis signaling pathway activation at the time point of6h,24h,72h and7d after reperfusion through immunohistochemisty contrast with the surgery group(P<0.05or P<0.01). RT-PCR showed that cytochrome c and caspase-3was reduced obviously compared with the surgery group (P<0.05or P<0.01) at the time point of24h and72h after reperfusion. Western blot analysis revealed the results were consistened with the previous results, and also, the caspase-9was decreased obviously in contrast with the surgery group (P<0.05or P<0.01). At the upstream gene of the network, the protein BCL-2and AKT expressed enhancely in the IP treated group while BAX was attenuated compared with the surgery group (P<0.05or P<0.01).Conclusion:These results suggest that pathophysiology of this animal model was approximately to MHLF procedure for clinical aorta dissection surgery and made a successful SD rat model of MHLF. The protective effects of IP were associated with a reduction of DNA fragmentation, and inhibition of mitochondrial cytochrome c release, caspase-9and caspase-3activation, which alleviated cerebral ischemia/reperfusion (I/R) injury by anti apoptosis after MHLF in rats. Meanwhile, we found the gene and protein BCL-2, BAX and pAkt, which is located at the upstream of the signaling pathway, was in variety during MHLF for IP group and surgery group, suggesting that the protein Akt suppresses the mitochondrial apoptosis signaling pathway. These findings highlight the potential of ischemic preconditioning as a neuroprotective therapy and AKT may be a potential molecular therapeutic target for brain protection during MHLF for surgery of aorta dissection. |
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