Effect Of Limb Remote Ischemic Postcondtioning On Expression Of HIF-1α And HSP70 In Rat After Cerebral Ischemical Reperfusion

ObjectiveSD rats model of focal cerebral ischemia(MCAO) was induced with a nylon monofilament suture. To observe the change of expression of hypoxia-inducible factor-1a(HIF-1α) and heat shock protein 70(HSP70) and the effect of LRIP on different time rats after focal cerebral ischemical reperfusion injury in SD rat. And to investigate the underlying peotecting mechanism of LRIP on neurons and astrocytes after cerebral ischemical reperfusion injury. MethodsSD rats model of focal cerebral ischemia reperfusion was induced by intraluminal 1-hour transient middle cerebral artery occlusion(MCAO) with a nylon monofilament suture.Ischemic animals were randomly assigned to 3 groups: sham group, I/R group and limb remote ischemic postcondtioning group(LRIP).The LRIP performed immediately after reperfusion(LRIP was generated by 3 cycles of 10 min occlusion/10 min release of the bilateral hind femoral artery used rubber band.). To determine whether the model of MCAO was successful or not, Zea longa score method was used. Rats were sacrificed respectively on 1d, 3d after reperfusion and to obtain the brain by decapitation. Rats were evaluated for neurological deficits just before sacrifice used Garcia. Brians were harvested for infarct size estimation used TTC. EB extravasation was used to evaluate the permeability of the blood-brain barrier(BBB). HE staining and Nissl staining were used to observe the histological changes of brain tissue. The level of HIF-1α m RNA was determined by Q-PCR. Western blotting was used to analyze the quantitative alterations of HSP70. Immunohistochemistry and double immunofluorescence histochemistry were used to observe the distribution, type and number of positive cells of HIF-1α and HSP70. Results1 Garcia JH showed that the score of the rats were clearly decreased(*P<0.05) in I/R group and LRIP group, compared with the sham group. While it is higher(#P<0.05) in LRIP group than that of the I/R group. 2 The result of TTC staining showed that there was significant infarction in rat brain tissue, involving the cortex and striatum in I/R and LRIP group. Compared with the I/R group, the infarct volume reduced obviously in the RIPC group(#P<0.05). 3 The EB result revealed that there were significant EB extravasation in I/R group and LRIP group in the ipsilateral cortex and striatum. Whereas, the Evans blue content in the LRIP group was significantly decreased compared with that of the I/R group. 4 HE staining showed that part nucleus karyopyknosis, smaller nuclei, stained dark after cerebral ischemia-reperfusion injury, whereas these alterations could be suppressed by LRIP administration. 5 Nissl staining showed that the number of nissl body was significantly decreased or even disappear, and shape patchy. The number of nissl body was increased and the morphological damage reduced when LRIP treatment was administrated.6 Q-PCR and immunohistochemistry showed that the level of HIF-1α m RNA and number of positive cells of HIF-1α increased significantly after cerebral ischemia-reperfusion injury. LRIP treatment downregulated the level of HIF-1α m RNA expression, there was no statistically significant difference between LRIP and I/R group at 3d(P>0.05) except for that of 1d(P<0.01). Additionally, LRIP treatment downregulated HIF-1α positive cells expression(P<0.01). HIF-1α were observed to be located in neurons, as well as in astrocytes in rat cortical infarct areas peripheral. 7 Western blotting and immunohistochemistry showed that HSP70 protein expression and positive cells increased significantly after cerebral ischemia-reperfusion injury. LRIP treatment upregulated HSP70 protein expression, there was no statistically significant difference between LRIP and I/R group at 1d(P>0.05)except for that of 3d(P<0.01). LRIP treatment upregulated HSP70 positive cells expression(P<0.01). HSP70 was localized predominantly in neurons and endothelia cells and astrocytes in ischemic areas peripheral. Conclusion1. The present study showed that 3 cycles of 10 minutes ischemia and 10 minutes reperfusion in bilateral hind femoral artery has the obvious protective function to ischemia-reperfusion rats. 2. The protective effects of LRIP on brain probablely by suppression the neurons and astrocytes expression of HIF-1α, increasing the neurons, endothelia cells and astrocytes expression of HSP70 in ischemic areas peripheral.