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肢体远程缺血后处理促进局灶性脑缺血再灌注大鼠皮质区热休克蛋白70的表达
Limb remote ischemic postcondtioning promoting the expression of heat shock protein 70 in the rat cortex after focal cerebral ischemia reperfusion injury
目的 观察肢体远程缺血后处理（LRIP）对局灶性脑缺血再灌注损伤大鼠皮质梗死区周围神经元、血管内皮细胞以及星形胶质细胞热休克蛋白70（Hsp70）的表达变化，探讨LRIP发挥脑保护作用的可能分子机制。方法 健康成年SD大鼠，随机分为假手术组（sham）、局灶性脑缺血再灌注模型组（I/R）、LRIP组。实验采用线栓法建立局灶性大脑中动脉脑缺血(1h)再灌注模型(MCAO)，大鼠脑缺血再灌注即刻行双下肢股动脉橡皮筋结扎10min，放松10min，重复3次建立LRIP组模型。于再灌注1d、3d分别断头取脑，Zea longa评分作为判断MCAO模型成功的标准，Garcia神经行为学评分方法检测大鼠神经损伤程度，TTC检测脑梗死体积，Western blotting检测Hsp70蛋白表达含量, 免疫组织化学和免疫荧光技术，用于检测皮质梗死区周围Hsp70阳性表达细胞的数目、部位以及类型。结果 应用LRIP后，LRIP组与I/R组比较，神经行为学评分明显增加（P<0.05）、脑梗死体积显著降低（P<0.05），Hsp70蛋白表达明显增加，其中1d组无统计学意义（P>0.05），3d组有显著统计学意义（P<0.01），Hsp70阳性表达主要在梗死区周围神经元、血管内皮细胞和星形胶质细胞突起。结论 LRIP可明显改善脑缺血后神经行为学功能、降低脑梗死体积，根据本实验结果我们推测此作用可能与LRIP上调皮质梗死区周围神经元、血管内皮细胞和星形胶质细胞Hsp70表达有关。
Objective To explore the underlying molecular mechanisms of limb remote ischemic postconditioning (LRIP) protective role in the brain by observing the localization and changes of positive cells expression of heat shock protein 70 (HSP70) in focal cerebral ischemia-reperfusion injury (I/R) of rat cortical infarct areas after remote ischemic postcondtioning treatment. Methods A SD rat model of focal cerebral ischemia reperfusion was induced by intraluminal 1 hour transient middle cerebral artery occlusion with a nylon monofilament suture.Ischemic animals were randomly assigned to 3 groups: sham group, I/R group and LRIP group, 5 animals each group.The LRIP performed immediately after reperfusion(LRIP was generated by 3 cycles of 10 minutes occlusion/10 minutes release of the bilateral hind femoral artery used rubber band). To determine whether the model of MCAO was successful, Zea longa score method was used. Rats were sacrificed on 1day or 3days after reperfusion and the brain was obtained by decapitation. Rats were evaluated for neurological deficits just before sacrifice by Garcia. Brains were harvested for infarct size estimation used 2, 3,5-triphenyl tetrazolium chloride(TTC). 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 HSP70. Results Compared with I/R group, LRIP treatment significantly improved neurological functions（P<0.05）and decreased infract size（P<0.05）as well as upregulated HSP70 expression. There was no statistically significant difference between LRIP and I/R group at 1day (P>0.05)except for that of 3days（P<0.01）. HSP70 was localized predominantly in neurons and endothelia cells and astrocytes in ischemic peripheral areas. Conclusion LRIP treatment could improve neurological functions as well as decrease infract size. According to the results, we speculate this protective effect likely by increasing the neurons, endothelia cells and astrocytes expression of HSP70 in ischemic peripheral areas.
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