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(I/R)大鼠皮质梗死区周围热休克蛋白70(HSP70)的表达定位及阳性细胞表达变化,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)、I/R组及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,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及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,2,3,5-氯化三苯基四氮唑(TTC)法检测脑梗死体积,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组各组差异无统计学意义(P>0.05),3d各组差异有显著统计学意义(P<0.01);HSP70阳性表达主要在梗死区周围神经元、部分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,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 posteonditioning （LRIP） protective role in the brain by observing the localization and changes of positive eells expression of heat shock protein 70 （HSPTO） in focal cerebral isehemia-reperfusion injury （I/R） of rat cortical infarct areas after remote ischemic posteondtioning treatment. Methods A SD rat model of focal cerebral ischemia reperfusion was induced by intraluminal 1 hour transient middle cerebral artery oeelusion 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 IRIP 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 1 day or 3days after reperfusion and the brain was obtained by decapitation. Rats were evaluated for neurological deficits just before saerifiee 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. Immunohistoehemistry and double immunofluorescence histochemistry were used to observe the distribution, type and number of positive eells of HSPT0. 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 nostatistically significant difference between LRIP and I/R group at lday （ P 〉 0.05） except for that of 3days （ P 〈 0.01 ）. HSP70 was localized predominantly in neurons and endothelia ceils 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.