缺血预处理及乐脉颗粒对增强局灶性脑缺血耐受的作用机制

Mechanisms of ischemic preconditioning and Lemai granules in enhancing focal cerebral tolerance

目的:观察局灶性的脑缺血预处理对胶质纤维酸性蛋白和神经元特异性烯醇化酶表达的影响,以及给予乐脉颗粒干预治疗后的变化。方法:实验于2003-12/2004-11在四川大学华西医院外科和眼科实验室进行。取SD大鼠30只,随机分为3组,每组10只。①预缺血组:二次线栓法建立脑缺血耐受模型,预缺血10min,3d后给予大脑中动脉完全阻塞2h,再灌注22h。②假手术组:未进行缺血预处理,而单纯暴露动脉处的解剖结构10min,余同预缺血组。③乐脉颗粒组:预缺血10min,给予乐脉颗粒(丹参,川芎,赤芍,红花,香附,木香等组成)1.5g/(kg·d)灌胃,3d后给予大脑中动脉完全阻塞塞2h,再灌注22h。各组大鼠处死前进行神经功能缺损评分(0～4分,0分为无神经功能缺失症状;4分为不能自发行走);在再灌注22h麻醉状态下处死大鼠,测定脑梗死体积;进行免疫组化染色和图像分析比较各组纤维酸性蛋白、神经元特异性烯醇化酶的表达,以评估脑组织中星形胶质细胞活化和正常神经元存活情况。结果:30只大鼠进入结果分析。①脑梗死体积:乐脉颗粒组明显小于假手术组与预缺血组[(96.84±4.99),(147.62±4.70),(114.33±7.81)mm3,P<0.01]。②神经功能缺损评分:乐脉颗粒组显著低于预缺血组(2.06±0.08,2.18±0.22,P<0.01),两组均低于假手术组(3.18±0.16,P<0.01)。③胶质纤维酸性蛋白阳性表达的IA值:预缺血组与乐脉颗粒组均高于假手术组(6610.83±741.43,11937.70±868.34,3500.53±143.34,P<0.05,0.01),但乐脉颗粒组增高更为明显。④神经元特异性烯醇化酶阳性表达的IA值:乐脉颗粒组高于预缺血组和假手术组(9773.58±614.77,5459.82±605.14,2666.12±359.72,P<0.01);预缺血组高于假手术组(P<0.01)。结论:①局灶性缺血预处理10min能够对3d后的SD大鼠脑再梗死提供脑保护,诱导缺血耐受的形成。②局灶性缺血预处理能够诱导缺血耐受的产生,其可能的机制之一是通过促进星形胶质细胞活化增加减少神经元的损伤,乐脉颗粒能够增强这一效应。

AIM： To observe the influence of focal cerebral ischemic preconditioning on the expressions of ghal fibriliary acidic protein and neuron-specific enolase, and the changes after intervention and treatment of Lemai granules. METHODS： The experiment was carried out in the surgery and ophthalmological departments of West China Hospital, Siehuan University from December 2003 to November 2004. Thirty SD rats were randomly divided into 3 groups： ① isehemic preconditioning group （n=10）： Isehemie preconditioning was induced by intraluminal filament middle cerebral artery for 10 minutes. Middle cerebral artery occlusion （MCAO） was induced by intraluminal filament for 2 hour and reperfusion for 22 hours.② sham-operated group （n=10）： The rats did not receive ischemie preconditioning, only treated with exposure of arterial anatomic structure for 10 minutes, other treatments were the same as those in the ischemic preconditioning group. ③ Lemai granules group （n=10）： The rats were treated with isehemic preconditioning for 10 minutes, and then were given gastric perfusion of Lemai granules （including dan-shen root, Szechwan lovge rhizome, red peony root, safflower, nutgrass galingale rhizome and costusroot, 1.5 g/kg per day）, and middle cerebral artery occlusion （MCAO） was induced by intraluminal filament for 2 hour and reperfusion for 22 hours after 3 days. The rats were tested with neurologic deficit score （NDS, 0-4 points, 0 as without loss of neurological function, 4 as could not walk independently） before being killed. At 22 hours after reperfusion, the rats were killed under anesthesia, the infarcted volume was determined. The expressions of ghal fibriliary acidic protein and neuron-specific enolase were compared with immunohistochemical staining and image analysis to evaluate the activation of astrocytes and survival of normal neuron in brain tissue, RESULTS： All the30 rats were involved in the analysis of results. ① The infarct volume was significantly smaller in the Lernai granules group than in the sham-operated group and ischemic preconditioning group [（96.84±4.99）, （147.62±4.70）, （114.33±7.81） mm^3, P 〈 0,01]. ② The NDS was significantly lower in the Lemai granules group than in the ischemic preconditioning group （2.06±0.08, 2.18±0.22, P 〈 0.01）, both were lower than that in the sham-operated group （3.18±0.16, P 〈 0.01）.③ The IA value of glial fibriliary acidic protein positive expression was higher in the ischemic preconditioning group and Lemai granules group than in the sham-operated group （6 610.83±741.43, 11 937.70±868.34, 3 500.53±143,34, P 〈 0.05, 0.01）, but the increase was more obvious in the Lemai granules group. ④ The IA value of neuron-specific enolase positive expression was higher in the Lemai granules group than in the ischemic preconditioning group and sham-operated group （9 773.58±614,77, 5 459.82±605.14, 2 666.12±359.72, P 〈 0.01）, also higher in the ischemic preconditioning group than in the sham-operated group （P 〈 0.01）, CONCLUSION： ① Focal cerebral ischemic preconditioning for 10 minutes could provide brain protection and induce ischemic tolerance in SD rats with cerebral infarction after 3 days. ② Activation of astrocytes might play an important role in the induction of endogenous neuroprotection in the ischemic tolerance, and Lemai granules can enhance the ischemic tolerance induced by focal cerebral ischemic preconditioning.