电针百会和神庭穴对脑缺血再灌注损伤大鼠认知功能的影响

Effect of Electroacupuncture at Baihui and Shenting on Cognitive Function in Cerebral Ischemia-reperfusion Injured Rats

目的:观察电针百会和神庭穴对脑缺血再灌注损伤大鼠认知功能的影响,探讨其作用机制。方法:将111只雄性SD大鼠按照随机数字表法分为假手术组36只和手术组75只。假手术组麻醉后只分离左侧颈总、颈内、颈外动脉,不结扎、插线。手术组采用Zea Longa线栓法制备大鼠大脑中动脉缺血再灌注模型。依据Zea Longa评分法和Barnes迷宫实验对造模后大鼠进行神经行为学评分和认知缺损筛查,75只大鼠最终纳入72只,采用随机数字表法分为模型组和电针组,每组各36只。电针组于术后2 h给予大鼠电针百会、神庭穴。模型组除不予电针治疗外,其余操作同电针组。采用Barnes迷宫实验评估术后1、7 d大鼠的认知功能;运用2,3,5-氯化三苯四唑溶液(TTC)染色法检测术后1、7 d大鼠的脑梗死体积;以Iba1阳性细胞作为海马区小胶质细胞/巨噬细胞极化的标记物,采用免疫组化法观察术后1、7 d大鼠海马区小胶质细胞/巨噬细胞极化状况;运用Western blot检测术后1、7 d大鼠海马区M1型小胶质细胞/巨噬细胞特异性标记蛋白MHCⅡ及M2型小胶质细胞/巨噬细胞特异性标记蛋白Arg-1的表达;采用双抗体/夹心酶联免疫吸附测定法检测术后1、7 d大鼠海马区脑组织匀浆中M1型小胶质细胞/巨噬细胞特异性免疫细胞因子TNF-α、IL-1β及M2型小胶质细胞/巨噬细胞特异性免疫细胞因子IL-10、TGF-β的表达。结果:①假手术组大鼠各时间段均未见神经功能缺损;造模术后2 h,与假手术组比较,模型组及电针组神经行为学评分明显增高(P<0.05),Barnes迷宫逃避潜伏期时间明显延长(P<0.05),进入错误洞口次数明显增多(P<0.05);造模术后1 d,电针组与模型组大鼠Barnes迷宫逃避潜伏期时间、进入错误洞口次数比较,差异均无统计学意义(P>0.05);术后7 d,电针组大鼠Barnes迷宫逃避潜伏期时间、进入错误洞口次数较模型组明显减少(P<0.05)。②假手术组大鼠各时间段TTC均未见染色;造模术后1 d,电针组与模型组大鼠脑梗死体积比较,差异无统计学意义(P>0.05);术后7 d,电针组脑梗死体积较模型组明显缩小(P<0.05)。③造模术后1、7 d,电针组和模型组Iba1阳性表达率较假手术组明显增多,差异有统计学意义(P<0.05);电针组与模型组Iba1阳性表达率比较,差异无统计学意义(P>0.05)。④造模术后1 d,电针组和模型组Iba1、MHCⅡ、TNF-α、IL-1β蛋白表达较假手术组明显增多(P<0.05),电针组和模型组Arg-1、IL-10、TGF-β蛋白表达与假手术组比较,差异均有统计学意义(P<0.05),电针组与模型组Iba1、MHCⅡ、TNF-α、IL-1β、Arg-1、IL-10、TGF-β蛋白表达比较,差异无统计学意义(P>0.05);造模术后7 d,电针组和假手术组MHCⅡ、TNF-α、IL-1β蛋白表达较模型组明显减少(P<0.05),电针组Arg-1、IL-10、TGF-β蛋白表达较模型组、假手术组明显增多(P<0.05)。结论:电针百会、神庭穴能有效改善脑缺血再灌注损伤大鼠的认知功能,其机制可能是抑制小胶质细胞/巨噬细胞M1型极化,促进M2型极化,一定程度调节神经炎症应答,促进认知功能的恢复。

Objective: To observe the effect of electroacupuncture(EA) at Baihui and Shenting on cognitive function in rats after cerebral ischmeia-reperfusion(IR) injury, and explore the possible mechanism. Methods: A total of 111 male adult SD rats weighting(300±20)g were selected and randomly divided into the sham operation group(n=36) and the operation group(n=75). The common carotid artery, internal carotid artery and external carotid artery of rats in the sham-operation group were separated after anesthesia without ligation or thread insertion. The left middle cerebral arteries of rats in the operation group were occluded and reperfused by Zea Longa sutured-occluted method. Neurobehavioral score and cognitive impairment screening were performed on rats with Zea Longa score and Barnes maze test at two hours after surgery. The 72 rats meeting the inclusion criteria were randomly divided into model group(n=36) and electroacupuncture group(n=36). Two hours after the operation, the rats in the EA group were acupunctured at Baihui and Shenting. In the model group, expect that electroacupuncture was not used, the rest of the process was the same as that in the EA group. The changes in cognitive function of the rats were evaluated with Barnes maze 1, 7 days after cerebral ischmeia-reperfusion.The infarct volume was determined using TTC staining at 1, 7 days after surgery. In addition, Iba1 positive cells were used as markers of microglias/macrophages polarization. The polarization of microglial/macrophages in the hippocampus was observed by immunohistochemistry 1, 7 days after surgery in each group. The expression of MHCII(M1 microglia/macrophage marker protein) and Arg-1(M2 microglia/macrophage marker protein) in the hippocampus of rats were detected by Western blot 1, 7 days after surgery. The expression of TNF-α, IL-1β(M1 microglia/macrophage marker immunocytokines) and IL-10, TGF-β(M2 microglia/macrophage marker immunocytokines) in the hippocampus of rats were detected by ELISA 1, 7 days after surgery. Results: ① No neurologic impairment was observed in the sham operation group;compared with the sham operation group at two hours after surgery, the neurobehavioral scores of the model group and the EA group were significantly increased(P<0.05), Barnes maze escape incubation period was significantly prolonged(P<0.05), and the number of entering the wrong hole was significantly increased(P<0.05);at one day after surgery,there was no significant difference in Barnes maze escape latency time and number of entering the wrong hole between the EA group and the model group(P>0.05);however, at seven days after surgery, compared with the model group, Barnes maze escape latency time and number of entering the wrong hole in the EA group were significantly reduced(P<0.05). ② No TTC staining could be observed in the sham operation group at each point. There was no significant difference in cerebral infarction volume between the EA group and the model group at one day after surgery(P>0.05), and the reduction of cerebral infarction volume in the EA group was better than that in the model group at seven days after surgery(P<0.05). ③ At 1, 7 days after surgery, the positive expression rate of Iba1 in the EA group and the model group was significantly higher than that in the sham operation group(P<0.05), and there was no significant difference in the positive expression rate of Iba1 between the EA group and the model group(P>0.05). ④ At one day after surgery, the protein expression of Iba1, MHCII, TNF-α and IL-1β in the EA group and the model group were significantly increased compared with the sham operation group(P<0.05), there were significant differences in the protein expression of Arg-1, IL-10 and TGF-β in the EA group and the model group compared with the sham operation group(P<0.05), and there was no significant difference in the protein expression of Iba1, MHCII, TNF-α, IL-1β, Arg-1, IL-10 and TGF-β between the EA group and the model group(P>0.05). At seven days after surgery, the protein expressions of MHCII, TNF-α and IL-1β in the EA group and the sham operation group were significantly decreased compared with the model group(P<0.05), while the protein expressions of Arg-1, IL-10 and TGF-β in the EA group were significantly increased compared with the model group and the sham operation group(P<0.05). Conclusion: Electroacupuncture at Baihui and Shenting can effectively improve the cognitive function of rats with cerebral ischemia reperfusion injury. The mechanism may inhibit the M1 polarization of microglia/macrophages, promote the M2 polarization, regulate the neuroinflammatory response to a certain extent, and promote the recovery of cognitive function.