督脉电针对脑缺血再灌注后肢体痉挛大鼠大脑皮层氯离子稳态的影响

Effects of Governor Vessel electroacupuncture on chloridion homeostasis in the cortex of rats with limb spasm after cerebral ischemia-reperfusion

目的:探讨督脉电针对脑缺血再灌注后肢体痉挛大鼠大脑皮层氯离子(Cl^(-))稳态及γ-氨基丁酸(GABA)和钠-钾-氯协同转运蛋白1(NKCC1)表达的影响,探讨督脉电针缓解脑卒中后肢体痉挛的作用机制。方法:SD大鼠随机分为正常组9只、假手术组9只,其余采用大脑中动脉栓塞法复制脑缺血后再灌注痉挛大鼠模型,造模成功大鼠随机分为模型组、督脉电针组和巴氯芬组,每组9只。督脉电针组电针“大椎”“脊中”“后会”,每次30 min;巴氯芬组灌胃巴氯芬(0.4 mg/kg),两组均每日1次,连续治疗7 d。应用神经功能缺损评分对各组大鼠神经功能进行评估,采用改良Ashworth量表和电生理描记检测各组大鼠肌肉反应对张力传感器的作用力;采用TTC染色检测各组大鼠脑梗死体积百分比;采用干湿重法检测各组大鼠脑含水量;采用比色法检测各组大鼠大脑皮层中Cl^(-)和GABA含量;采用Western blot法和实时荧光定量PCR法分别检测大脑皮层中NKCC1蛋白和mRNA表达。结果:造模后,模型组、督脉电针组、巴氯芬组较正常组和假手术组神经功能缺损评分和改良Ashworth评分升高、肌肉反应对张力传感器的作用力降低(P<0.01)。治疗后,督脉电针组、巴氯芬组较同组治疗前神经功能缺损评分和改良Ashworth评分降低、肌肉反应对张力传感器的作用力升高(P<0.05,P<0.01);与同时间点模型组比较,督脉电针组、巴氯芬组神经功能缺损评分和改良Ashworth评分降低、肌肉反应对张力传感器的作用力升高(P<0.05)。与正常组和假手术组比较,模型组脑含水量、脑梗死体积百分比升高(P<0.01);与模型组比较,督脉电针组、巴氯芬组脑含水量、脑梗死体积百分比降低(P<0.01)。与正常组和假手术组比较,模型组大脑皮层中Cl^(-)含量升高(P<0.01),GABA含量降低(P<0.01);与模型组比较,督脉电针组、巴氯芬组Cl^(-)含量降低(P<0.05,P<0.01),GABA含量升高(P<0.05,P<0.01)。与正常组和假手术组比较,模型组大脑皮层NKCC1蛋白和mRNA表达均上调(P<0.01);与模型组比较,督脉电针组、巴氯芬组NKCC1蛋白和mRNA表达下调(P<0.01,P<0.05);督脉电针组较巴氯芬组NKCC1蛋白和mRNA表达上调(P<0.01,P<0.05)。结论:督脉电针可改善脑卒中后大鼠的肢体痉挛程度,其机制可能与调控大脑皮层中Cl^(-)稳态、增加GABA含量、下调NKCC1蛋白和mRNA表达有关,从而减轻脑水肿和梗死程度,改善神经功能缺损症状,降低肌张力。

Objective To observe the effect of electroacupuncture(EA)stimulation of Governor Vessel on chloridion(Cl^(-))homeostasis and the expression ofγ-aminobutyric acid(GABA)and Na+-K+-Cl^(-)cotransporter 1(NKCC1)in the cerebral cortex of cerebral ischemia-reperfusion injury(CIRI)model rats,so as to explore its mecha⁃nism underl-ying alleviating limb spasm after stroke.Methods Forty-five male SD rats were randomly divided into nor⁃mal,sham-operation,model,EA and baclofen groups,with 9 rats in each group.The CIRI model was established by occlusion of the middle cerebral artery and reperfusion.EA(100 Hz)was applied to“Dazhui”(GV14),“Jizhong”(GV6)and“Houhui”for 30 min.Rats of the baclofen group received gavage of baclofen solution(0.4 mg/kg,1 mL/100 g),once daily for 7 consecutive days.Neurological deficit score was assessed according to Zea Longa’s method.The mus⁃cular tone of quadriceps femoris of the limb was evaluated by modified Ashworth scale and electrophysiological recording methods,separately.TTC staining was used to detect cerebral infarction volume,and the brain water content of rats in each group was determined by wet and dry weight method.The contents of Cl^(-)and GABA in the cerebral cortex were detected by colorimetric method,and the expression levels of NKCC1 mRNA and protein in the cerebral cortex were detected by quantitative real-time PCR and Western blot,separately.Results No significant differences were found between the normal and sham-operation groups in all the indexes.Compared with the normal and sham-operation groups,the neurological deficit score,modified Ashworth muscle tone score,brain water content,cerebral infarct volume percent,Cl^(-)content and expression levels of NKCC1 mRNA and protein were all evidently increased(P<0.01),and muscle tone of electrophyiological electromyogram(EMG)signal and GABA content were strikingly decreased(P<0.01)in the model group.Compared with the model group,both EA and baclofen groups had an obvious increase in EMG signal displayed muscle tone,and GABA content(P<0.05,P<0.01),and a marked decrease in the neurological deficit score,modified Ashworth score,brain water content,cerebral infarct percent,Cl^(-)content and expression levels of NKCC1 mRNA and protein(P<0.05,P<0.01).Conclusion EA stimulation of acupoints of the Governor Vessel can improve the degree of limb spasm and reduce the degree of cerebral edema and infarction in rats with stroke,which may be related to its functions in protecting Cl^(-)homeostasis,up-regulating GABA concentration,and down-regulating the expression of NKCC1 protein and mRNA in the cerebral cortex.