电刺激促进脑梗死大鼠星形胶质细胞与神经元可塑性的重建

背景对实验性卒中模型有目的进行瘫痪肢体的功能训练后脑组织修复过程中组织结构变化需借助相应方法观察其特征.目的用易卒中型肾血管性高血压鼠复制大脑中动脉闭塞模型,探讨电刺激在脑梗死康复中对星形细胞与神经元可塑性的修复.设计随机对照实验.单位中山大学动物中心和电镜室.材料实验于2002-01/2004-12在中山大学中山医学院动物中心和附属第一医院神经科实验室完成.健康雄性SD大鼠200只,3个月龄,体质量90～110 g.所选的易卒中型肾血管性高血压鼠的收缩压必须达到180 mmHg(1 mm Hg=0.133 kPa)以上,复制的大脑中动脉闭塞模型经走横木试验评分为1分者.选出180只随机分为电刺激组和对照组,各90只.方法电刺激组在瘫痪肢体取4个穴位,进行电刺激,6 d为1个疗程,在电刺激治疗第1,3,6,9个疗程末对大鼠进行运动功能评分及取脑梗死灶边缘区组织进行以下检测1]走横木试验评分法(1分为严重障碍,7分为正常).2]电镜观察.3]胶质酸性蛋白及神经丝蛋白和微管相关蛋白2测定采用免疫组织化学染色,两组标本的每张切片随机选5个视野,统计阳性细胞数;选取30个胶质纤维酸性蛋白阳性细胞,测其阳性胞浆平均吸光度(A值).4]观察神经元凋亡采用原位末端标记法.5]脑微血管扩张标准为视野下为完整的微血管记数1个.主要观察指标1]大鼠运动功能评分.2]大鼠脑神经元与星形胶质细胞的超微结构.3]大鼠脑胶质酸性蛋白、神经丝蛋白和微管相关蛋白2表达情况.4]大鼠脑神经元凋亡结果.5]大鼠脑微血管舒张情况.结果大鼠进入结果分析180只,余20只在随机分组时超过1分退出实验.1]两组大鼠走横木试验结果从3～9个疗程末瘫痪肢体功能恢复电刺激组均好于对照组,第9疗程达6分大鼠电刺激组显著多于对照组(42,26,χ2=15.4,P＜0.01).2]大鼠脑胶质纤维酸性蛋白阳性吸光度值第3,6,9个疗程电刺激组显著高于对照组(52.97±0.59)%比(46.40±0.56)%;(49.44±0.80)%比(46.40±0.56)%;(43.25±0.48)%比(34.20±0.50)%,P＜0.05].3]大鼠脑神经丝蛋白测定结果第6和第9个疗程电刺激组显著高于对照组(22.9±2.7)%比(11.9±2.3)%;(26.5±1.7)%比(11.7±1.5)%,P＜0.05].4]大鼠脑微管相关蛋白2测定结果第6和第9个疗程电刺激组显著高于对照组(21.7±1.3)%比(11.3±1.1)%;(24.4±2.1)%比(11.9±2.3),P＜0.05].5]大鼠脑神经元凋亡数两组无显著差别.6]大鼠脑微血管开放数目检测结果第1,3,6,9个疗程电刺激组显著多于对照组(33比19;48比31;45比25;46比23,Z=-2.309,P＜0.05).结论电刺激治疗可以促进瘫痪肢体功能恢复.可能是电刺激治疗可使星形胶质细胞的缺血性水肿消退,增强神经元活性,激发了脑微血管的舒张,从而改善脑循环.

Effect of electro-stimulating therapy on the repair of astrocytes and neurons in the rehabilitative course of cerebral infarction

BACKGROUND: At present, there is few reports about using middl ecerebral artery obstraction (MCAO) model to determine the repair course of cerebral infarction during functional training.OBJECTIVE: To determine the effect of electro-stimulating therapy on promoting the rehabilitation of cerebral infarction and its mechanism.DESIGN: Randomized controlled study.SETTING: Animal Center and Electron Microscope Laboratory of Zhongshan University.MATERIALS: The experiment was carried out in the Animal Center of Zhongshan Medical College and Neurological Laboratory of the First Affiliated Hospital of Zhongshang University from January 2002 to December2004. A total of 200 healthy males SD rats, aged 3 months and weighing 90-110 g, were selected. According to the following criteria: SBP＞180mmHg (1 mmHg=0.133 kPa), BWT score of MCAO models which were reproduced by RHRSP was 1, totally 180 RHRSP were admitted to the research and divided into electro-stimulating therapy group (n=90) and control group (n=90).METHODS: Electro-stimulating was given to four accupuncture points of the paralyzed limbs of rats. The electro-stimulating treatment was given about 30 minutes once a day. And a therapy course was 6 days, and between two therapy courses there was one-day break. At the end of 1st, 3rd,6th and 9th therapy courses, the brain of motor function and tissue in marginal zone of cerebral infarction were assayed as follow: 1] The beam walking test (BWT, 1 as severe disorder and 7 as normal). 2] Electron microscope. 3] Astrpcyte glial fibriliary acidic protein, neurofilament protein and microtubule-associated protein-2 were assayed with immunohistochemistry. Five fields of each slice in the two groups were randomly selected to add up the positive cell number. Totally 30 positive cells of glial fibriliary acidic protein was selected to assay average absorbency (A) of positive cellular plasm. 4] Apoptosis of neurons were observed with in situ end-labeling (ISEL). 5] Brain-micro vasodilatatio was observed according to the criteria of one complete microvessel account under the field.MAIN OUTCOME MEASURES: 1] Scores of motor function; 2] Ultramicrostructure of cranial neurons and astrocyte; 3] Cranial glial fibriliary acidic protein, neurofilament protein and microtubule-associated protein-2;4] Apoptosis of neurons; 5] Diastole of cerebral microvessel.RESULTS: Totally 180 rats were eligible while 20 rats were excluded because of their BWT score＞1 after MCAO operation. 1] Results of beam walking test (BWT): Functional recovery of paralysis limbs in electric stimulation group was better than that in control group from the third to the ninth course. In the ninth course, 6 points of rats in electric stimulation group was more than that in control group (42, 46, χ2=15.4, P ＜ 0.01). 2]Positive absorbency of cerebral glial fibriliary acidic protein: That in electric stimulation group was higher than that in control group in the 3rd, 6th,and 9th (52.97±0.59)% vs (46.40±0.56)%; (49.44±0.80)% vs (46.40±0.56)%;(43.25±0.48)% vs (34.20±0.50)%, P ＜ 0.05]. 3] Assay of neurofilament protein: That in electric stimulation group was higher than that in control group in the 6th and 9th course (22.9±2.7)% vs (11.9±2.3)%; (26.5±1.7)%vs (11.7±1.5)%, P ＜ 0.05]. 4] Assay of microtubule-associated protein-2:That in electric stimulation group was higher than that in control group in the 6th and 9th course (21.7±1.3)% vs (11.3±1.1)%; (24.4±2.1)% vs(11.9±2.3)%, P ＜ 0.05]. 5] Apoptosis of neurons: There was not significantly different between the two groups. 6] Results of open number of cerebral microvessel: That in electric stimulation group was higher than that in control group in the 1st, 3rd, 6th and 9th course (33 vs 19; 48 vs 31;45 vs 25; 46 vs 23, Z=-2.309, P ＜ 0.05).CONCLUSION: Electro-stimulating treatment can promote motor function of paralyzed limbs, which was due to that electro-stimulating treatment may promote extinction of the swollen feet of astrocytes, reinforce neurons activity and arouse the dilatation of cerebral capillary which promote the microvascular dilatation in order to improve cerebral blood circulation.