针刺预处理脑组织提取液对抗大鼠脑缺血再灌注损伤的作用

背景:根据中医"治未病"理论,近年来提出了针刺预处理扶正学说.目的:证实针刺预处理脑组织提取液的抗脑缺血再灌注损伤作用.设计:随机对照实验.单位:湖北中医学院针灸推拿研究所及解剖组胚教研室.材料:实验于2003-09/2004-07在湖北中医学院针灸推拿研究所完成;选用成年Wistar大鼠102只.20只用于脑组织提取液制备,82只用于后续实验. 方法:以"肾俞"、"百会"穴针刺预处理大鼠,制备正常及针刺预处理脑组织提取液.82只大鼠随机分6组.空白对照组5只行空白对照,假手术对照组15只行假手术,脑缺血再灌注对照组16只行脑缺血再灌注造模,生理盐水对照组16只先行生理盐水腹腔注射,再行脑缺血再灌注造模,正常脑组织提取液对照组15只先行正常脑组织提取液腹腔注射,再行脑缺血再灌注造模,针刺预处理脑组织提取液组15只先行针刺预处理脑组织提取液腹腔注射,再行脑缺血再灌注造模.腹腔注射分别于脑缺血造模前2 h和1 h进行,每只大鼠共注射2次,1 mL/次.上述除空白对照组外,其他各组大鼠分别于术后1,3,7 d取材(即各分3小组:每小组5只).各组动物分别于相应时间段取材,脑组织石蜡包埋切片,光镜下进行脑组织病理学观察,在400倍镜下进行存神经元计数,计数区域为各片顶皮质Ⅰ区Ⅴ层(内锥体层).主要观察指标:①脑组织病理学观察.②脑顶皮质Ⅰ区Ⅴ层幸存神经元计数.结果:实验过程中共有2只动物死亡,脑缺血再灌注对照组和生理盐水对照组各1只,100只大鼠实验数据进入结果分析.①脑组织病理学观察结果:除空白对照组和假手术对照组外,其他各组各时间段脑片镜下均可见弥散性神经元缺血缺氧性病理改变,但均无灶性坏死区.②脑顶皮质Ⅰ区Ⅴ层幸存神经元计数:再灌注1 d,脑缺血再灌注对照组幸存神经元密度较空白对照组显著降低(338.8±31.2),(753.4±60.8)个/mum2,F=129.36,P＜0.05];再灌注3,7 d神经元变性进-步加剧,但两者间无差异(F=1.76,P＞0.05).各时间段生理盐水对照组、正常脑组织提取液对照组与脑缺血再灌注对照组间的正常及针刺预处理脑组织提取液差异不显著(F=1.76,P＞0.05).在3个时间段针刺预处理脑组织提取液组幸存神经元密度均显著高于脑缺血再灌注对照组(438.1±41.0),(338.8±31.2)个/mm2;(296.4±27.1),(124.8±13.4)个/mm2;(269.5±30.4),(132.4±15.7)个/mm2,F=129.36,P＜0.05].结论:以肾俞、百会穴针刺预处理脑组织提取液行大鼠腹腔注射,可使随后发生的脑缺血再灌注引起的神经元丢失数量显著减少,针刺预处理脑组织提取液已产生明显的对抗脑缺血再灌注损伤作用.

Effect of brain tissue extract after acupuncture preconditioning on cerebral ischemia-reperfusion injury in rats

BACKGROUND: According to the thought "prevention of diseases", a conception of "strengthening the vital by acupuncture preconditioning (AP)" is suggested recently.OBJECTIVE: To investigate the effect of brain tissue extract after AP on cerebral ischemia-reperfusion injury.DESIGN: Random control experiment.SETTING: Institute of Acupuncture & Moxibustion and Massage, and Department of Anatomy and Embryology, Hubei College of Traditional Chinese Medicine.MATERIALS: Totally 102 adult Wistar rats were selected during the experiment, which was completed in the Institute of Acupuncture & Moxibustion and Massage of Hubei College of Traditional Chinese Medicine from September 2003 to July 2004. Among them, 20 rats were used to prepare cerebral tissue extract, and another 82 were used in the subsequent experiment.METHODS: The brain tissue extract was obtained from the rats which were given electroacupuncture at Shenshu (BL-23) and Baihui (DU-20).Totally 82 rats were randomly divided into 6 groups. Five rats in blank control group were taken as blank control, 15 in sham-operation control group were performed with sham operation, 16 in cerebral ischemia-reperfusion control group with cerebral ischemia-reperfusion, 16 in saline control group with the injection of saline intravenously firstly and then cerebral ischemia-reperfusion modeling, 15 in normal cerebral tissue extract control group with the injection of normal cerebral tissue extract intravenously firstly and then cerebral ischemia-reperfusion modeling, and 15 in AP cerebral tissue extract group with the injection of cerebral tissue extract intravenously firstly and then cerebral ischemia-reperfusion modeling.Intravenous injection was performed 2 hours and 1 hour before cerebral ischemic modeling, and each rat was injected twice with 1 mL/time. Brain tissue of the rats was taken ont 1, 3, 7 days after reperfusion respectively (or each group was divided into 3 subgroups with 5 rats in each) except those in blank control group. The blain tissue of rat in each group was selected at the relevant time points, and embedded with paraffin and cut into pieces. Cerebral histopathology was observed under the light scope (× 400)and the survival neurons were counted whose area was layer y of region Ⅰ in parietal cortex (inner cone).cortex.RESULTS: Two rats died during the experiment in cerebral ischemiareperfusion control group and saline control group respectively. Another Except blank control group and sham operation group, the brain sections of different time points in other groups showed scattering ischemic anoxic Count of survival neurons in layer Ⅴ of area I in parietal cortex: One day after reperfusion, survival nerve density of the brain ischemic reperfusion model group (338.8±31.2)/mm2] was significantly lower than that of blank control group (753.4±60.8)/mm2] (F=129.36, P ＜ 0.05); degeneration of the nerves became worse after reperfusion for 3 days and 7 days, but with no significant difference (F=1.76, P ＞ 0.05). There was no significant difference between the saline control group, normal brain tissue extract group and brain ischemic reperfusion model group at different time points (F=1.76, P ＞ 0.05). Survival neuron density in group of brain tissue extract after AP at the three time points was significantly higher than that in brain ischemic reperfusion model group (438.1±41.0), (338.8±31.2)/mm2,(296.4±27.1), (124.8±13.4)/mm2; (269.5±30.4), (1.324±0.157)/mm2;F=129.36, P ＜ 0.05].CONCLUSION: Injection of the brain tissue extract after AP at Shenshu (BL-23) and Baihui (DU-20) into the celiac cavity of rats could obviously reduce the subsequent neuron loss induced by brain isehemia-reperfusion and protect brain from ischemia-reperfusion injury.