【作者】 曾光伟；

【导师】 吴爱群； 汤善钧；

【作者基本信息】 郑州大学 ， 人体解剖与组织胚胎学， 2004， 硕士

【摘要】 目的:缺氧缺血性脑损伤(Hypoxic-ischemic brain damage,HIBD)是严重威胁新生儿健康的常见病因,发病机制复杂,存活患儿中部分留有脑性瘫痪、癫痫、智力低下等不同程度中枢神经系统后遗症。传统理论认为哺乳动物中枢神经发育成熟后不具有产生新生神经元的能力。近年来,哺乳动物受损脑组织自身神经修复的证据不断有报道,哺乳动物发育成熟的中枢神经系统产生新生神经元的有限性,限制其受损后的修复能力。干细胞被认为是能够提供新生细胞和组织的工具,用来修复或替换损伤的细胞和组织。Mckay首次将存在于哺乳动物脑内具有自我更新和多分化潜能的细胞定义为神经干细胞。神经干细胞是否在受损脑组织的自身修复中扮演重要角色,研究表明哺乳动物发育及成熟的脑在不同时期和不同区域均有神经干细胞存在。文献报道MCAO大鼠脑缺氧缺血区神经元内DNA和RNA储量显著下降,神经元数量减少,针刺后可见神经元内DNA和RNA储量不同程度恢复,神经元数量增多,提示针剌能够较早从细胞和分子水平扭转变性与坏死病变过程。目前多数研究是关于电针对急性脑缺血缺氧成年大鼠的干预,有关电针对新生大鼠HIBD治疗及其远期效果则鲜有报道。本实验采用新生大鼠脑缺氧缺血动物模型,观察电针同时刺激“百会”和“大椎”两穴位后,应用免疫组织化学检测神经上皮干细胞蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)和5—溴—2—脱氧脲嘧啶(Brdu)在脑缺氧缺血新生大鼠海马结构中的表达,以进一步明确电针对脑缺氧缺血后神经发生水平的干预如何,为电针穴位疗法的临床应用提供实验依据。方法:7日龄健康Wistar大鼠100只,体重12-15克,完全随机为4组:空白对照组郑州大学以〕O生午硕丁学位论文电针对脑缺氧缺血新生大鼠海马结构Nestin、Brd、t和GFA尸表达的影响(25只,存活23只):颈部正中切口,不结扎左侧颈总动脉,不做低氧处理。模型判定组(25只,存活20只)、缺氧缺血组(25只,存活22只)和缺氧缺血后电针治疗组(25只,存活24只),各组动物分别结扎左侧颈总动脉,然后将动物置于8%02一92N2混合气中2h后取出。模型判定组动物在缺氧缺血48h处死,制备脑片及脑组织切片,应用TTC及HE染色判定模型是否成功。脑缺氧缺血后电针组在缺氧缺.血后恢复Zd,开始.刺激“百会”和‘。大椎,’两穴。将针柄分别连接至电刺激仪的电极L(电针一刺激参数:频率16Hz,强度10mV),留针10min,治疗2个疗程,每个疗程IOd,两疗程间间隔Zd。24d后除模型判定组外各组动物分别灌注取左侧脑组织,常规石蜡包埋、切片一,行Nestin、Brdu和GFAp免疫组化染色,光镜下观察海马结构的阳性细胞分布、记数定量分析,采用SPSS10.O软件对数据进行统计学处理。结果:1.TTC组织化学染色:模型判定组动物的脑片经TTC功能性酶组织化学染色后,其左侧大脑半球的大部分脑实质着色浅呈苍白色,而右侧大脑半球染成深红色,提示模型成功。2.HE染色:模型组判定组动物的左侧大脑半球HE染色可见脑水肿和脑室及蛛网膜下腔出血等病理改变;脑组织灶性溶解坏死,间质及血管周围明显水肿,胶质细胞增生;部分神经元固缩、变性和坏死。3.Nestin免疫组织化学染色:各组新生大鼠脑组织海马结构内的海马CAI,CAZ及CA3区和齿状回等显示出Nestin免疫阳性细胞分布。脑缺氧缺血组海马CAI区Nestin免疫阳性细胞数(25.72士3.21)高于正常对照组(19.86士3.83,P<0.05);脑缺氧缺血后电针组海马CAI区Nestin免疫阳性细胞数(30.85士3.79)高于脑缺氧缺血组(尸<0.05)。4.Brdu免疫组织化学染色:Brdu免疫组织化学染色显示各组Wistar幼鼠脑组织内侧脑室的室管膜下层、齿状回颗粒细胞层的下层及脐眠体等显示出Brdu免疫阳性细胞分布;脑缺氧缺血组齿状回Brdu免疫阳性细胞数(19.78士2.86)高于正常刘一照组(10.22士2.21,尸<o、05);脑缺氧缺血后电针组齿状回Brdu免疫阳性细胞数(24.04士2.58)稍高于脑缺氧缺血组〔尸<0.05)。4.GFAP免疫组织化学染色:各组新生大鼠脑组织海马结构内的海马CAI,CAZ郑州大学卫)(Jl生硕士学位论交电针对脑缺氧缺JIn新生大鼠海马结构N二tll:、阶山:和G队尸表达钩影响及CA3区、齿状回脱和眠体等显示出GFAP细胞分布口脑缺氧缺血组海马CAI!X_GFAP免疫阳性细胞数(63 .56士3 .08)高于正常对照组(48 .59士3 .35,尸<0.05);脑缺氧缺血后电针组海马CAI区GFAI〕免疫阳性细胞数(64.68士3.03)同脑缺氧缺!fll组相比较无显著性差异(尸>0.05)。结论:脑缺氧缺血可诱导新生大鼠海马CAI区星形胶质细胞反应性增生和Nestin免疫阳性神经丁一细胞数增高。电/ff一delJ激脑缺氧缺血新生人鼠“百会”、“人椎”两穴位:1.一可促进脑缺孰缺血新生大鼠海马CAI区Nestin免疫阳性神经干细胞表达。2.一可促进脑缺氧缺血新生人鼠海马结构齿状回亚颗粒细胞增殃。提小电针穴位治疗可明显提高(加强)脑缺戴缺l位后海马结构神经发生水(能力)更多还原

【Abstract】 Objective: Hypoxic-ischemic brain damage(HIBD) plays an importment role in perinatal medicine because of the high mortality rate and neurological and intellectual impairment in the surviving infants .At present ,despite scholors have done lots of investigation in the treatment of HIBD, no effective therapeutic and neuroprotective strategies after hypoxic-ischemia (HI) have yet been available in clinic therefore , one of the most urgent tasks for obstetricians and neurologist is to develop therapeutic strategies.The primary theory conceived that the developed CNS of mammalian has no ability to generate new neurons. Evidence for neuronal self-repair following insults to the mammalian brain has been scarce until very recently. The limited ability of the mature CNS to generate new neural cells limits the self-repair capacity of the damaged brain. Stem cells are believed to provide a tool by which new cells and tissues can be made and by which damaged ones can be replaced or repaired. Do neural stem cells play an importment role in the self-repair of damage brain. Mcaky first defined neural stem cells that possess two potentials such as self-renewal and multidirectional differentiation in the mammalian brain Neural stem cells have been isolated from various regions and various development periods of the mammalian brain, recently, eperimental study indicated that electroacupuncture could high levels of DNA and RNA in neurons and increase the number of new neurons in the ischemic area of a model of middle cerebral artery occlusion .the result demonstrated that EA had the capacity to promote the recovery of brain damage fromthe molecular and cellular level. However, there were fewer reports about the treatment and the long-term effects of EA on HIBD in neonatal rats. The present study was done to observe the influence of electroacupuncturing "BaiHui" and "DaZhui" points on the neurogenesis which was reflected with immunoreactive Nesti^ Brdu and GFAP expressions in the hippocampal formation in neonatal rats after hypoxia-ischemia .so as to confirm the effects of EA on HIBD in neonatal rat, investigate the possible mechanism of neurogenesis, and provide the theoretical basis for clinical application.Methods:7-d-old Wistar rats(n= 100) weighing 12~15g were used in neurogenesis study and randomly divided into four groups. The sham-operation rats(n=25, survival n=23) were save as normal control group only received the operation without hypoxia-ischemia treatment.The animal medols (n=75, survival=66) of cerebral hypoxia-ischemia brain damage (HIBD) were prepared by left common carotied artery ligation and systemic hypoxia (8% oxygen in nitrogen) for 2h in model group(survival=20) , HIBD group(survival=22) and HIBD treated with electroacupucture group(survival=24) . Pathological changes of the hypoxia-ischemia area were observed by the TTC (2,3,5-Triphenyltetrazolium bromide) and HE staining till the rats of model group decapitated after HIBD 2 days .After 2 d of recovery, the rats of HIBD treated with electroacupucture group were treated electroacupucture ("baihui" and "dazhui"points) every day till the rats were decapitated after HIBD 22 days, the needle handles were respectively connected to stimulative machine( 16Hz, lOmV), the rats received 10 min treatment each day, each course contained 10 treatments, 2 d interval between two courses.The 42 rats (Brdu was injected into the peritoneal cavities of all rats to label the proliferating cells before decapitation) were subjected to perfusion-fixation 22 d later except the rats of model group , the left brains were regularly paraffin wax embedded, sectioned and stained by Nestle Brdu and GFAP immunohistochemical staining. The distribution and number of the postive cells were observed for quantitative and statistical analysis .Results: ?HE staining: brain edema .cerebromalacia and hemorrhage in ventricles of brain and in arachnoid were observed in the left of cerebral hemisphere of themodel group. The neuron was contract, degeneration and necrosis, the focal lysis of the brain tissue nec更多还原