【作者】 李宛青；

【导师】 吴爱群； 汤善钧；

【作者基本信息】 郑州大学 ， 人体解剖学与组织胚胎学， 2003， 硕士

【摘要】 目的：缺血缺氧性脑损伤（hypoxic-ischemic brain damage，HIBD）是严重威胁新生儿健康甚至致残的常见原因，其发病机制复杂，且目前仍无特效治疗。近来有实验研究报道电针治疗可促进窒息性脑瘫幼鼠脑部血液循环，提高脑血流及降低耗氧量，减轻一氧化氮（nitric oxide，NO）对神经细胞的损伤作用，减少后遗症的发生。电针治疗还可减轻脑缺血后细胞水肿及线粒体肿胀程度。这说明电针刺激可提高脑的抗损伤能力、加快损伤修复。但多数研究是关于电针对急性脑缺血成鼠的干预，有关电针对新生鼠HIBD治疗及其远期效果则较少报道。本实验采用新生大鼠脑缺血缺氧模型，观察同时电针“百会”、“大椎”两穴对脑缺血缺氧后海马CA1区胆碱乙酰转移酶（choline acetyl transferase，ChAT）、大脑皮层及海马CA2、CA3区脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）表达的影响，以及对海马神经元、神经胶质细胞超微结构和神经元尼氏体改变的影响，以进一步明确电针治疗缺血缺氧性脑病的疗效，探索其可能性机制，为电针治疗的临床应用提供理论依据。 方法：61只出生7d的健康Wistar大鼠，体重12～15g，随机分为三组。Ⅰ假手术对照组（n=16只）：动物仅接受手术处理，不结扎血管，不缺氧。Ⅱ缺血缺氧组（n=24，成功22只）：结扎左侧颈总动脉，然后将动物置于8％O₂-92N₂混合气中2h，不加任何治疗。Ⅲ缺血缺氧后电针治疗组（n=21只，成功19只）：缺血缺氧处理同Ⅱ，第二天开始，同时刺激“百会”、“大椎”两穴，将针柄分别连接至电刺激仪的电极上（频率16Hz，强度10V），留针10min，每天一次，lOd为 郑州大学加03年硕士学位论文电针对缺血缺氧新生大鼠脑组织C扮汀和BDN下表达及超微结构的影响一疗程，两疗程间隔Zd。I和11组大鼠于缺血缺氧后24h随机各抽取3只，取左侧脑组织作TTC（2，3，5一triphenyltetrazonlium.ehloride，2，3，5一氯化三苯基四氮哇）染色。22d后，每组各取3只，分别处死，取左侧海马，4%戊二醛固定，常规脱水，环氧树脂618#浸透包埋，制成超薄切片，醋酸双氧铀和构椽酸铅双重染色，H-7500型透射电镜观察。其余42只，22d后常规灌注取材，石蜡包埋、切片，行I王E、Nissl染色、ChAT和BDNF免疫组化染色，光镜下观察、记数，并用图象分析仪测量各组尼氏体灰度值，最后采用SPSS10.O软件对数据进行统计学处理。结果:1.HE染色:I组:脑组织结构及细胞层次清楚。H组:脑组织水肿明显，神经元有变性坏死现象，细胞结构模糊，染色质分布不均匀。ili组:脑水肿减轻，神经细胞变性与坏死减轻，细胞形态结构接近正常。2.ChAT免疫组织化学染色:I组海马CAI、CAZ、CA3、齿状回均有大量ChAr免疫反应阳性神经元分布（CAI区:29.74士4.85）;11组CAI区ChAT阳性神经元数量显著减少（13.96士7.62），与I组相比差异有统计学意义（尸<0 .05），CAZ、CA3区和齿状回阳性神经元分布基本同I组;m组CAI区阳性神经元数目明显增加件5.54士5.05)，与n组相比，差异有统计学意义（尸<0 .05），与I组相比，差异无统计学意义。3.BDN下免疫组织化学染色:I组大脑皮层及海马CAZ、CA3区BDN[F免疫阳性细胞数，皮层:14.14士6.n，海马:13.4肚5.56;而11组较I组明显增加，皮层:24.49士8.31，海马:21.93巧.12，两组相比差异有统计学意义（尸<0 .05）;m组较11组显著增加，皮层:31.35土9.91，海马:27.63土7.15，差异有统计学意义（尸<0 .05）。4.海马区超微组织结构改变:I组海马神经元和神经胶质细胞细胞核和细胞质轮廓规整，无水肿及损伤现象。核膜结构清晰、光滑，核内染色质均匀分布;核周细胞器丰富，结构完整;线粒体内峭清晰可见，膜完整;内质网核糖体丰富。n组海马神经元及神经胶质细胞损伤明显，细胞水肿，线粒体减少，部分线粒体肿胀、峭断裂，结构欠完整，粗面内质网表面核糖体脱落，染色质凝集成块状。m组多数神经元形态正常，可见细胞核和胞质细胞器接近正常，线粒体增多，膜结构完整，少数胞质轻度水肿，胶质细胞超微结构受损程度也明显轻于n组。5 .Nissl染色:I组海马CAI区神经元胞浆中布满深蓝色颗粒状的尼氏体（灰度值: 郑州大学加03年硕士学位论文电针对缺血缺氧新生大鼠脑组织ChAr和BDNF表达及超微结构的影响122.90士12.10）;H组海马cAI区神经元内尼氏体大量消失，与I组比较灰度值呈升高趋势（18831士10.43），两组之间差异有统计学意义（尸动.05）;m组海马CAI区神经元内尼氏体较I组有轻度脱失，但较11组明显减轻，灰度值较11组呈降低趋势（135.19士7.00），两组间差异有统计学意义（尸动.05）。结论:电针缺血缺氧新生大鼠“百会”、“大椎”两穴后能:1.促进缺血缺氧新生大鼠海马CAI区Cll八J的表达。2.促进缺血缺氧新生大鼠大脑皮层和海马CAZ、CA3区BD到[F的表达。3.明显改善缺血缺氧新生大鼠海马神经元和神经胶质细胞超微结构的退行性改变。4.明显减轻缺血缺氧新生大鼠海马CAI区神经元尼氏体的脱失现象。 以上结果提示电针治疗对缺血缺氧性脑损伤有明显的神经保护作用。更多还原

【Abstract】 Objective: Hypoxic-ischemic brain damage(HIBD) could result in neonatalmorbidity and mortality, some patients with HIBD developed into cerebral palsy and mental retardation. So far no specific neuroprotective intervention after hypoxia-ischemia (H-I) has been available. Recently, the experimental studies indicated that electroacupuncture(EA) could improve the brain blood circulation, attenuate the neurotoxicity of nitric oxide(NO) and the incidence of sequela in asphyxial neonatal rats with cerebral palsy; EA could extenuate cellular edema and mitochondrial swelling in ischemic rats. These results demonstrated that EA had the capacity to promote the recovery of brain damage. A great deal of studies were to treat the acute cerebral ischemia on adult rats with EA, 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 expression of ChAT and BDNF, the ultrastructural changes, Nissl body changes in hippocampus and cerebral cortex in neonatal rats as the model of cerebral H-I; so as to confirm the effects of EA on HIBD , investigate the possible mechanism, and provide the theoretical basis for clinical application.Methods: 61 healthy 7-d-old Wistar rats weighing 12-15g were randomly dividedinto three groups . Group I , Sham-operated control group(n=16): the normal rats only received the operation without H-I treatment. Group II, H-I group(n=23, only 19 rats up to the standard): the rats were subjected to permanent ligation of left side artery and exposured to hypoxia(8% oxygen in nitrogen) for 2 hours. Group III, H-I + EA group(n=21, only 19 up to the standard): from the second day after H-I, the rats were electroacupunctured on "Bai Hui" and "DaZhui" points synchronously, the needle handles were respectively connected to stimulative machine( 16Hz, 10V), the rats received 10 min treatment each day, each course contained 10 treatments, 2 d interval between two courses. 24h after hypoxia , three rats in group I and group II respectively were random put to death for TTC(2,3,5-Triphenyltetrazolium bromide) staining. 22 d Later, three rats were selected from each group and killed . The left side hippocampus from each rat was dissected out, the specimens were fixed in 4% glutaraldehyde solution, then routinely dehydrated, and embedded into epoxy resin 618#, ultrathin sections were prepared and stained by uranyl acetate and lead citrate, an electron microscope H-7500 was used for observation. The 42 rats left were subjected to perfiision-fixation 22 d later, the brains were regularly paraffin wax embedded, sectioned and stained by HE staining, Nissl staining, ChAT and BDNF immunohistochemical staining. The neuron structures were observed, the number of positive neurons counted under microscope and the grey scale values were measured, the data were statistically analysed with SPSS 10.0.Results:1. HE staining: the structure of neurons was clear in group I ; however, in group II, there were marked tissue swelling , neuronal degeneration and necrosis , the cellular structure was clouding, the nuclear chromatin distribution was not homogeneous; group III revealed decreased tissue swelling , neuronal degeneration and necrosis, the cellular structure was almost normal.2. ChAT immunohistochemical staining: ChAT positive neurons distributed widely in CA1, CA2, CA3 and dentate gyms subfields of group I (CA1: 29.74+4.85); the number of them declined markedly in the CA1 subfield of group II(13. 96+7.62),and as compared with group I , there was significant difference( p<0.05); increased markedly in group 111(25.54± 5.05), as compared with group II(p<0.05). There was no significant difference between group III and group I.3. BDNF immunohistochemical staining: BDNF positive neurons were less in the cortex and CA2 and CA3 subfields of group I(cerebral cortex: 14.14±6.11 , hippocampus: 13.42更多还原