【作者】 严乐勤；

【导师】 魏尔清；

【作者基本信息】 浙江大学 ， 生理学， 2002， 博士

【摘要】 脑缺血疾病是多发的中枢神经病变，是常见的致死致残原因，在人口老龄化的现代社会，其防治意义越来越重要，本类疾病的早期发现和治疗可以极大程度地降低致死致残率。故研究其损伤／抗损伤机制，探索其防治措施，具有重大的意义。在过去的10～15年越来越多的证据表明兴奋性毒性是缺血性脑损伤的最主要发病机理。NMDA受体在介导急性谷氨酸兴奋性毒性中起了关键性作用，NMDA受体的拮抗剂的使用能降低缺血性或低糖脑损伤动物模型中的神经元死亡。许多实验室都证明阻断了NMDA受体能降低短暂或永久性局灶性缺血模型的梗死面积。 本实验室在脑缺血损伤研究中，已建立了整体局灶性脑缺血(大脑中动脉阻塞，MCAO)和全脑缺血(四血管阻断，4VO)模型。在局灶性脑缺血模型中，已经发现半胱氨酰白三烯受体拮抗剂ONO-1078(pranlukast)对脑缺血损伤有保护作用。为了更加深入地研究脑缺血损伤机制，探讨脑缺血损伤中起主要作用的兴奋性氨基酸毒性 一 机制及其治疗药物，本研究进一步建立了脑片和神经元水平的体外脑缺血模型；探讨 了新类型N’MD损伤对抗药物；还对NMD的信号转导特点作了分析。希望通过本 研究，能够阐明体外NMDA损伤特点及发现抗脑缺血新药的途径；并且，为今后药 物干预兴奋性氨基酸信号通路打下必要的基础。0 l．离体脑片损伤模型（OGD和 NMDA）及原代培养神经元 NMDA损伤 大鼠离体海马脑片缺氧缺糖（OGD）损伤 大鼠离体海马脑片制备后，在通氧混合气的正常脑脊液（nACSF）中恢复 60 min， 然后移入通氮混合气的无糖脑脊液（吵化SF）中缺氧缺糖，取出脑片与2％TTC避光 37’C温浴 60 dll，染色后根据每克湿重加入 20 ml抽提液（乙醇：二甲亚矾一 50：50）， 在密闭容器内避光置 24 h，测量前摇匀后取 200 PI至 96孔板，在 490 urn波长，酶 标仪测定各孔OD＿值。另一组平行实验测得各组脑片上清和匀浆LDH，计算LDH 释放率。结果发现，随着缺氧缺糖时间的延长脑片hC染色逐渐下降，LDH释放率 也逐渐增高，TTC染色下降百分率和LDH释放百分率呈显著负相关（r。－0．933）。 提示了本实验首次建立的分光光度法检测hC还原产物来评价大鼠海马脑片的缺氧 缺糖性损伤的方法，是一种简便快速、切实可行的方法，可用于评价神经保护药物的 作用。 小鼠皮层－纹状体离体脑片OGD模型及NMDA损伤模型的建立 小鼠皮层．纹状体离体脑片制备后，恢复 60 min，然后进行缺氧缺糖处理或加入 NMDA处理。取出脑片与2％TTC避光37’C温裕60 min，经甲醛固定后，在体视显 微镜下以CCD摄像头摄取并输入计算机保存24位RGB彩色图像。用本实验室开发 的脑缺血图像分析软件计算皮层、纹状体的nC染色面积，并计算染色面积百分率。 二 — — 然后将RGB图像转换为8－位的平均灰度模式，计算皮层、纹状体区的hC染色平均 灰度，并计算平均灰度百分率。结果发现，OGD时间越长，皮层、纹状体TTC染色 的平均灰度和面积越小。NMDA剂量依赖性损伤脑片，导致TTC染色平均灰度明显 下降。提示了用TTC染色方法结合计算机图像分析的评价方法为离体脑片急性损伤 及神经保护药作用的评价提供了另一种简便、客观、定量和可行的评价方法，在脑缺 血或其他脑损伤研究中，有推广应用的价值。 大鼠皮层神经元NMDA损伤模型的建立 原代培养的皮层神经元体外10天（DfV10）时，用神经元特异的烯醇化酶抗体进 行免疫组化鉴定，证明所培养的是神经元。神经元用不同浓度的NMDA，处理不同 时间，观察神经元的损伤情况。我们观察到NMDA对皮层神经元的毒性作用有明显 的剂量依赖关系，随着N’MDA浓度的增加和作用时间的延长，其毒性作用增强。我 们选择了 50 u mol／L NMDA处理 15 nun为损伤模型。 2．药物对OGD和NMDA损伤的保护作用 用大鼠离体海马脑片缺氧缺糖（OGD）模型和分光光度法检测 nC还原产物的评 价方法，我们首先对几种常用的抗脑缺血药物：尼莫地平、地塞米松、氯肢酮作了观 察，发现尼莫地平、氯胺酮和地塞米松都能显著提高缺氧缺糖海马脑片的TTC染色。 但整体动物实验有效的半肮氨酞白三烯受体桔抗剂ONO－1078对脑片OGD损伤无保 护作用。我们还首次发现了经典的抗精神病药氟吸陡醇对大鼠海马脑片缺氧缺糖损伤 的保护作用，同时也发现了氟嗓唆醇对大鼠海马脑片NMDA损伤的保护作用。但多 巴胺不影响正常脑片的活性，及D。多巴胺受体特异的桔抗剂多潘立酮对大鼠海马脑 片缺氧缺糖损伤没有保护作用提示了氟赈唆醇的保护作用与多巴胺D更多还原

【Abstract】 Cerebral ischemia is one of common CMS diseasie with high incidence and is the cause leading to death and disablement. Its prevention and treatment is becoming more and more important in the modern society because of the increasing of old people. Effective treatments at early stage of this disease may decline the rate of death and disablement to a minimum. So, it is very important to study the damage/anti-damage mechanisms and to explore the preventive ways. Over the past 10-15 years, substantial evidence has accumulated implicating excitotoxicity in the pathogenisis of ischemic brain injury. NMDA receptors are crucial in acute glutamate neurotoxicity, NMDA-antagonists could reduce neuronal death in animal models of ischamic or hypoglycemic brain injury. Many laboratories havedemonstrated that NMDA-receptor blockade reduces infarct volume in both lissencephalic and gyrencephalic animal models of transient or permanent focal ischemia.Permanent focal cerebral ischemia and global ischemia models have been established in our laboratory recently. Also, we have found that ONO-1078, a selective cysteinyl-leukotriene receptor antagonist, is protective against ischemic brain injury due to the permanent focal cerebral ischemia. To study the mechanisms of ischemic brain injury the excitotoxicity more thoroughly, and the therapeutic approach of ischemic brain injury, we in this research established the rational in vitro models of brain slice and primary neuronal cultures, searched new NMDA receptor antagonists, and analyzed the characteristics of signal transduction triggered by NMDA. We hope to elucidate the characteristics of injury induced by NMDA in vitro and find potential important targets for therapeutic invention for cerebral ischemia.1. In vitro injury models of brain slice (OGD and NMDA insult) and primary neuronal cultures(NMDA insult)Oxygen/glucose deprivation (OGD)-induced injury of rat hippocampal slice in vitroThe rat hippocampal slices prepared were allowed to recover in the normal artificial cerebrospinal fluid(ACSF) bubbled with gas mixture of 95% O2+5% CO2 for 1 h, then they were thansfered to glucose-free nACSF which was bubbled with gasmixture of 95%N2+5%CO2. After treatment with OGD, the slices were placed into 2% TTC solution in dark and incubated at 37*Cfor 1h. The slices were weighted and a 50 : 50 mixture of ethanol/dimethyl sulfoxide was then added to extract the formazan in dark for 24 h. For analysis, 200 y I aliquots of the red solvent extract were placed in 96 well plate. Average absorbance was read at x 490nm in Automated Microplate Reader. In a second experiment, the released LDH due to OGD insult was measured. We found that with the time prolongation of OGD, TTC staining of the hippocampal slices was reduced and LDH release rate was increased consistently. The percent decrease of TTC staining was significantly correlated (r = -0.933) to the percent increase of LDH release due to OGD insults. Our results indicate that the solvent extraction and spectrophotometric quantification of formazan has potential utility as an objective way to index OGD-induced injury of brain slices, it is a simple and reliable method for screening neuroprotective drugs.OGD and NMDA-induced injury of mouse corticostriatum slice in vitro The mouse corticostriatum slices prepared were allowed to recover for 1 h, then subjected to OGD or NMDA. After treatment, the slices were incubated in 2% TTC solution in dark at 37"Cfor 1h, then fixed in fomaldehyde, taken photographs with CCD camera under microscope, the computer imaging was utilized to assess the intensity and area of TTC staining. We found that with the time prolongation of OGD, the average intensity and area of TTC staining decreased consistently. The average area of TTC staining also decreased consistently after NMDA insult, and inmixture of 95%N2+5%CO2. After treatment with OGD, the slices were placed into 2% TTC solution in dark and incubated at 37*Cfor 1h. The slices were weighted and a 50 : 50 mixture of ethanol/d更多还原