【作者】 张凯；

【导师】 何志义；

【作者基本信息】 中国医科大学 ， 神经病学， 2002， 硕士

【摘要】 前言 脑梗死严重威胁着人类健康，探讨脑梗死的病理生理学机制，寻求有效的治疗手段成为研究的热点。随着认识的不断深入，人们提出了脑梗死边缘地带(半暗带)的概念和缺血再灌注损伤级联反应学说。脑缺血再灌注损伤机制包括相互承启、交互作用的四个机制：兴奋性毒性作用、梗死周围去极化、炎症、细胞凋亡。半暗带是无灌注的中心区和正常组织间的移行区，它不是静止的，随时间和治疗其大小发生改变。半暗带是级联反应起作用的地方。在以上研究中，脑源性神经营养因子(BDNF)的作用引起人们的重视。作为一种具有多种生物学功能的细胞因子，BDNF广泛参与脑梗死的病理生理过程。许多研究表明BDNF对限制脑缺血后分解代谢产物所产生的损伤级联反应有重要作用。国外一些学者研究了大鼠MCAO后BDNFmRNA的表达规律，但实验结果不尽相同。本实验采用大鼠大脑中动脉栓塞模型研究脑局灶缺血再灌注后BDNF的表达规律并探讨其意义。 材料与方法 健康雄性Wistar大鼠30只，体重280-320g，随机分成10组。正常对照组、假手术组各3只，缺血再灌注组24只，即缺血90分钟，再灌注15min、30min、60min、2h、4h、6h、12h、24h各3只。缺血再灌注组，按照改良的栓线法建立大脑中动脉缺血再灌注动物模型；假手术组，除不栓线外其余操作同缺血再灌注组；正常对照组不施行手术。上述30只动物分别在所需时间点给予10％水合氯醛1.5ml腹腔麻醉，左心室插管，肝素化盐水冲洗后，以4％多聚甲醛磷酸缓冲液(PH7.4)250ml左心室灌注，断头、取脑，自额极至枕叶分为A在人D在五等份。取C脑片，置4℃4％多聚甲醛缓冲液固定 24 ／J’时，常规脱水、浸蜡、包埋，并制成 7 pm厚冠状切片。采用过氧化酶标记的链霉卵白素染色法uP法人用免疫组化方法检测BDNF在脑组织中的表达情况，同时相应时间点的片子还做了HE染色及尼氏染色。 结 果 参考onsa及ne大rson的方法，于大鼠苏醒后zh进行5分制评分。评分标准对分，无神经功能缺损症状；1分，不能完全伸展对侧前爪；2分，向对侧划圈；3分，向对侧倾倒；4分，不能自发行走，意识清晰度下降；5分，死亡。为保证缺血再灌注损伤程度的一致性，取评分1上分者人选，其余舍弃。脑组织形态学观察：正常对照组和假手术组大脑半球外观正常8E染色镜下见双侧半球组织结构正常。缺血再灌注组：大体见额、领顶部颜色苍白J胀、脑表静脉充血，镜下见尾状核、大脑皮质呈不同程度的缺血改变，核深染固缩，胞体皱缩、胶质细胞增生、血管扩张，少量白细胞渗出，缺血中心区组织疏松，形似海绵状。尼氏染色见尼氏体溶解，虎斑消失。免疫组化染色结果显示：正常对照及假手术组仅在大脑皮层神经元有微弱的BDNF免疫阳性表达；缺血再灌注组在梗死中心区和梗死灶边缘带有显著增强的阳性表达，尤其在梗死灶边缘带增加更明显。梗死灶对侧的脑组织中仅有少量表达。所有脑组织血管中几乎都无表达。BDNF阳性细胞表达，于再灌注15min开始增多，再灌注lh明显增多，Zh达到高峰，4h开始下降，24h恢复至对照水平。 讨 论 本实验免疫组化染色显示：正常对照组及假手术组脑组织的大脑皮层仅有微弱BDNF阳性表达，缺血再灌注组在梗死中心区 ·2· 及梗死边缘带免疫阳性细胞显著增多，梗死边缘带增加尤为显著。 梗死灶对侧脑组织中则表达更少。提示正常脑组织中BDNF表达 很少，单纯手术操作对其表达无显著影响；但在遭受如缺血缺氧等 刺激时表达明显增加，表明BDNF参与了脑组织缺血缺氧损伤后 的病理生理过程。梗死边缘带高水平的BDNF表达可能是神经元 在低氧及代谢产物的刺激下诱导分泌增多之故。但在梗死中心 区，损伤过于严重，细胞不能发生有效反应，故梗死中心区 BDNF 低水平表达。本实验还发现梗死边缘带免疫阳性细胞主要是神经 元。瓶神经元是脑梗死后BDNF的主要来源。 脑缺血缺氧产生燃性氨基酸（主要是谷氨酸入激活NMDA 受体依赖性的Ca入通道，Ca卜大量内流另！起细胞内 Ca入超载，进 而损伤线粒体，促进自由基生成。上述反应被认为是脑缺血／再灌 注损伤的主要过程，最终导致神经元及其他细胞的坏死和凋亡。 BDNF表达后，在梗死区特别是梗死边缘带以自分泌和旁分泌方 式发挥生物学作用。BDNF能够对抗高浓度氨基酸的神经毒性。 BDNF能够下调NMDA受体功能，以此抵抗兴奋性氨基酸的毒性。 BDNF可通过诱导钙结合蛋白的表达而稳定细胞内 Cah浓度，从 而保护神经元抵抗损伤。BDNF’能使体外培养的神经元内超氧化 物歧化酶、谷眈甘肽过氧化物酶含量明显增加，可以使自由基积累 减少，减轻自由基对神经元的损伤。而且BDNF抑制脑缺血后诱 导型一氧化氮合酶的表达，并抑制胶质细胞的活性和巨噬细胞的 浸润，即能减少自由基的生成，还具有抗炎症作用。BDNF能通过 阻止Caspase－3的活更多还原

【Abstract】 Today in most countries ischemic stroke does harm to people’ s health seriously. It becomes a hot topic to study the pathophysiologic mechanisms of ischemic stroke and search for efficient way to thera-peut it. Following the study carrying out , some people has pointed out that there is a rim of mild to moderately ischemic tissues ( ische-mic penumbra ) between the normal perfused brain and the evoking infarct. Its size varies according to the time and the therapy. The the-ory of ischemia - reperfusion injury contains four reactive mecha-nisms: excitotoxic injury and perrinfarct deplarization and inflamma-tion and apoptosis as well. The cascade of damage takes place mainly in the ischemic penumbra.In the course of these studies, many people pay attention to Brain -Derived Neurotrophic Factor( BDNF ) with multifunction, which is associated with lots of physiologic and pathophysiologic processes in bodies.Many studies suggested that BDNF can limit the cascade of dam-age following brain ischemia, up to now, people have studied the ex-pression of BDNF mRNA in MCAO models. But the results did not a-gree with each other.In our experiment we studied the expression of BDNF protein inMCAO rat model and discussed the protective machenisms.Material and MethodThere are 30 male Wistar rats, and they weight 280 - 320g re-spectively. They were divided randomly into three groups. Normal control group: three rats, sham - operated group three rats, ischemic - reperfusion group: twenty ?four rats, they were further divided into eight subgroups evenly at different time point such as 15min, 30min, 1h, 2h, 4h, 6h, 12h, 24h reperfused after suffering ischemia ninety minutes. The rats in ischemia - reperfusion group were operated ac-cording to the improved MCAO. The rats in sham - operation were op-erated as the rats in ischemia - reperfusion, except of not pulling the mononalon line into the EGA. Rats in normal control group were not operated at all.The brain was divided into five portions evenelly. The third chop were fixed. The method of SP immunostaining was used to study the expression of BDNF protein.ResultOnly a few neurons in the cerebral cortex show a weak labeling after staining with BDNF antiserum in normal controls and sham ?ope rated controls. In MCAO Group: the most marked up -regulation oc-cus in neurons with the infarcts and the border zone . Neurons in the border zones showed a moderately intense expression. Signs of BDNF up - regulation also seemed to occur in the contralateral hemisphere. 15 min after reperfusion, a diffuse expression of BDNF was found, which further intensified until Ih. The peak levels of BDNF were found 2h. Beginning to decline at 4h. The levels of BDNF hadreached control level at 24h.DisscussionIn our study immunohistochemical findings demonstrated that only a few neurons show a weak labeling after staining with BDNF antise-rum in normal controls and sham - operated controls. BDNF was in-creased in infarct or penumbra ( especially in the border zones) versus normal contralateral hemisphere. It suggests that few BDNF expression in normal brain tissues and that operation dont have effect on BDNF expression. BDNF expression up - regulates evidently following suffer-ing from hypoxia - ischemia stimulation. It demonstrated that BDNF protein is related to the pathphysiologic processes after the hypoxia -ischemia injury. An explanation for the high levels of BDNF in the pe-numbra might be that hypoxia ?induced stress of neurons increase BDNF expression. If the stress is too severe, cells might not respond as efficiently (lower levels observed in infarcts). We found that the most immunochemical staining positive cells are neurons. The fact suggests that BDNF mainly derives from neurons.Hypoxia - ischemia injury induced a great deal of excitotoxicity glutamate. Inappropriate activation of glutamate receptors is thought to be responsible for the death of neurons following brain insults such as ischemia. It is widely believed that the deleterious effect更多还原