【作者】 苗晶；

【导师】 冯加纯；

【作者基本信息】 吉林大学 ， 神经病学， 2014， 博士

【摘要】 缺血性白质损伤可以引起认知功能障碍，严重损害人类健康，其主要的发生机制有免疫炎性损害、氧化应激损伤、突触结构及功能异常等。目前研究的较多的为免疫炎性损伤机制。小胶质细胞作为中枢神经系统免疫吞噬细胞发挥着双重作用，少量被激活时，可以释放神经营养因子、吞噬废弃的组织碎片发挥保护性作用；大量被激活则产生炎性损害作用。CX3CR1作为小胶质细胞表面的一种受体分子，在生理条件下，使小胶质细胞保持静息状态、维持内环境的稳定状态；在急性缺血、变性疾病、神经创伤时发挥着损害性作用。在缺血性白质损伤时，如何变化、起什么样的作用尚未有研究。p38MAPK是MAPK家族中重要成员，主要参与炎性应激反应。p38MAPK是否参与CX3CR1介导的缺血性白质损伤的发病过程目前还不清楚。目的：观察CX3CR1是否参与了慢性缺血性白质损伤发病过程；CX3CR1对慢性缺血性白质损伤是否通过p38MAPK途经实现。方法：雄性Wistar大鼠320只，随机分为正常组、假手术组、缺血组、阴性对照组、干预组，Morris水迷宫观察造模后28d学习记忆功能。造模后不同时间点（1d、3d、7d、14d、28d），HE染色及Luxol Fast Blue（LFB）染色法评价胼胝体、内囊、视神经纤维变化；Western Blot观察CX3CR1、P-p38/p38蛋白表达；免疫荧光双标技术观察CX3CR1与小胶质细胞关系；免疫组织化学染色评价小胶质细胞变化规律；侧脑室内注射不同浓度CX3CR1中和抗体、p38抑制剂进一步观察缺血后上述指标的变化。结果：（1）造模后28d，缺血组逃避潜伏期、空间探索路径较正常组延长，跨越平台次数显著减少（P<0.01）。（2）随着缺血时间的延长，HE染色观察到胼胝体、内囊及视神经区域白质纤维疏松，排列紊乱且空泡样改变；LFB染色可见上述区域髓鞘崩解范围扩大，分层明显，部分髓鞘出现空泡状；CX3CR1表达量持续增加，与CD11b标记的小胶质细胞数目持续增多相一致且高于假手术组和正常组（P<0.01）。（3）随着缺血时间的延长，P-p38/p38比值在缺血3d时出现明显变化，与CX3CR1变化具有相关性。（4）不同浓度CX3CR1中和抗体对缺血性白质损伤的认知功能、纤维损伤程度、CD11b小胶质细胞数目及P-p38/p38比值均有改善作用，且呈现剂量依赖性。应用p38抑制剂后，缺血性白质的纤维损伤程度及小胶质细胞数目均有不同程度改善。结论：（1）CX3CR1过表达通过介导小胶质细胞的变化对缺血性白质产生损伤作用，进而影响空间学习记忆功能。（2）CX3CR1/p38MAPK信号通路是缺血性白质损伤发生机制之一。更多还原

【Abstract】 White matter lesions (WMLs) induced by chronic cerebral hypoperfusion (CCH) havebeen suggested to contribute to cognition impairment, which causes serious damage to humanhealth. Inflammation, oxidative stress and dysfuction of synatic structure are involved in thepathologic progress. For many years, increasing evidence indicates that inflammation plays acritical role in the pathogenesis of WMLs. In WMLs, microglia plays a dual role in diseaseprogression, being essential for clearing necrotic tissues and releasing of neurotrophic factors.Once the number of actived microglia is high, it can damge the normal tissue by producingsome inflammatory factors. CX3CR1, exclusively expressed by microglia in the brain,contributes to the ability of microglia to maintain a resting phenotype. CX3CR1has an effecton some disorders, such as acute ischemia, degenarative diseases and nerve damages.However, there is no study conferring to CX3CR1involved in the WMLs after CCH.p38MAPK, one of the important members in the MAPK families, is mainly involved ininflammation. It is unclear whether p38MAPK is involved in the process of WMLs inducedby CX3CR1after CCH.Objective: The purpose of our study was to examine the relationship of CX3CR1withWMLs in CCH; to further study p38MAPK in the pathogenesis of CX3CR1invovled inWMLs. Methods: CCH was induced by permanent occlusion of bilateral common carotidarteries (BCCA) in male Wistar rats.320rats were randomly divided into five groups asfollows:(i)normal;(ii)sham-operation group;(iii)rats exposed to BCCA contribute to CCH;(iv)rats received control anti-IgG antibody or DMSO then exposed to BCCA;(v) rats receivedlateral ventricle stereotaxic injection of anti-CX3CR1neutralizing antibody or a p38inhibitorthen exposed to BCCA. Performance of Morris water maze task and the relationship ofCX3CR1with CD11b are analyzed at28d after CCH. Hematein Eosin(HE) and Luxol FastBlue(LFB) for white matters(corpus callosum, internal capsule and optic nerve), Western Blotfor CX3CR1and p38MAPK, immunohistochemistry for the progress of microglia areanalyzed at different times(1d,3d,7d,14d,28d) after post-hypoperfusion. All of above objectives were measured after post-hypoperfusion with lateral intraventricular injection ofdifferent concentration CX3CR1neutralizing antibodies or a p38inhibitor. Results：(1) Onthe28thafter surgery, compared with the control group, BCCA group significantly increasedthe mean escape latency, path length and reduced the numbers that the rats crossed over theposition where the platform located(P<0.01).(2) HE staining showed fibers lossen,disorganization and vacuolus in the corpus callosum, internal capsule and optic nerve. LFBstaining showed that increased number of vacuoles and demyelinated fibers in the abovewhite matters. A gradual increase in CX3CR1protein levels was observed in white mattersand CD11b marked activated microglia was almost at similar levels in these areas(P<0.01).(3)The ratio of P-p38/p38in white matters was significantly higher at the3rdafterpost-hypoperfusion, which had relationship with the alternation of CX3CR1.(4)Theadministration of anti-CX3CR1neutralizing antibodies, in which therer is an optismconcentrtion, alleviated behavior, white matters, microglia activation and the alternation ofP-p38/p38in rats with CCH. The administration of a p38inhibitor alleviated white mattersand microglia activation. Conclusion：(1) CX3CR1over expression has a bad effect byaffecting microglia on white matters after CCH, which plays an important role in learning andmemory.(2) CX3CR1/p38MAPK signaling is one of the pathogenesis in the WMLs afterCCH.更多还原