【作者】 黄明；

【导师】 胡波；

【作者基本信息】 华中科技大学 ， 神经病学（专业学位）， 2015， 博士

【摘要】 [目的]缺血性脑卒中后,CXCL12/CXCR4信号通路在炎症细胞趋化至损伤区域过程中起着关键性的作用。小胶质细胞/巨噬细胞作为卒中后重要的炎症细胞,被激活后的不同极化表型对炎症反应的影响受到了广泛的关注。我们在此基础上探讨,调控CXCL12/CXCR4轴是否可通过影响不同极性的小胶质细胞/巨噬细胞,从而抑制卒中后的炎症反应。[方法]采用成年雄性(Sprague-Dawley, SD)大鼠建立永久性大脑中动脉闭塞(permanent middle cerebral artery occlusion, pMCAO)模型。pMCAO后1小时及随后的每12小时注射AMD3100,共5次。然后,在pMCAO后不同时间点对梗死体积进行了评估。免疫荧光染色观察pMCAO后AMD3100对脑组织中不同极性的小胶质细胞/巨噬细胞的影响。糖氧剥夺(Oxygen-glucose deprivation, OGD)模型用来模拟体外缺血条件,应用Transwell实验来研究调控CXCL12/CXCR4信号通路对不同的极性小胶质细胞迁移的影响。实时荧光定量PCR法检测缺血周边区(IBZ)分泌的相关细胞因子。nRNA的表达。应用MTT比色法和定量MAP-2酶联免疫吸附实验,检测不同极性小胶质细胞对神经细胞活性的影响。[结果]体内实验中,急性期给予AMD3100治疗减少了pMCAO舌的梗死体积。显著降低了活化的小胶质细胞/巨噬细胞数量的增加,而并未影响其活化状态。缺血损伤激活了几乎所有受影响区域的小胶质细胞/巨噬细胞,并诱导其M1和M2两种不同的极化表型的增加。缺血边界区(ischemic border zone, IBZ)大多数活化的小胶质细胞/巨噬细胞为M1型,经急性期给予AMD3100治疗后显著减少了迁移至IBZ的M1型的数量。同时,降低了脑组织缺血周边区域促炎细胞因子,包括肿瘤坏死因子(Tumor necrosis factor α, TNF-α)、诱导型一氧化氮合酶(inducible Nitric Oxide Synthase, iNOS)等的基因表达水平。此外,缺血损伤后,几乎所有的小胶质细胞/巨噬细胞均有表面受体CXCR4的表达。在体外实验中,OGD条件下AMD3100通过拮抗CXCL12/CXCR4通路对M1和M2型小胶质细胞的迁移,产生了不同程度的抑制作用。M1型小胶质细胞可能通过分泌相关细胞因子而抑制神经元的存活。[结论]缺血性脑卒中急性期给予AMD3100,通过拮抗CXCL12/CXCR4言号通路,主要抑制了M1型的小胶质细胞/巨噬细胞迁移至缺血周边区域,从而减轻炎症反应并改善神经功能恢复。调控招募至缺血周边区域的M1和M2型小胶质细胞/巨噬细胞之间的平衡,有可能为缺血性脑卒中提供一种潜在的治疗策略。[目的]为研究长期给予CXCR4受体拮抗剂AMD3100的治疗,是否会进一步改善脑卒中的预后。[方法]采用雄性SD (Sprague Dawley, SD)大鼠永久性大脑中动脉闭塞(permanent middle cerebral artery occlusion, pMCAO)建立脑卒中动物模型。用特定的抗体标记EPCs。连续7天给予AMD3100处理,观察对内源性内皮祖细胞(endothelial progenitor cells, EPCs)募集的影响。F1TC-葡聚糖染色用于毛细血管密度测定,神经行为学功能通过神经功能缺损评分进行评估。[结果]pMCAO后,经连续7天给予AMD3100处理后,缺血边界区(Ischemic boundary zone, IBZ)的内源性EPCs数量以及毛细血管密度显著减少,而且神经行为学功能的恢复,较生理盐水组更差。[结论1我们的研究表明,pMCAO后内源性的EPCs:经由CXCL12/CXCR4轴招募至IBZ。而且,经长期给予AMD3100处理后,可能会通过减少EPCs的募集,抑制血管新生,并不能改善神经功能预后,反而会加重组织损伤。更多还原

【Abstract】 [Purpose] CXCL12/CXCR4signal inhibitor AMD3100plays a vital role in inflammatory cell migration into the central nervous system (CNS) after ischemic stroke. Microglia/macrophages play a critical role in cerebral ischemia, and the effect of their polarization on inflammatory responses has received extensive discussions. We determine whether and how acute AMD3100treatment modulates polarized microglia/macrophages via CXCL12/CXCR4signaling to affect inflammation after ischemic stroke.[Methods] Adult male Sprague-Dawley (SD) rats underwent permanent middle cerebral artery occlusion (pMCAO) and AMD3100was injected at1hour and next12hours after pMCAO for five times. Infarct volume were assessed after pMCAO. Immunofluorescence was utilized to determine the action of AMD3100on the polarized microglia/macrophages in brain tissue after pMCAO. Oxygen-glucose deprivation (OGD) model was used to imitate the ischemic condition in vitro and the role of CXCL12/CXCR4signaling on migration of different polarized microglia was evaluated using a transwell system. Real-time PCR was used to detect mRNA expressions of cytokines secreted in the ischemic area. MTT colorimetric assay and quantitative MAP-2Enzyme-linked immunosorbent assay (ELISA) were used to evaluate the effects of microglia/macrophage phenotype on the fate of ischemic neurons.[Results] In vivo, acute AMD3100administration attenuated infarct volume and improved neurological outcome after pMCAO. AMD3100treatment in acute ischemia significantly attenuates the number of activated microglia/macrophages rather than impacting their activated state.Ischemic insults activates almost all microglia/macrophages in the attectea area and induces increase in M1and M2polarization. Also, in vivo, M1phenotype constitutes the majority of activated microglia/macrophages and AMD3100treatment significantly attenuates migration of M1phenotype into the ischemic border zone (IBZ), and declines the gene levels of pro-inflammatory cytokines including TNF-a, iNOS in ischemic region after pMCAO. In addition, ischemic injury largely up-regulates the expression of CXCR4in microglia/macrophages and CXCR4is primarily expressed in microglia/macrophages. While under OGD condition, AMD3100significantly suppresses the migration of both M1and M2microglia through inhibition of CXCL12/CXCR4signaling under ischemic condition. Ml microglia inhibited neuronal survival under pathological conditions via soluble factors.[Conclusions] Treatment with AMD3100in acute phase of ischemic stroke suppresses M1microglia/macrophages migration to ischemic tissue through inhibition of CXCL12/CXCR4signaling, and, attenuates inflammation, improves neurological outcome after ischemic stroke. Balancing M1and M2microglia/macrophages recruited to ischemic region may provide potential therapies after ischemic injury. [Purpose] To study whether prolonged CXCR4antagonist AMD3100treatment in acute phase of stroke could improve the outcome after injury.[Methods] Male SD rats were used to establish the permanent middle cerebral artery occlusion (pMCAO) models and specific markers were utilized to identify EPCs. CXCR4antagonist AMD3100was administrated for consecutive seven days just after pMCAO and then its role in the recruitment of endogenous EPCs was evaluated. Capillary density was determined with FITC-dextran staining, and neurobehavioral function was assessed by neurological function.[Results] After AMD3100treatment for seven consecutive days, the number of endogenous EPCs and capillary density in ischemic boundary zone (Ischemic boundary zone, IBZ) of rats after pMCAO were both significantly reduced, and neurobehavioral function recovery was worse than the saline group.[Conclusions] Our study suggested that after pMCAO, endogenous EPCs were recruited to the IBZ via CXCL12/CXCR4axis. And prolonged administration of AMD3100at acute phase could inhibit angiogenesis in the IBZ and rather aggravate the outcome after stroke.更多还原