远隔缺血后适应治疗缺血性卒中的骨髓干细胞动员机制

As a therapeutic strategy, remote ischemic postconditioning holds great promise for ischemic stroke, but the underlying mechanisms yet remain unclear, hindering its translation from bench to beside. Here, we will specifically explore the role of repeated remote ischemic conditioning in therapeutic mobilization of bone marrow stem cells for brain remodeling after ischemic stroke in mice.. Two hours after ischemic stroke caused by distal middle cerebral artery occlusion (dMCAo), mice will receive remote bilateral limb ischemic postconditioning (RIPC), or granulocyte colony-stimulating factor (G-CSF) injection as positive control, or half-dose of G-CSF combined with RIPC, and daily afterward for 7 days. Behavior tests and cerebral blood flow by laser speckle will be monitored daily. On post-ischemic consecutive days (d1~d7), circulating bone marrow stem cells in the peripheral blood, and associated chemoattractants in the bone marrow/peripheral blood/brain will be measured by FACS and ELISA respectively. In addition, the recruitment of bone marrow cells into the brain tissue, as well as the number and proliferation ratio of bone marrow nucleated cells per femur will be assessed by immunohistochemistry. On d7, the brain infarct size, the vascular neural network remodeling including angiogenesis and neurogenesis, and the neurotrophic factors in cerebral homogenate will be determined.. Altogether, from the perspective of mobilization of bone marrow stem cells, we aim to reveal the mechanism and benefit of limb ischemic postconditioning for behavioral recovery and brain plasticity in ischemic stroke, thereby providing key laboratory data for future application of remote ischemic postconditioning in stroke patients.

作为一种抗缺血损伤的内源性保护策略，远隔缺血后适应为治疗缺血性脑卒中提供了新方法。机制未明是阻碍其转化的关键问题。本项目将从骨髓干细胞动员的视角探讨远隔缺血后适应脑修复机制。. 我们采用大脑中动脉远端闭塞法制备小鼠急性脑缺血模型。动物脑缺血后立即给予首次远隔肢体缺血适应，每天1次，共7天。同时设置粒细胞集落刺激因子（G-CSF）阳性对照组，半剂量G-CSF联合缺血适应组，模型对照组和假手术组等。观察指标：1、每天动物神经行为学评分，激光散斑检测脑血流；2、每天流式细胞术检测外周血中骨髓干细胞数量，ELISA检测骨髓、外周血、脑的趋化因子，免疫化学法检测脑区骨髓干细胞定植，股骨骨髓细胞计数及增殖检测；3、第7天检测动物脑梗死体积，免疫病理检查脑血管新生和神经发生，ELISA检测脑匀浆神经营养因子。. 本课题试图揭示重复远隔缺血后适应的骨髓细胞动员作用及脑修复效果，为临床转化提供实验依据。

作为一种强大的机体内源性对抗缺血损伤的保护策略，远隔缺血后适应为防治缺血性脑血管病提供了新思路。但机制未明，是阻碍其临床转化的关键问题。本项目将研究：1,远隔缺血适应对急性脑缺血损伤后软脑膜侧枝血供代偿的影响；2,从骨髓干细胞动员的视角探讨肢体缺血后适应脑保护的起效机制。. 我们采用三血管闭塞小鼠缺血性脑卒中模型( 3VO, 15 min短暂夹闭双侧颈总动脉合并永久电凝右侧大脑中动脉远端)模拟人类缺血性脑卒中。实验组动物造模即刻起给予双下肢缺血适应(RIPC)，每日1次，共28天。同时设置粒细胞集落刺激因子（G-CSF）阳性对照组，以及模型对照组和假手术组。. 在动物脑缺血24h我们发现：(1)RIPC和G-CSF治疗组小鼠较模型对照小鼠的行为学评分有明显改善(-0.2 and -0.1 vs -0.7)，脑梗死体积明显减小 (31.8% and 26.4% vs 45.4%)；(2)RIPC和G-CSF治疗促进单核/巨噬细胞在软脑膜的浸润和侧枝血运的代偿；(3)预先 4 天腹腔注射 5-氟尿嘧啶(5-FU) (150 mg/kg)消耗掉单核/巨噬细胞后，RIPC和G-CSF的脑保护作用被削弱/消除；(4)ELISA发现脑缺血后小鼠外周血和患侧脑组织的 mG-CSF含量都有明显增高，而 RIPC和G-CSF干预消除了这个趋势；(5)蛋白免疫印迹发现脑缺血损伤后促血管新生因子Ang-2表达明显升高，而RIPC和G-CSF干预组与模型组比较未显示明显差异。以上数据提示，远隔缺血后适应能够提高急性脑缺血损伤后缺血脑区的侧枝血液供应，从而产生脑保护作用。. 此外，本课题尚在依任务书进行肢体缺血后适应的骨髓干细胞动员作用及规律相关实验，所获结果将丰富远隔缺血后适应在缺血后中风后治疗性血管新生方面的应用价值，为肢体缺血适应的临床转化提供实验依据。