Protective mechanisms of ischemic postconditioning

 描述（由申请人提供）：中风是美国的主要死亡原因，中风后幸存的人往往会出现严重的长期残疾。由于没有神经保护剂可用于治疗中风，因此迫切需要可以恢复失去的功能的新疗法。中风的主要类型是局灶性脑缺血，这是由大脑中的动脉阻塞引起的。原发性脑损伤在缺血发作后立即发生，继发性损伤由再灌注引起，此时血流恢复到阻塞的动脉。再灌注损伤的特征在于炎症，其涉及缺血脑中巨噬细胞（MΦ）的募集。缺血后处理（IPostC），或再灌注的机械中断，是一种新兴的和有前途的神经保护策略，但IPostC的潜在保护机制在很大程度上是未知的。由于再灌注损伤涉及MΦs，并且IPostC中断再灌注，因此研究者假设MΦs可能参与IPostC的保护作用。此外，缺血性脑中的MΦ包括驻留的小胶质细胞衍生的MΦ（MiMΦ）和血液单核细胞衍生的MΦ（MoMΦ），但尚未研究它们在IPostC中的潜在独特作用。此外，MΦ被极化成促炎性M1形式和抗炎性M2形式，但尚未研究M1和M2极化的MΦ如何参与IPostC。因此，研究人员将使用新的方法来研究MΦs的抑制和M1/M2极化的改变如何有助于IPostC的保护作用。首先，他们在小鼠中建立了一种新的IPostC模型，使他们能够使用各种遗传修饰的小鼠品系和抗体研究MΦ。其次，他们将使用荧光激活细胞分选（FACS）技术来识别，定量和分选MoMΦ和MiM Φ。第三，他们将使用尖端的高通量Fluidigm（r）BioMark HD系统，一种实时PCR技术，来测量纯化的MoMΦs和MiMΦs的基因表达。初步数据已经表明：（1）IPostC抑制MoMΦs的积累，但对MiMΦs的影响较小;（2）外源性MoMΦs，而不是常驻MiMΦs，具有最强的M1和M2基因表达;（3）MoMΦ耗竭导致野生型（WT）小鼠中较小的梗死;（4）通过CCR 2/CCL 2抑制剂或通过CCR 2基因缺陷抑制缺血脑中MoMΦ的募集，分别减轻了WT和CCR 2基因敲除（KO）小鼠的脑损伤;（5）M1极化增大，而M2极化抑制梗死面积;（6）M2的损伤，特别是在MΦ条件性IL 4 R α基因KO小鼠中，导致更大的梗死面积。基于这些初步发现，研究人员因此提出了3个具体目标：（1）研究IPostC对急性缺血性脑损伤中MoM β和MiM β积累和极化的影响;（2）研究MoM β积累的抑制是否对IPostC对抗卒中的保护作用至关重要;（3）研究M β极化在IPostC介导的对抗卒中的保护作用中的作用。的 长期目标是推进IPostC和MΦ策略在卒中患者中的临床转化。

 DESCRIPTION (provided by applicant): Stroke is a leading cause of death in the United States, and those who survive stroke often live with serious long-term disabilities. With no neuroprotectants available to treat stroke, there is an urgent unmet need for new therapeutics that can recover lost function. The major type of stroke is focal cerebral ischemia, which is caused by a blocked artery in the brain. Primary brain injury occurs immediately after ischemic onset, and secondary injury is caused by reperfusion, when flow is restored to the blocked artery. Reperfusion injury is characterized by inflammation that involves the recruitment of macrophages (MΦs) in the ischemic brain. Ischemic postconditioning (IPostC), or the mechanical interruption of reperfusion, is an emerging and promising neuroprotective strategy, but the underlying protective mechanisms of IPostC are largely unknown. Because reperfusion injury involves MΦs, and IPostC interrupts reperfusion, the investigators hypothesize that MΦs could be involved the protective effects of IPostC. In addition, MΦs in the ischemic brain comprise both resident microglia-derived MΦs (MiMΦs) and blood monocyte-derived MΦs (MoMΦs), but their potential unique roles in IPostC have not been studied. Moreover, MΦs are polarized into a pro-inflammatory M1 form and an anti-inflammatory M2 form, but how M1 and M2-polarized MΦs are involved in IPostC has not been studied. The investigators will therefore use novel approaches to study how the inhibition of MΦs and the alteration of M1/M2 polarization contribute to IPostC's protective effects. First, they have established a new IPostC model in mice that enable them to study MΦs using various genetically-modified mouse strains and antibodies. Second, they will use the fluorescence-activated cell sorting (FACS) technique to identify, quantify and sort MoMΦs from MiMΦs. Third, they will use the cutting-edge high-throughput Fluidigm(r) BioMark HD system, a real-time PCR technique, to measure the gene expression of purified MoMΦs and MiMΦs. Pilot data have already shown that: (1) IPostC inhibited the accumulation of MoMΦs, but had less effect on MiMΦs; (2) exogenous MoMΦs, but not resident MiMΦs, had the strongest M1 and M2 gene expression; (3) MoMΦ depletion resulted in smaller infarctions in wild-type (WT) mice; (4) inhibition of MoMΦ recruitment in the ischemic brain by CCR2/CCL2 inhibitors or by CCR2 gene deficiency attenuated brain injury in WT and CCR2 gene knockout (KO) mice, respectively; (5) M1 polarization enlarged while M2 polarization inhibited infarct sizes; and (6) impairment of M2, specifically, resulted in larger infarctions in MΦ conditional IL4Rα gene KO mice. Based on these preliminary findings, the investigators therefore propose 3 Specific Aims: (1) to study the effects of IPostC on MoMФ and MiMФ accumulation and polarization in acute ischemic brain injury; (2) to study whether inhibition of MoMФ accumulation is critical for the protective effect of IPostC against stroke; an (3) to study the role of MФ polarization in IPostC-mediated protective effects against stroke. The long-term goal is to advance the clinical translation of IPostC and MΦ strategies for stroke patients.