Protective mechanisms of ischemic postconditioning

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 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.