Protective mechanisms of ischemic postconditioning

Stanford University, Stanford CA

Investigators

Linked publications, trials & patents

Abstract

? DESCRIPTION (provided by applicant): Stroke is a leading cause of death in the United States, and those who survive stroke often live with serious disability, but no neuroprotectants are available to treat stroke. The major type of stroke is focal cerebral ischemia caused by a blocked artery. Primary brain injury occurs immediately after ischemia onset, and secondary injury is caused by reperfusion, when flow is restored. Reperfusion injury is characterized by inflammation that involves recruitment of macrophages (MFs) in the ischemic brain. Ischemic postconditioning (IPostC), or the mechanical interruption of reperfusion, is an emerging and promising neuroprotective strategy, but little is known about its underlying protective mechanisms. Because macrophages (MFs) are critical for reperfusion injury, and IPostC interrupts reperfusion, the investigators hypothesize that MFs could be important for the protective effects of IPostC. MFs in the ischemic brain comprise resident microglia-derived MFs (MiMFs) and blood monocyte-derived MFs (MoMFs), but their roles are controversial. Recruitment of exogenous MoMFs is modulated by the chemokine receptor CCR2 and its ligand CCL2. Functionally, both types of MFs polarize to two extreme forms, a pro-inflammatory M1 form and an anti-inflammatory M2 form, and many subsets exist between these two extremes. Thus, MF phenotypes and subsets are complex, and with few techniques to distinguish between them, progress toward understanding their role in ischemic brain injury has been slow. The investigators will use novel approaches to overcome these barriers. First, they have constructed a new mouse IPostC model that will enable the study of MFs using various genetically-modified mouse strains. Second, they will use flow cytometry to identify, quantify and sort MoMFs and microglia/MiMFs (As microglia and MiMFs are indistinguishable by flow cytometry, they are referred together as microgia/MiMFs). Third, they will use the cutting-edge high-throughput Fluidigm(r) BioMark HD system, a real- time PCR technique, to measure for the first time gene expression of MoMFs and microglia/MiMFs at purified or single cell levels. The investigator's pilot data has shown that: (1) exogenous MoMFs, but not resident MiMFs/microlglia, are the major cell types with strong M1 and M2 gene expression; (2) MoMF depletion resulted in smaller infarctions in wild-type mice; (3) inhibition of CCR2/CCL2 and deficiency of CCR2 attenuated brain injury in wild-type and CCR2 gene knockout (KO) mice, respectively; and (4) M1 polarization enlarged while M2 polarization inhibited infarct sizes. Taken together, exogenous MoMFs and M1/M2 polarization have crucial roles in ischemic brain injury. The investigators therefore propose three Specific Aims: (1) to investigate the effects of IPostC on MoMF and microglia/MiMF accumulation in the ischemic brain and their gene expression/polarization profiles; (2) to test if CCR2/CCL2-modulated MoMFs are involved in the protective effects of IPostC; and (3) to investigate how M1/M2 polarization affects IPostC's protective effects. The long-term goal is to advance the clinical translation of IPostC and MF strategies to stroke patients.