Abstract WP98: Microglia/macrophage Polarization Dynamics Reveal Novel Mechanism Of Injury Expansion After Focal Cerebral Ischemia

INTRODUCTION: Mononuclear phagocytes are highly plastic cells that assume diverse phenotypes in response to microenvironmental signals. The phenotype-specific roles of microglia/macrophages in ischemic brain injury are poorly understood. METHODS: Cerebral ischemia was induced in mice by unilateral middle cerebral artery occlusion (MCAO) for 60 min; animals were sacrificed on Day 1 to Day 14 of reperfusion. RT-PCR and immunohistochemical staining for M1 and M2 microglia/macrophage markers were performed to characterize phenotypic changes. In vitro experiments using a transwell system, a conditioned medium transfer system, or a coculture system allowing cell-to-cell contacts were employed to further elucidate the effect of neuronal ischemia on microglia polarization and, conversely, the effect of microglia phenotype on the fate of ischemic neurons. RESULTS: The levels of M1 specific genes (iNOS, CD11b, CD16, CD32 and CD86) gradually increased over time from day 3 onwards and remained elevated for at least 14 days after ischemia. In contrast, the expression of M2 genes (CD206, Arg1, CCL22, Ym1/2, IL-10 and TGFβ) was induced since day 1, began to drop at day 7 and returned to pre-injury levels by day 14 after MCAO. Double immunofluorescent staining of microglia/macrophage marker Iba-1 with CD206 or CD16/32 confirmed that Iba-1+ cells assume the M2 phenotype at early stages of ischemic stroke but gradually transformed into the M1 phenotype in peri-infarct regions. In vitro experiments revealed that ischemic neurons prime microglial polarization towards M1 phenotype. M1-polarized microglia or M1-conditioned media exacerbated oxygen glucose deprivation (OGD)-induced neuronal loss. In contrast, maintaining the M2 phenotype of microglia protected neurons against OGD. CONCLUSIONS: Our results suggest that microglia/macrophages respond dynamically to ischemic injury, experiencing an early "healthy" M2 phenotype followed by a transition to a "sick" M1 phenotype. These dual and opposing roles of microglia/macrophages suggest that stroke therapies should be shifted from simply suppressing microglia/macrophage towards adjusting the balance between beneficial and detrimental microglia/macrophage responses.