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Introduction: Ischemic stroke results in activation of microglia, which may polarize towards a pro-inflammatory (M1) phenotype and/or an anti-inflammatory (M2) phenotype. Enhancer of zeste homolog (EZH) 2 is a histone-lysine N-methyltransferase enzyme, a key modulator of microglia polarization. We here investigated whether microglial-specific deletion of EZH2 leads to a beneficial protective effect in stroke and microglial over-expressing microglial EZH2 exacerbates stroke outcome in vivo . Methods: Aged male mice were subjected to 60-minutes middle cerebral artery stroke. Tamoxifen administration was started 30 days prior to stroke to induce genetic deletion of microglial EZH2 in CX3CR1-creER/EZH2-floxed mice. EZH2 floxed mice were used as controls. Microglial EZH2 over-expressing was performed using lentiviral vectors. Mice were sacrificed for immunohistochemistry and crystal violet staining (brain infarct assay) after behavior tests at 3 days after stroke. Results: The expression of microglial EZH2 was significantly abrogated with tamoxifen injection in KO mice compared to the control floxed mice (144±15.43 vs. 50.65±4.99 cells/mm 2 , P2 , P2 , P Conclusions: Genetic deletion of EZH2 in microglia improved stroke outcome in aged while overexpression of EZH2 exacerbated stroke outcome. The effect of protective effect of EZH2 inhibition may be due to limiting microglial M1 polarization and enhancing M2 polarization.

Introduction: Ischemic stroke results in activation of microglia, which may polarize towards a pro-inflammatory (M1) phenotype and/or an anti-inflammatory (M2) phenotype. Enhancer of zeste homolog (EZH) 2 is a histone-lysine N-methyltransferase enzyme. Accumulating evidence has suggested EZH2 is key modulator of microglia polarization by epigenetically regulating gene expression. We here investigated whether microglial-specific deletion of EZH2 leads to a beneficial protective effect in stroke in vivo . We further tested the therapeutical potential of an EZH2 inhibitor after stroke. Methods: Aged male mice were subjected to 60-minutes middle cerebral artery stroke. Tamoxifen administration was started 30 days prior to stroke to induce genetic deletion of microglial EZH2 in CX3CR1-creER/EZH2-floxed mice. EZH2 floxed mice were used as controls. EZH2 inhibitor GSK343 was I.P. injected (once a day for two consecutive days), starting three hours after stroke onset. Mice were sacrificed for immunohistochemistry and crystal violet staining (brain infarct assay) after behavior tests at 3 days after stroke. Results: The expression of microglial EZH2 was significantly abrogated in KO mice compared to the control floxed mice (144±15.43 vs. 50.65±4.99 cells/mm 2 , N=5/each group, P2 , N=4/each group, P2 , N=4/each group, P Conclusions: Both genetic deletion of EZH2 in microglia and pharmacological inhibition of EZH2 improved stroke outcome in aged. The effect may be due to limiting microglial M1 polarization and enhancing M2 polarization.

Introduction: Cerebral angiogenesis is correlated with post-stroke prognosis yet its mechanism is far from clear. Accumulating evidence shows that Rac1, a Rho-related GTPase, plays a central role in promoting neovascularization via activating Pak 1 in endothelial cells. We investigated whether activating endothelial Rac1 improved functional recovery and angiogenesis after ischemic stroke. Methods and Results: Young male mice were subjected to a 60-minute middle cerebral artery (MCAO) stroke. Endothelial Rac1 was overexpressed by brain injection of lentiviral vectors encoding Rac1 with endothelial promoter ENG the day after MCAO. Treatment improved the recovery of sensorimotor and cognition function assessed by single pellet reaching (day 7: p=0.042; day 14: p=0.012, n=7/group), adhesive removal (day 7: p=0.013, n=7/group) and novel object recognition test (day 21: p=0.045, n=7/group) compared to the control vector group. Follow-up immunofluorescence revealed that ENG-Rac1 promoted angiogenesis as measured by the endothelial proliferation (CD105+BrdU, p=0.016, n=5/group) and the elevation of pericyte (NG2, p=0.008, n=5/group), which is indispensable for stable vessel structure, in the peri-infarct zone, with no effect on brain cavity size. We further explored Rac1 signaling, involving Pak1, in mediating angiogenesis in vitro. Human brain endothelial cell line-5i (HEBC 5i) was subjected to a 16-hour oxygen-glucose deprivation (OGD) followed by re-oxygenation. Rac1 (NSC23766 at 30 μM) and/or Pak1 (IPA3 at 10 μM) inhibitors were incubated with HEBC 5i 24 hours after OGD. Delayed inhibition of Rac1 reduced endothelial proliferation (CCK-8 assay) at 48 hours (p=0.039, n=4/group) and the ability of migration (scratch assay) at 72 hours (p Conclusions: Activation of endothelial Rac1 improves post-stroke recovery and angiogenesis and this pro-angiogenetic effect is mediated by Pak1.

Axonal plasticity is critical for functional recovery after stroke. Our previous study suggested that Rac1, a small GTPase, contributes to post-stroke axonal regeneration. However, the specific cellular and molecular mechanisms mediating its effects remain unknown. The two major factors controlling axonal plasticity in the injured brain are the neuronal intrinsic property for outgrowth and astrocytic inhibition of axonal growth. We investigated the selective roles of neuronal and astrocytic Rac1 in post-stroke recovery and axonal regeneration. Stroke was induced by middle cerebral artery occlusion for 60 minutes in 7 to 8 week old male mice. Brain injection of lentivirus encoding Rac1 with neuronal (NSE) or astrocytic (GFAP) promotors was performed 7 days after stroke in wild type mice to overexpress neuronal or astrocytic Rac1. Tamoxifen administration was started 7 days after stroke in Thy1-cre/Rac1 floxed mice to induce genetic deletion of neuronal Rac1. Delayed overexpression of neuronal Rac1 promoted sensorimotor recovery assessed using a single pellet reaching test (% success rate) from 21 to 28 days after stroke (Day 28: GFP control 51.25±4.41 vs. NSE-Rac1 66.67±2.36, n=8-9, p

Brain endothelial cells play an essential role in BBB integrity and vascular recovery after injury. Accumulating evidence shows that Rac1, a Rho-related GTPase, are central in promoting endothelial vascular development. However, the contribution of endothelial Rac1 in ischemic stroke has not been directly investigated. We hypothesized that Rac1 signaling protects endothelial cells and promotes angiogenesis after stroke. Immortalized human brain endothelial cells (HBEC-5i) were challenged by oxygen-glucose deprivation (OGD) for 18 hours followed by re-oxygenation. For cell viability test, NSC 23766 (NSC, 3, 15 and 30 μM), a specific Rac1 inhibitor, was added at re-oxygenation (immediately after the18 hours ODG). The viability of endothelial cells was assessed by CCK-8 (% normoxia control) 24 hours after the18 hours OGD. For migration and tube formation assays, NSC at 30 μM was added 24 hours after the 18 hours OGD to avoid potential effects on cell survival. Cell migration using scratch assay (wound area compared to initial time %) was examined 72 hours after the 18 hours OGD. Tube formation, the ability of cells to align into tube-like structures (number of junctions), was measured 28 hours after the 18 hours OGD. Data are presented as mean±SEM. OGD reduced endothelial cell viability (control 100±3.23 vs. OGD 69.97±1.11, n=6-7, p0.05; 15 μM: OGD+vehicle 69.97±1.11 vs. OGD+NSC 60.77, n=6, p