Abstract WP287: Dissecting Microglial Activation and Inhibition Windows to Optimize Stroke Recovery

Introduction: Acute ischemic stroke (AIS) caused by large vessel occlusion (LVO) is a leading cause of disability worldwide. Even with rapid recanalization, nearly half of patients remain impaired at 90 days, underscoring the need for new strategies. Innate immune responses, particularly microglia (MG), are critical drivers of outcomes. MG support debris clearance but, when persistently activated, can worsen injury. Their spatiotemporal roles in LVO stroke remain poorly defined, limiting development of immunomodulatory therapies. Methods: We employ transient middle cerebral artery occlusion (tMCAO) in mice with chemogenetic strategies for temporally precise, region-specific MG modulation. Using Cx3cr1-Cre mice, excitatory (hM3Dq) or inhibitory (hM4Di) DREADDs are stereotactically delivered to the caudoputamen, the infarct core. Clozapine-N-oxide (CNO) enables phase-specific activation or inhibition. Behavioral outcomes are assessed with a custom bottom-view open field test, optimized with deep learning to capture gait, paw placement, tail arch, and locomotion. Post-mortem analyses include immunohistochemistry and confocal imaging, to characterize myelination, synaptic remodeling, and inflammatory states. Results: Το date, we have validated the viral constructs were successfully delivered to the caudoputamen of Cx3cr1-Cre mice, confirming feasibility of targeted chemogenetic manipulation, and animals have subsequently undergone tMCAO. Longitudinal testing with the open field assay showed significant gait length reductions from baseline to post-stroke, demonstrating sensitivity to functional impairments. Immunohistochemistry revealed a dynamic MG response, with phagocytic microglia increasing through day 14 and declining thereafter. These findings support our model that early MG activation aids repair, whereas prolonged activation is detrimental, suggesting temporally precise activation and delayed inhibition could optimize recovery. Impact: This project will generate the first temporal map of MG contributions to stroke recovery, addressing a key barrier to phase-specific immunotherapy. By identifying windows when MG activation or suppression is beneficial, the study will inform rational immunomodulation strategies for LVO stroke and may extend to other CNS injuries. Ultimately, these findings could transform rehabilitation by advancing precision immunotherapy that maximizes repair while minimizing chronic inflammation.