Abstract DP120: The Spatial Distribution of Innate Immune Cells is Directed by Temporal Dynamics and Reciprocal Cell Recruitment

Introduction: Microglia (MG), macrophages (MΦ), and polymorphonuclear neutrophils (PMNs) are among the first immune cells to respond in ischemic stroke and ultimately influence long-term outcomes. The complex roles of these cells in the immune response are in part governed by their position, but these spatial patterns are not well defined as preclinical studies are often limited to representative regions of the infarct. We used light sheet microscopy (LSM) to fully map the spatiotemporal patterns of MG/MΦ and PMNs post-stroke and investigate how these innate immune cells act to reciprocally mediate cell localization. Methods: The transient middle cerebral artery occlusion (tMCAO) stroke model was used in a Ly6G-tdTomato x Csf1r-EGFP transgenic mouse line expressing fluorescent markers for PMNs and MG/MΦ. In a subset of animals, PMN adhesion was inhibited via ICAM-1 antibody blockade. Whole brains were collected at days 1, 2, and 3 post-tMCAO, cleared, and imaged using LSM. Brains were co-labeled with for neurons and vasculature and registered to the Allen Brain Atlas. Results: MG/MΦ and PMNs exhibited cell type-specific spatial patterning that persisted longitudinally throughout acute infarct evolution and was not confined to the boundaries of the infarcted tissue ( Figure 1 ). Specifically, PMN infiltration was most pronounced around the middle cerebral artery root and peaked at D3, while MG/MΦ also peaked at D3 but accumulated in more rostral regions of the dorsal striatum. Further, MG/MΦ patterning was influenced by PMN presence, as evidenced by a shift in MG/MΦ localization away from the vasculature in response to PMN adhesion blockade ( Figure 2 ). Conclusions: Our data demonstrate that MG/ΜΦ and PMNs follow longitudinally consistent spatial patterns that are independent of precise infarct topology. Instead, MG/ΜΦ position relative to the vasculature was mediated by PMN infiltration, suggesting a reciprocal relationship between these immune cell populations. These data highlight the relevance of the entire post-stroke immune milieu in the investigation of novel immunomodulatory strategies for stroke.