Ischemic Duration is More Critical than Reperfusion Efficacy in Driving Early Neuroinflammation and Motor Deficits after Transient Middle Cerebral Artery Occlusion

Ischemic stroke remains a leading cause of long-term disability, despite more patients undergoing vessel recanalization. Ischemia/reperfusion injury (I/RI), the inflammatory cascade triggered by ischemia and reperfusion, contributes to infarct evolution and functional outcomes. Clinical stroke presentation varies due to differences in ischemic duration and reperfusion success, yet preclinical models rarely account for this physiologic contribution. This preclinical-clinical disconnect may underlie the translational failure of acute therapies targeting the myeloid cell-mediated immune response (specifically neutrophils and microglia/bone marrow-derived macrophages). To address this physiological heterogeneity, we employed variations of the transient middle cerebral artery occlusion model in myeloid-reporter mice, systematically altering ischemic duration and reperfusion success. Using longitudinal perfusion imaging and behavioral testing, we found that infarct pathology, locomotor deficits, and innate immune responses were significantly influenced by ischemic duration, and to a lesser extent, reperfusion status. Microglia/macrophage and neutrophil morphology, as well as the spatial association of these myeloid cells, were the most strongly affected cellular features. Further, neutrophil density, morphology, and spatial patterning correlated with acute locomotion and motor recovery across the entire cohort. These findings highlight the differential roles of ischemic duration and reperfusion efficacy in driving neuroinflammation and stroke outcomes, emphasizing the importance of incorporating physiologic heterogeneity into preclinical I/RI models to better guide translational strategies.