Surgical protocol and ischemic duration determine myeloid cell features and locomotor deficits in preclinical...

Ischemic stroke remains a leading cause of disability, despite increasing patient eligibility for undergoing vessel recanalization. Ischemia/reperfusion injury (I/RI), the inflammatory cascade triggered by ischemia and subsequent reperfusion, contributes to infarct evolution and functional outcomes. However, investigation of mechanisms underlying I/RI is hindered by variable outcomes reported across preclinical studies, which can largely be attributed to differences in the stroke models used. Although the transient middle cerebral artery occlusion (tMCAO) model has been widely adopted to recapitulate I/RI, interchangeable use of the Longa and Koizumi surgical protocols and a range of durations of occlusion have resulted in inconsistent reports of pathological and functional outcomes. To evaluate the effects of tMCAO model variability, we employed the Longa and Koizumi tMCAO protocols across increasing ischemic durations. Using multimodal imaging and behavioral testing, we report that infarct pathology, locomotor deficits, and innate immune responses were significantly influenced by ischemic duration and surgical protocol at 24 and 72 h post-tMCAO. Myeloid cell morphology and spatial relationships were the most robustly affected cellular features but were differentially affected by ischemic duration and surgical protocol. These findings provide a comprehensive assessment of the consequences of variable tMCAO dynamics to guide experimental design for future preclinical studies.