Investigating Ischemic Stroke Recurrent Injury in a Secondary Middle Cerebral Artery Occlusion Model in Mouse.

Stroke is the second leading global cause of mortality and disability, with recurrent strokes contributing significantly to poor outcomes and increased neurological burden. Despite advances in acute clinical stroke management, experimental models for recurrent ischemic stroke remain limited due to their procedural complexity and high mortality rates. In this study, we aimed to establish a recurrent ischemic stroke in a mouse model and evaluate the brain pathological changes and functional deficits using a two-stage middle cerebral artery occlusion (MCAO) protocol. Adult male C57BL/6J mice underwent transient MCAO under Laser Doppler blood flow monitoring. A secondary MCAO was performed 14 days after the initial surgery to simulate stroke recurrence. Postoperative assessments included locomotor activity analysis, neurobehavioral deficits scoring by using a six-point scoring system, inflammatory markers including hypoxia-inducible factor-1α (HIF-1α) and tumor necrosis factor-α (TNF-α), and histopathological evaluation using hematoxylin and eosin (H&E) staining. After recurrent MCAO administration, mice exhibited significantly reduced locomotor activity, decreased movement speed, and diminished neurobehavioral scores, as well as increased inflammatory markers, compared to control groups. Histopathological detection revealed moderate to severe neuronal cell necrosis, neuropil vacuolation, and localized hemorrhage in the cortex and hippocampus of recurrent MCAO mice. The inflammatory markers were also induced in this recurrent MCAO procedure. This two-stage MCAO mouse model presents, for the first time, a method that effectively simulates recurrent ischemic stroke with consistent neurological and pathological outcomes and improves postoperative survival. This model could provide a reliable platform for mechanistic studies and may be applied in the future evaluation of neuroprotective therapies for recurrent stroke.