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

Yi-Je Chen; Cheng-Tien Wu; Chang-Mu Chen; Shing-Hwa Liu

doi:10.3791/68806

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Method Article

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

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DOI:

10.3791/68806

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September 16th, 2025

Yi-Je Chen^*¹ , Cheng-Tien Wu^*² , Chang-Mu Chen³ , Shing-Hwa Liu⁴^,⁵^,⁶

¹Department of Pharmacology, University of California, Davis, ²Department of Nutrition, China Medical University, ³Division of Neurosurgery, Department of Surgery, College of Medicine and Hospital, National Taiwan University, ⁴Institute of Toxicology, College of Medicine, National Taiwan University, ⁵Department of Medical Research, China Medical University Hospital, China Medical University, ⁶Department of Pediatrics, College of Medicine and Hospital, National Taiwan University

^* These authors contributed equally

Erratum Notice

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Summary

Here, we present a method for secondary middle cerebral artery occlusion in mice to investigate the progression of recurrent ischemic stroke. The surgical procedures, postoperative care, neuronal pathological alterations, and survival rate are described.

Abstract

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.

Introduction

Stroke remains a leading cause of death and long-term disability worldwide, posing a significant clinical and socioeconomic burden. More than 30 million people worldwide are affected by stroke and its consequences, including mobility loss, cognitive impairment, and the increased risk of vascular diseases¹^,². Despite advances in acute treatment such as thrombolysis and endovascular thrombectomy, many survivors remain at high risk of stroke recurrence, which is associated with worsened prognosis, greater disability, and increased mortality rates. Recurrent strokes occur in approximately 10-20% of patients within....