Standardization of The Rat Intraluminal MCAO Model: A Detailed Protocol with Quality Control Measures

The rat middle cerebral artery occlusion (MCAO) model remains the reference standard for elucidating ischemic stroke pathophysiology and validating neuroprotective therapies. Subtle surgical deviations, however, can profoundly alter infarct volume and behavioral outcomes. Using a "visualize-then-systematize" framework, we re-examined every step of model establishment and validation. Detailed photographic documentation of key surgical landmarks and critical steps was performed to standardize anatomical orientation and filament placement. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to precisely map the volume and topology of infarcts at 24 h and 72 h after reperfusion. Neurological deficits were graded with the Zea-Longa, Bederson, and modified Garcia scales. Twenty-four male Sprague-Dawley rats underwent 60 min transient MCAO; infarct volume and neurological scores were assessed at 24 h and 72 h. Approximately 87.50% of animals achieved successful cerebral blood-flow blockade (Zea-Longa ≥ 1 and unequivocal TTC-defined infarct), with an overall mortality of 12.50%, attesting to the model's high fidelity. This standardized protocol provides a technically refined, reproducible foundation for preclinical stroke research, facilitating inter-laboratory consistency in studies investigating stroke pathophysiology and therapeutic interventions.