Isbatan, A.; Lee, S.; Damen, F. C.; Chen, S.; Maslon, M. M.; Du, X.; Minshall, R. D.; Cai, K.; Chen, J.

Background: The intraluminal middle cerebral artery occlusion (MCAO) model is widely used in preclinical stroke research, but outcomes remain inconsistent because of variable occlusion times and inadvertent filament placement into the pterygopalatine artery (PPA). Methods: We applied a modified version of the Koizumi method to directly visualize the internal carotid artery (ICA) PPA bifurcation and systematically compare targeted ICA versus PPA occlusion. We combined multiple complementary methods - survival studies, neurological scoring, 2,3,5 Triphenyltetrazolium chloride (TTC) infarct staining, Evans blue blood brain barrier (BBB) permeability assays, laser speckle cerebral blood flow imaging, small-animal magnetic resonance imaging (MRI), and evaluation of inflammatory gene expression through qPCR to rigorously characterize injury patterns, survival, and inflammatory responses. Results: ICA occlusion approximately caused a rapid 70% reduction in cerebral blood flow, progressive infarction (15 min: smaller infarct with approximately 50% 7-day survival; 30 min: moderate infarct but >50% mortality), and cortical edema. In contrast, PPA occlusion produced only a gradual 44% decline in blood flow, small infarcts at 60 min) and preserved survival (80% at 7 days). Finally, magnetic resonance imaging in 15-minute ICA occluded mice identified the development of cortical edema which was not observed in sham, 15-minute PPA, or 60-minute PPA mice. Conclusion: These findings identify 15-minute ICA occlusion as the most practical balance between reproducible infarction and survival for long-term studies, while 30 minutes is best reserved for short-term infarct induction when survival is less critical. By resolving two critical sources of MCAO variability, occlusion duration and vascular targeting, this study provides a standardized protocol and evidence-based occlusion benchmarks that will improve reproducibility in preclinical stroke studies