Use of Laser Speckle Contrast Imaging for Distribution of Animals by Severity of Brain Tissue Damage in a Neonatal Hypoxia-Ischemia Model in Mice.

Background/Objectives: Inter-individual variability in injury severity represents a major barrier to reproducibility in neonatal hypoxia-ischemia (HI) models. Objective early postoperative stratification of animals is therefore essential for standardized group allocation and reliable assessment of experimental outcomes. This study aimed to evaluate whether laser speckle contrast imaging (LSCI) can be used as a rapid, noninvasive tool for early post hoc stratification of ischemic brain damage severity in neonatal mice following HI. Methods: Neonatal CD-1 mice (postnatal day 9; n = 60) underwent hypoxia-ischemia using a modified Rice-Vannucci protocol. Cerebral perfusion was assessed by laser speckle contrast imaging at baseline, 3 h, and 7 days after HI. The difference in mean perfusion between ipsilateral and contralateral hemispheres at 3 h (Δ perfusion) was used to stratify animals into severity groups. Brain injury was quantified by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 24 h and 7 days. Survival was monitored for 7 days and analyzed using Kaplan-Meier curves and the log-rank (Mantel-Cox) test. Results: LSCI-derived Δ perfusion at 3 h enabled the formation of distinct injury-severity groups (no visible damage, mild, moderate, and severe) with significant between-group differences (p < 0.0001). TTC-based lesion area increased stepwise across severity groups, and Δ perfusion correlated with lesion size when all animals were analyzed together (r = 0.688, p = 0.0011). No significant correlations were observed within individual severity groups, indicating that the overall association was driven primarily by between-group differences. Survival analysis revealed 75% mortality in the severe injury group (p < 0.0001). Conclusions: LSCI represents a robust and practical approach for early, objective, group-level stratification of neonatal mice by HI injury severity, thereby improving reproducibility and statistical validity in preclinical studies. However, its ability to predict outcomes within individual severity categories is limited, and repeated long-term measurements may pose technical challenges.