Optimizing therapeutic hypothermia conditions in a translational preclinical model of neonatal hypoxia-ischemia in rats

BackgroundTherapeutic hypothermia is the only clinically approved treatment for neonatal hypoxia-ischemia (NHI), although its efficacy remains partial. In preclinical research, hypothermia is widely used as a reference therapy; however, its protocol is highly variable across studies, limiting robust comparisons with emerging neuroprotective strategies. This study aimed to define an optimal and standardized hypothermia protocol in the Rice-Vannucci model, not to challenge clinical practice, but to establish a reliable benchmark for preclinical therapeutic development. MethodsNHI was induced in postnatal day 7 (P7) rat pups, followed by normothermia or hypothermia for 2, 3, or 5 hours. Short- and long-term outcomes were assessed using lesion volume measurements by MRI, neurological scoring, behavioral tests, and histological analyses. The impact of immediate hypothermia initiation was also examined. ResultsAcross analyses, both 2- and 3-hour hypothermia durations provided greater neuroprotection than 5 hours--including brain lesion volume, motor and cognitive performances, and markers of neuronal preservation and neuroinflammation. However, for several parameters, 2 hours of hypothermia showed superior efficacy compared with 3 hours. Immediate initiation further modestly improved outcomes. ConclusionA 2-hour hypothermia protocol represents the most robust and reproducible preclinical reference, enabling meaningful comparison with novel therapies in the Rice-Vannucci model. IMPACTO_LIBy establishing an optimized hypothermia protocol in the Rice-Vannucci model, this study offers a consistent and robust reference for preclinical evaluation of emerging therapies. C_LIO_LIIt does not question clinical hypothermia protocols, but addresses variability in preclinical literature C_LIO_LIOptimizing the hypothermia reference protocol is mandatory to reliably identify new effective treatments in preclinical studies and to enhance their likelihood of successful and efficient clinical translation C_LI