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

ABSTRACT Background Therapeutic 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. Methods NHI 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. Results Across 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. Conclusion A 2-hour hypothermia protocol represents the most robust and reproducible preclinical reference, enabling meaningful comparison with novel therapies in the Rice–Vannucci model. IMPACT By establishing an optimized hypothermia protocol in the Rice–Vannucci model, this study offers a consistent and robust reference for preclinical evaluation of emerging therapies. It does not question clinical hypothermia protocols, but addresses variability in preclinical literature Optimizing 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