Background: Hypoxic ischemic encephalopathy remains a significant cause of developmental disability 1 , 2 . The standard of care for term infants is hypothermia, which has multifactorial effects 3 – 5 . Therapeutic hypothermia upregulates the cold-inducible protein RNA binding motif 3 (RBM3) that is highly expressed in developing and proliferative regions of brain 6 , 7 . The neuroprotective effects of RBM3 in adult are mediated by its ability to promote translation of mRNAs such as reticulon 3 (RTN3) 8 . Methods: Hypoxia ischemia or control procedure was conducted in Sprague Dawley rat pups on postnatal day 10 (PND10). Pups were immediately assigned to normothermia or hypothermia at the end of the hypoxia. In adulthood, cerebellum-dependent learning was tested using the conditioned eyeblink reflex. The volume of the cerebellum and magnitude of cerebral injury were measured. A second study quantified RBM3 and RTN3 protein levels in the cerebellum and hippocampus collected during hypothermia. Results: Hypothermia reduced cerebral tissue loss and protected cerebellar volume. Hypothermia also improved learning of the conditioned eyeblink response. RBM3 and RTN3 protein expression were increased in the cerebellum and hippocampus of rat pups subjected to hypothermia on PND10. Conclusions: Hypothermia was neuroprotective in male and female pups and reversed subtle changes in the cerebellum after HI.

Choline is an essential nutrient with many roles in brain development and function. Supplementation of choline in early development can have long-lasting benefits. Our experiments aimed to determine the efficacy of choline supplementation in a postnatal day (PND) 10 rat model of neonatal hypoxia ischemia (HI) at term using both male and female rat pups. Choline (100 mg/kg) or saline administration was initiated the day after birth and given daily for 10 or 14 consecutive days. We determined choline’s effects on neurite outgrowth of sex-specific cultured cerebellar granule cells after HI with and without choline. The magnitude of tissue loss in the cerebrum was determined at 72 h after HI and in adult rats. The efficacy of choline supplementation in improving motor ability and learning, tested using eyeblink conditioning, were assessed in young adult male and female rats. Overall, we find that choline improves neurite outgrowth, short-term histological measures and learning ability in males. Surprisingly, choline did not benefit females, and appears to exacerbate HI-induced changes.