Supplementary Material for: The Neuroprotective Effects of Hypothermia on Bilirubin-Induced Neurotoxicity in vitro

<b><i>Background:</i></b> In high-risk newborns indirect hyperbilirubinemia can lead to acute bilirubin encephalopathy and kernicterus. Despite the current therapeutic modalities, preventing or reversing the neurotoxicity cannot be achieved in all infants. <b><i>Objective:</i></b> To investigate the neuroprotective effects of hypothermia on bilirubin-induced toxicity in primary mouse neuronal cell cultures. <b><i>Methods:</i></b> Hippocampal cell cultures, isolated from newborn mouse brains, were incubated with unconjugated bilirubin (UCB) at 3 days in vitro (DIV) and immediately exposed to varying degrees of hypothermia. Neuronal viability and mitochondrial health were compared between the normothermia (37°C), mild (34°C), moderate (32°C) and severe (29°C) hypothermia groups. Confocal microscopy and fluorescent dyes (propidium iodide and JC-1) were used for cell evaluation. To determine the late effects of hypothermia, the cultures were also examined at 7 DIV after returning to normothermic conditions. <b><i>Results:</i></b> Induction of any degree of hypothermia increased the neuronal survival after 24 h of UCB treatment. Neuronal death rate and mitochondrial membrane potential loss were lowest in the neurons exposed to moderate hypothermia. We also observed that mild to moderate hypothermia had late protective effects on neuronal cell viability, whereas deep hypothermia did not improve neuronal survival. <b><i>Conclusions:</i></b> We conclude that hypothermia reduces the cell death induced by bilirubin toxicity in neuronal cells. Although moderate hypothermia has a better outcome than mild hypothermia, deep hypothermia as low as 29°C has adverse effects on neuronal cell viability.