Selective vulnerability of rat brain regions to unconjugated bilirubin.

Hippocampus is one of the brain regions most vulnerable to unconjugated bilirubin (UCB) encephalopathy, although cerebellum also shows selective yellow staining in kernicterus. We previously demonstrated that UCB induces oxidative stress in cortical neurons, disruption of neuronal network dynamics, either in developing cortical or hippocampal neurons, and that immature cortical neurons are more prone to UCB-induced injury. Here, we studied if immature rat neurons isolated from cortex, cerebellum and hippocampus present distinct features of oxidative stress and cell dysfunction upon UCB exposure. We also explored whether oxidative damage and its regulation contribute to neuronal dysfunction induced by hyperbilirubinemia, considering neurite extension and ramification, as well as cell death. Our results show that UCB induces nitric oxide synthase expression, as well as production of nitrites and cyclic guanosine monophosphate in immature neurons, mainly in those from hippocampus. After exposure to UCB, hippocampal neurons presented the highest content of reactive oxygen species, disruption of glutathione redox status and cell death, when compared to neurons from cortex or cerebellum. In particular, the results indicate that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein implicated in the maintenance of cellular oxidation status. However, longer neuronal exposure to UCB caused down-regulation of DJ-1 expression, especially in hippocampal neurons. In addition, a greater impairment in neurite outgrowth and branching following UCB treatment was also noticed in immature neurons from hippocampus. Interestingly, pre-incubation with N-acetylcysteine, a precursor of glutathione synthesis, protected neurons from UCB-induced oxidative stress and necrotic cell death, preventing DJ-1 down-regulation and neuritic impairment. Taken together, these data point to oxidative injury and disruption of neuritic network as hallmarks in hippocampal susceptibility to UCB. Most importantly, they also suggest that local differences in glutathione content may account to the different susceptibility between brain regions exposed to UCB.