Neonatal hyperbilirubinemia : in vivo characterization of mechanisms of bilirubin neurotoxicity and pharmacological treatments

Neonatal jaundice or hyperbilirubinemia is the result of alterations in the bilirubin metabolism. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced neurological dysfunction and, if untreated, eventually death by kernicterus. Severe hyperbilirubinemia results in the saturation of the bilirubin binding capacity of plasma albumin, with the consequent increase in the fraction of unconjugated bilirubin (UCB) not bound to albumin (free bilirubin, Bf) that, due to its lipophilicity, crosses the blood brain barrier accumulating in the brain and triggering the neuronal injury. In the developing central nervous system, a wide range of cellular functions are affected and the concerted disruption of their regulation results in cellular damage. Patients experiencing prolonged toxic bilirubin levels are characterized by a number of neurological deficits, such as abnormalities in motor, sensitive and cognitive functions. Despite intensive studies, several aspects of the mechanisms operating at the onset of the disease are still partially understood. To study severe neonatal hyperbilirubinemia and possible therapies, I took advantage of the Ugt1-/- mouse model previously generated in my laboratory. Homozygous mutant mice develop jaundice and accumulate bilirubin in the brain due to the lack of the Ugt1a1 enzyme. If untreated, mutant mice show neurological deficits leading to early neonatal lethality. The work performed in this Thesis explores three different aspects of bilirubin neurotoxicity: 1. The role of Bf in vivo by the administration of human serum albumin; 2. The events preceding death by a time-course analysis of mutant pups; 3. The investigation of the role of neuroinflammation by the administration of a neuroprotective and anti-inflammatory drug. The obtained results showed that increasing plasma bilirubin-binding capacity by albumin supplementation decreases bilirubin neurotoxicity. In fact, daily albumin administration avoided the accumulation of Bf in the brain by its mobilization from tissues to plasma, resulting in the complete rescue of bilirubin-induced brain impairment and lethality. Moreover, this study highlighted the reliability of Bf as the best marker to predict neurotoxicity risk. The time-course investigation of the events occurring in the cerebellum of Ugt1-/- pups leading to bilirubin-brain damage showed the prevalence of ER stress, oxidative stress and neuroinflammation at the onset of neonatal hyperbilirubinemia that, in turn, affected brain integrity by their concerted effect resulting in neurodegeneration. Finally, the administration of minocycline (MNC), an antibiotic with neuroprotective and anti-inflammatory properties, partially prevented lethality by hyperbilirubinemia and rescued animals by significantly reducing neurodegenerative and neuroinflammatory features that characterized the untreated Ugt1-/- mouse model. This study demonstrates the contribution of ER stress and inflammation in the onset of the disease, the relevance of these mechanisms during the attenuation of neurodegeneration by MNC administration, and that albumin supplementation is a potential therapeutic alternative to treat bilirubin neurotoxicity in acute cases in which prompt exchange transfusion is required.