36 Combined Neurotoxicity of Bilirubin and Hyperoxia in Cortical Neuronal Culture

Abstract

Background. Hyperbilirubinemia in newborn infants and especially in premature infants enhances the risk for developing cerebral palsy. In the developing rat brain and cell culture hyperoxia has been identified to cause neurodegeneration. We therefore examined the effects of bilirubin in combination with hyperoxia in cultured rat neuronal cultures.Methods. Primary cortical neuronal cultures were prepared from Wistar rat embryos at 18 days gestation and exposed to bilirubin (0,1–250 μM) in combination with hyperoxia (80%) for 48 h. Control cells were kept in normoxia. Cell viability was assessed by the methyltetrazolium method (MTT) and lactate dehydrogenase release (LDH). Cells showed morphologic changes consistent with apoptosis.Results. In normoxia bilirubin reduced cell viability by 58 % vs controls (p< 0,05) above a concentration of 5 μM whereas LDH release signifficantly increased from 20% to 94% above bilirubin concentration from 25 μM. Combination with hyperoxia caused additional loss of MTT cleavage and LDH release was increased in all concentrations of bilirubin from 0.1–250 μM.Conclusion. We conclude that a combination of bilirubin and hyperoxia results in stronger detrimental effects on neurons than bilirubin or hyperoxia alone. Our results suggest that in premature infants exposed to higher oxygen concentrations hyperbilirubinemia may exacerbate neuronal damage and contribute to neurological impairment.