Combined effects of bilirubin and hypoxia on cultured neurons from the developing rat forebrain

Hyperbilirubinemia and hypoxia are common causes of brain injury in the newborn. To determine the effects of free bilirubin associated with transient hypoxia on developing rat neurons, the cells were exposed to bilirubin (0.25 to 5 micromol/L) and/or to hypoxia for 3 or 6 hours (95% N2-5% CO2). Glutamate receptor antagonists were added to some cultures. Cell death characteristics, energy metabolism, and protein synthesis were analyzed for 96 hours. Bilirubin increased apoptotic cell death. When associated with hypoxia, the neuronal loss was worsened. Bilirubin reduced energy metabolism, whereas a 6-hour exposure to hypoxia increased it for at least 24 hours, with no influence of additional bilirubin. Bilirubin with or without hypoxia induced 2 increases in protein synthesis, at 1 and 72 hours. In this model, bilirubin may promote programmed neuronal death. When bilirubin is associated with hypoxia, the deleterious effects are enhanced. The suppression of bilirubin induced neuronal damage by the NMDA (N-methyl-D-aspartate) receptor antagonist MK801 suggests the involvement of glutamate.