[Combined neuronal toxicity of bilirubin and hypoxia. Study of cultured rat neurons].
Clinical observations suggest that bilirubin encephalopathy is often seen in newborn infants presenting not only with hyperbilirubinemia but also with alterations in oxygen transport like in severe anaemia. Since bilirubin and hypoxia have been shown to be detrimental to the central nervous system, the present study was designed to test the additional effects of the two insults on the outcome of cultured neurons from the forebrain of 14 day-old embryos. After 6 days in vitro, neurons were exposed either to bilirubin (0.5 microM) or to bilirubin and hypoxia for 6 hours. Thereafter, cells were reoxygenated for 96 hours in standard conditions. Control cells were kept in normoxia. Cell viability was assessed by the methyltetrazolium method. Cell death (apoptosis or necrosis) was characterized by fluorescent nuclear staining with DAPI. Rates of protein synthesis and energy metabolism in neurons were measured by [3H]leucine and [3H]2-deoxyglucose incorporation, respectively. Data are reported as percentages of change as compared to controls. Each experiment involved 5 to 10 dishes per time point and was repeated 2 to 4 times.


RESULTS
Bilirubin reduced cell viability by 24.5% vs controls (p < 0.001) at 96 h while 16% of the neurons exhibited morphological features of apoptosis (p < 0.001). The combination of hypoxia with bilirubin induced a 34% decrease in cell viability (p < 0.001) and the percentage of apoptotic cells was higher than after the exposure to hypoxia or to bilirubin alone. The rate of protein synthesis increased significantly in all experimental conditions as early as 1 h after the onset of the insult and at 48 h post reoxygenation. It increased again at 72 h in the cells exposed to bilirubin or to bilirubin and hypoxia. These sequential changes in synthesis of specific proteins seem to be involved in delayed neuronal death. Bilirubin decreased significantly [3H]2-deoxyglucose incorporation at 24 h while it increased when the neurons were exposed to both bilirubin and hypoxia (+60%, p < 0.001) and decreased thereafter. These data confirm the deleterious effects of bilirubin on neuronal viability, on protein synthesis and metabolic rates. The combination of bilirubin with hypoxia resulted in stronger detrimental effects on neurons than bilirubin alone.
Vert
P
P
Université de Nancy 1, Faculté de Médecine, Service de Néonatologie, Maternité Régionale Universitaire-54042 Nancy-France.
Grojean
S
S
Daval
J L
JL
D000818:Animals; D000963:Antimetabolites / Q000378:metabolism; D001663:Bilirubin / Q000009:adverse effects*; D018929:Cell Culture Techniques; D003847:Deoxyglucose / Q000378:metabolism; D004195:Disease Models, Animal; D006801:Humans; D006932:Hyperbilirubinemia / Q000150:complications*; D002534:Hypoxia, Brain / Q000503:physiopathology*; D007231:Infant, Newborn; D007232:Infant, Newborn, Diseases; D007647:Kernicterus / Q000503:physiopathology*; D009474:Neurons / Q000473:pathology*; D014176:Protein Biosynthesis
D000963:Antimetabolites; D003847:Deoxyglucose; D001663:Bilirubin