Bilirubin Toxicity and Differentiation of Cultured Astrocytes

Abstract

OBJECTIVE:To study the toxicity of bilirubin in primary cultures of newborn rat cerebral cortical astrocytes. STUDY DESIGN:Primary cultures of newborn rat astrocytes were incubated at bilirubin concentrations of 0, 1, 5, 10, 25, 50, 100, 200, and 2000 M, at a bilirubin:albumin molar ratio of 1.7. Bilirubin toxicity was determined by changes in cellular morphology, trypan blue staining, and lactate dehydrogenase (LDH) release into the culture medium at various times of incubation. To determine if differentiation of astrocytes affects bilirubin toxicity, cultures were treated with dibutyryl cyclic adenosine monophosphate. RESULTS:All three indices of toxicity showed a bilirubin concentration dependence. LDH release in experimental cultures was significantly elevated (p Ͻ 0.05) above that of control cultures by 24 hours at bilirubin concentrations of Ն100 M. The absolute amount of LDH release differed significantly between the 200 and 2000 M cultures from 1.5 to 24 hours, after which duration of exposure appeared to take over and all cultures approached maximum. LDH release for the lower concentrations all reached maximum by 120 hours, except for the 1 M cultures, which showed no significant elevation above control throughout the study period. At 100 and 200 M bilirubin, LDH release by untreated cells was significantly higher (p Ͻ 0.05) than release by treated cells by 36 hours. CONCLUSION:Undifferentiated astrocytes appeared to be more sensitive to bilirubin toxicity, which may correlate with the greater susceptibility of newborns to kernicteric injury. Studies with primary astrocyte culture may provide insight into how bilirubin sensitivity changes with brain development as well as the cellular and biochemical mechanisms of bilirubin encephalopathy.Hyperbilirubinemia-induced encephalopathy, i.e., kernicterus, has been well described. [1][2][3][4] However, the principal underlying mechanisms of bilirubin neurotoxicity have been much more difficult to determine despite extensive study. Currently, bilirubin is postulated to have a multifactorial role in cellular damage. Exposure to bilirubin has been associated with changes in energy metabolism; changes in carbohydrate, amino acid, and lipid metabolism; alteration in membrane function; decreased membrane potential; alteration in enzymatic function; and inhibition of protein and DNA synthesis. [1][2][3][4] Models that have been used to study these mechanisms include in vivo models such as hyperosmolar opening of the blood-brain barrier 5 and congenitally jaundiced rats, 3 or in vitro models, e.g., perfused brain sections, 6 synaptosome preparations, 7,8 neural-derived transformed cell lines, 9 and glial cell cultures. 10 -12 We chose to study bilirubin toxicity in primary culture of astrocytes.Investigation with primary culture of astrocytes has several advantages. It involves normal diploid cells with biochemical and morphologic features close to those found in vivo. 13 It is a simple system, consisting of a highly enriched preparation of astrocytes, 14 which elim...

RHD maternal–fetal genotype incompatibility and schizophrenia: extending the MFG test to include multiple siblings and birth order

“…The hypothesized role of Rh incompatibility disease in schizophrenia risk is consistent with the neurodevelopmental 24 and glial asthenia 13 hypotheses of schizophrenia, as Rh incompatibility disease can lead to fetal hypoxia and an increase in unconjugated bilirubin, 8,9 a neurotoxin than can damage undifferentiated glial cells. 10,11 A previously published analysis of the same set of nuclear families that used only the youngest affected child in each nuclear family reported a one-sided P-value of 0.027. 7 An analysis reported here using multiple siblings had a onesided P-value of 0.014.…”

“…2,6,7 neurotoxin that can damage undifferentiated glial cells. 10,11 Hypoxia and glial cell damage have been associated with schizophrenia. 12,13 Earlier studies could not distinguish between direct child or maternal RHD genotype effects and the effect of RHD maternal -fetal genotype (MFG) incompatibility because they employed study designs that did not allow for explicit modeling of these distinct effects.…”

Biological risks for neurological abnormalities associated with hyperbilirubinemia

“…13,14 In addition, there is a current opinion that immaturity is associated with a greater sensitivity to neuronal sequelae by UCB and that cell viability was shown to be directly related to aging-in-culture. 15 Studies performed by us 16 and others 17 in nerve cell culture models point out that UCB induces greater apoptosis in more immature cells when compared with older ones. In fact, we have shown that astrocytes cultured for 5 days in vitro (DIV), as well as 4 DIV neurons exposed to 86 mM UCB in the presence of 29 mM human serum albumin (HSA), reveal approximately twofold higher levels of apoptosis when compared with more differentiated astrocytes and neurons.…”

The protective effect of docosahexaenoic acid on the bilirubin neurotoxicity

“…UCB toxicity to neurons has been reported in brain sections, cultured cell lines, and isolated nerve terminals 9 . Recent studies have described that astrocyte functions are compromised following UCB exposure [9][10][11] . Astrocytes are glial cells that provide metabolic support to neurons and contribute to formation of the blood-brain barrier.…”