Choline supplementation mitigates effects of bilirubin in cerebellar granule neurons in vitro.

BACKGROUND: Premature infants may suffer from high levels of bilirubin that could lead to neurotoxicity. Bilirubin has been shown to decrease L1-mediated ERK1/2 signaling, L1 phosphorylation, and L1 tyrosine 1176 dephosphorylation. Furthermore, bilirubin redistributes L1 into lipid rafts (LR) and decreases L1-mediated neurite outgrowth. We demonstrate that choline supplementation improves L1 function and signaling in the presence of bilirubin. METHODS: Cerebellar granule neurons (CGN) were cultured with and without supplemental choline, and the effects on L1 signaling and function were measured in the presence of bilirubin. L1 activation of ERK1/2, L1 phosphorylation and dephosphorylation were measured. L1 distribution in LR was quantified and neurite outgrowth of CGN was determined. RESULTS: Forty µM choline significantly reduced the effect of bilirubin on L1 activation of ERK1/2 by 220% (p = 0.04), and increased L1 triggered changes in tyrosine phosphorylation /dephosphorylation of L1 by 34% (p = 0.026) and 35% (p = 0.02) respectively. Choline ameliorated the redistribution of L1 in lipid rafts by 38% (p = 0.02) and increased L1-mediated mean neurite length by 11% (p = 0.04). CONCLUSION: Choline pretreatment of CGN significantly reduced the disruption of L1 function by bilirubin. The supplementation of pregnant women and preterm infants with choline may increase infant resilience to the effects of bilirubin. IMPACT: This article establishes choline as an intervention for the neurotoxic effects of bilirubin on lipid rafts. This article provides clear evidence toward establishing one intervention for bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into the mechanism of the ameliorative effect of choline on bilirubin neurotoxicity.

Sexual dimorphic vulnerability of respiratory control in a neonatal rodent model of fetal alcohol spectrum disorder.

Fetal alcohol spectrum disorder (FASD) has been linked to numerous poor neurological outcomes as well as impairments in respiratory neural control. Females are known to metabolize ethanol (EtOH) differently than males suggesting a sexual dimorphic sensitivity to EtOH exposure. We used a rodent model of FASD to investigate whether EtOH disrupts respiratory neural control. Rat pups received a single intraperitoneal injection of 2 different doses (0.8 mg/g or 4.4 mg/g) of EtOH. Whole-body plethysmography was used ∼24 h later to assess ventilatory responses to acute hypoxia (HVR) and hypercapnia (HCVR). Females treated with 4.4 mg/g of EtOH exhibited an attenuated HVR and HCVR, but there was no effect on males, and no effect of 0.8 mg/g on either sex. There was unexpected mortality of unknown causes, especially in females, that occurred 2-3 days after EtOH administration. These data suggest that important ventilatory defense responses in females are impaired following developmental EtOH exposure, and this may be associated with increased risk of later death.

Neonatal hypoxia ischemia redistributes L1 cell adhesion molecule into rat cerebellar lipid rafts.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a devastating disease with lifelong disabilities. Hypothermia is currently the only treatment. At term, the neonatal cerebellum may be particularly vulnerable to the effects of HIE. At this time, many developmental processes depend on lipid raft function. These microdomains of the plasma membrane are critical for cellular signaling and axon extension. We hypothesized that HIE alters the protein content of lipid rafts in the cerebellum. METHODS: Postnatal day (PN) 10 animals, considered human term equivalent, underwent hypoxic-ischemic (HI) injury by a right carotid artery ligation followed by hypoxia. For some animals, LPS was administered on PN7, and hypothermia (HT) was conducted for 4 h post-hypoxia. Lipid rafts were isolated from the right and left cerebella. The percent of total L1 cell adhesion molecule in lipid rafts was determined 4 and 72 h after hypoxia. RESULTS: No sex differences were found. HI alone caused significant increases in the percent of L1 in lipid rafts which persisted until 72 h in the right but not the left cerebellum. A small but significant effect of LPS was detected in the left cerebellum 72 h after HI. Hypothermia had no effect. CONCLUSIONS: Lipid rafts may be a new target for interventions of HIE. IMPACT: This article investigates the effect of neonatal exposure to hypoxic-ischemic encephalopathy (HIE) on the distribution of membrane proteins in the cerebellum. This article explores the effectiveness of hypothermia as a prevention for the harmful effects of HIE on membrane protein distribution. This article shows an area of potential detriment secondary to HIE that persists with current treatments, and explores ideas for new treatments.

Bilirubin inhibits lipid raft dependent functions of L1 cell adhesion molecule in rat pup cerebellar granule neurons.

BACKGROUND: The mechanism of bilirubin neurotoxicity is poorly understood. We hypothesize that bilirubin inhibits the function of lipid rafts (LR), microdomains of the plasma membrane critical for signal transduction. To test this hypothesis, we measured the effect of free bilirubin (Bf) between 7.6 and 122.5 nM on LR-dependent functions of L1 cell adhesion molecule (L1). METHODS: Cerebellar granule neurons (CGN) were plated on poly-L-lysine overnight, and neurite length was determined after 1 h treatment with L1 alone or L1 and bilirubin. L1 activation of ERK1/2 was measured in CGN in the presence or absence of bilirubin. The effect of bilirubin on L1 distribution in LR was quantitated, and the localization of bilirubin to LR was determined. RESULTS: The addition of bilirubin to CGN treated with L1 significantly decreased neurite length compared to L1 alone. L1 activation of ERK1/2 was inhibited by bilirubin. Bilirubin redistributed L1 into LR. Bilirubin was associated only with LR-containing fractions of a sucrose density gradient. CONCLUSION: Bf significantly inhibits LR-dependent functions of L1 and are found only associated with LR, suggesting one mechanism by which bilirubin may exert neurotoxicity is through the dysfunction of protein-LR interactions. IMPACT: This article establishes lipid rafts as a target for the neurotoxic effects of bilirubin. This article provides clear evidence toward establishing one mechanism of bilirubin neurotoxicity, where little is understood. This article paves the way for future investigation into lipid raft dependent functions, and its role in neurodevelopmental outcome.

Choline supplementation prevents the effects of bilirubin on cerebellar-mediated behavior in choline-restricted Gunn rat pups.

BACKGROUND: Bilirubin is produced by the breakdown of hemoglobin and is normally catabolized and excreted. Neurotoxic accumulation of serum bilirubin often occurs in premature infants. The homozygous Gunn rat lacks uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), the enzyme needed to biotransform bilirubin. This rodent model of hyperbilirubinemia emulates many aspects of bilirubin toxicity observed in the human infant. We demonstrate that choline supplementation in early postnatal development is neuroprotective in the choline-restricted Gunn rat, when hyperbilirubinemia is induced on postnatal day 5. METHODS: We first compared behaviors and cerebellar weight of pups born to dams consuming regular rat chow to those of dams consuming choline-restricted diets. Second, we measured behaviors and cerebellar weights of pups born to choline-restricted dams, reared on a choline-restricted diet, supplemented with or without choline, and treated with or without sulfadimethoxine (SDMX). RESULTS: A choline-restricted diet did not change the behavioral outcomes, but cerebellar weight was reduced in the choline-restricted group regardless of genotype or SDMX administration. SDMX induced behavioral deficits in jj pups, and choline supplementation improved most behavioral effects and cerebellar weight in SDMX-treated jj rats. CONCLUSIONS: These results suggest that choline may be used as a safe and effective neuroprotective intervention against hyperbilirubinemia in the choline-deficient premature infant. IMPACT: This article investigates the effect of neonatal jaundice/bilirubin neurotoxicity on cerebellar-mediated behaviors. This article explores the potential use of choline as an intervention capable of ameliorating the effect of bilirubin on the choline-restricted developing brain. This article opens the door for future studies on the action of choline in the presence of hyperbilirubinemia, especially in preterm neonates.

Association of fatty acid ethyl esters in meconium with behavior during childhood.

OBJECTIVE: To examine associations between amounts of fatty acid ethyl esters (FAEEs) in meconium and behavior in school aged children exposed to alcohol and drugs in utero. METHODS: A secondary analysis of a prospective cohort of cocaine, polydrug exposed children, primarily African-American, low socioeconomic status, recruited at birth into a longitudinal study. FAEEs were quantified with gas chromatography via a flame ionization detector. Meconium was analyzed for FAEEs for 216 newborns of whom 194 were assessed with the Child Behavior Checklist (CBCL) at ages 4, 6, 9, 10, 11, and 12. Generalized estimating equation analyses were used to assess the relationship of quantity of FAEEs to outcomes, controlling for maternal psychological distress. RESULTS: Higher concentrations of FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, ethyl linoleate, and ethyl linolenate) were associated with caregiver reported aggressive and/or delinquent behavior at ages 10 and 12. After control for caregiver psychological distress, and age, significant (p < 0.05) FAEE by age interactions were found for ethyl myristate for aggression and for ethyl oleate, ethyl linoleate and ethyl linolenate for delinquency. Thus, higher concentrations of FAEE were related to more caregiver reported aggressive and delinquent behaviors of clinical significance at ages 10 and 12. CONCLUSION: Higher concentrations of FAEEs in meconium are potential markers for children at risk for aggressive and delinquent behaviors related to the effects of prenatal alcohol exposure.

Fatty acid ethyl esters in meconium and substance use in adolescence.

Prenatal alcohol exposure (PAE) continues to be a serious public health problem, yet no reliable clinical tools are available for assessing levels of drinking during pregnancy. Fatty acid ethyl esters (FAEEs), the nonoxidative metabolites of ethanol measured in meconium, are potential biomarkers to quantify the level of PAE. The association between the concentrations of FAEEs from meconium and adolescent substance use and related problems was examined in a prospective birth-cohort of adolescents exposed to alcohol and drugs in utero. FAEEs were quantified with gas chromatography via a flame ionization detector. Meconium was analyzed for FAEEs in 216 newborns; 183 of them (81 boys, 102 girls) were assessed at age 15 for alcohol, tobacco, and marijuana use using biologic assays and self-report. Substance use problems were assessed using the Problem Oriented Screening Instrument for Teenagers. Findings from multivariable logistic regression analyses indicated that, after controlling for other prenatal drug exposure and covariates, higher concentrations of FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, ethyl linoleate, ethyl linolenate, and ethyl arachidonate) were related to a greater likelihood of marijuana use and experiencing substance use problems, but not tobacco or alcohol use, at age 15. Elevated levels of FAEEs in meconium may be promising markers for PAE, identifying newborns at risk for early substance use and developing substance use problems.