We performed histologic and immunohistochemical analysis of cerebellar sections from a preterm infant (32 weeks 5 days) dead on the 4th day of life with the diagnosis of kernicterus and compared the results with 1 age-matched nonicteric patient. Poorer Luxol fast blue–periodic acid Schiff and Bodian-Luxol fast blue stainings as well as neurofilament expression were observed in the kernicterus case, indicating loss of axon neurites and myelin fibers. Elevated claudin-5 and cluster of differentiation 34 expression associated with increased blood vessel density suggests bilirubin-induced angiogenic sprouting. Upregulation of vascular endothelial growth factor and its receptor 2 was observed in nucleus dentatus and Purkinje neurons. Although upregulation of multidrug resistance–associated protein 1 was increased in cerebellar neurons, it was not able to prevent bilirubin-induced neurotoxicity. These data add new insights into the pathophysiology of kernicterus, revealing vascular endothelial growth factor and its receptor 2, as well as angiogenic sprouting, as new players in neurologic damage by unconjugated bilirubin.
BritoMASilvaRFMBritesD. Cell response to hyperbilirubinemia: a journey along key molecular events. In ChenFJ, ed. New Trends in Brain Research. New York: Nova Science; 2006:1–38.
2.
SmithermanHStarkARBhutaniVK. Early recognition of neonatal hyperbilirubinemia and its emergent management. Semin Fetal Neonatal Med. 2006;11(3):214–224.
BhutaniVKJohnsonL. Prevention of severe neonatal hyperbilirubinemia in healthy infants of 35 or more weeks of gestation: implementation of a systems-based approach. J Pediatr (Rio J). 2007;83(4):289–293.
5.
EbbesenF. Recurrence of kernicterus in term and near-term infants in Denmark. Acta Paediatr. 2000;89(10):1213–1217.
6.
HansenTWR. Kernicterus in term and near-term infants—the specter walks again. Acta Paediatr. 2000;89(10):1155–1157.
7.
PalmerRHKerenRMaiselsMJYeargin-AllsoppM. National Institute of Child Health and Human Development (NICHD) conference on kernicterus: a population perspective on prevention of kernicterus. J Perinatol. 2004;24(11):723–725.
8.
FalcãoASBellarosaCFernandesA. Role of multidrug resistance-associated protein 1 expression in the in vitro susceptibility of rat nerve cell to unconjugated bilirubin. Neuroscience. 2007;144(3):878–888.
9.
FalcãoASSilvaRFMPancadasSFernandesABritoMABritesD. Apoptosis and impairment of neurite network by short exposure of immature rat cortical neurons to unconjugated bilirubin increase with cell differentiation and are additionally enhanced by an inflammatory stimulus. J Neurosci Res. 2007;85(6):1229–1239.
10.
BritoMABritesD. Effect of acidosis on bilirubin-induced toxicity to human erythrocytes. Mol Cell Biochem. 2003;247(1-2):155–162.
11.
FernandesASilvaRFMFalcãoASBritoMABritesD. Cytokine production, glutamate release and cell death in rat cultured astrocytes treated with unconjugated bilirubin and LPS. J Neuroimmunol. 2004;153(1-2):64–75.
12.
VazARSilvaSLBarateiroA. Pro-inflammatory cytokines intensify the activation of NO/NOS, JNK1/2 and caspase cascades in immature neurons exposed to elevated levels of unconjugated bilirubin. Exp Neurol. 2011;229(2):381–390.
13.
DenneryPASeidmanDSStevensonDK. Neonatal hyperbilirubinemia. N Engl J Med. 2001;344(8):581–590.
14.
WatchkoJFMaiselsMJ. Jaundice in low birthweight infants: pathobiology and outcome. Arch Dis Child Fetal Neonatal Ed. 2003;88(6):F455–F458.
15.
BritoMARosaAIFalcãoAS, et al.Unconjugated bilirubin differentially affects the redox status of neuronal and astroglial cells. Neurobiol Dis. 2008;29(1):30–40.
16.
FernandesAFalcãoASAbranchesE. Bilirubin as a determinant for altered neurogenesis, neuritogenesis, and synaptogenesis. Dev Neurobiol. 2009;69(9):568–582.
17.
SilvaSLVazARBarateiroA. Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation. Neurobiol Dis. 2010;40(3):663–675.
18.
PalmelaICardosoFLBernasM. Elevated levels of bilirubin and long-term exposure impair human brain microvascular endothelial cell integrity. Curr Neurovasc Res. 2011;8(2):153–169.
19.
DulakJŁobodaAZagórskaAJózkowiczA. Complex role of heme oxygenase-1 in angiogenesis. Antioxid Redox Signal. 2004;6(5):858–866.
20.
CardosoFLBritesDBritoMA. Looking at the blood-brain barrier: molecular anatomy and possible investigation approaches. Brain Res Rev. 2010;64(2):328–363.
21.
PerlmanJMRogersBBBurnsD. Kernicteric findings at autopsy in two sick near term infants. Pediatrics. 1997;99(4):612–615.
22.
GkoltsiouKTzoufiMCounsellSRutherfordMCowanF. Serial brain MRI and ultrasound findings: relation to gestational age, bilirubin level, neonatal neurologic status and neurodevelopmental outcome in infants at risk of kernicterus. Early Hum Dev. 2008;84(12):829–838.
23.
SchuttaHSJohnsonL. Bilirubin encephalopathy in the Gunn rat: a fine structure study of the cerebellar cortex. J Neuropathol Exp Neurol. 1967;26(3):377–396.
24.
LinSWeiXBalesKR. Minocycline blocks bilirubin neurotoxicity and prevents hyperbilirubinemia-induced cerebellar hypoplasia in the Gunn rat. Eur J Neurosci. 2005;22(1):21–27.
25.
AronicaEGorterJAJansenGH. Expression and cellular distribution of multidrug transporter proteins in two major causes of medically intractable epilepsy: focal cortical dysplasia and glioneuronal tumors. Neuroscience. 2003;118(2):417–429.
26.
BoerKTroostDJansenF. Clinicopathological and immunohistochemical findings in an autopsy case of tuberous sclerosis complex. Neuropathology. 2008;28(6):577–590.
27.
BellarosaCBortolussiGTiribelliC. The role of ABC transporters in protecting cells from bilirubin toxicity. Curr Pharm Des. 2009;15(25):2884–2892.
28.
WennbergRP.The blood-brain barrier and bilirubin encephalopathy.
Cell Mol Neurobiol. 2000;20(1):97–109.
29.
BurgessGHOhWBratlidD. The effects of brain blood flow on brain bilirubin deposition in newborn piglets. Pediatr Res. 1985;19(7):691–696.
30.
LiJStuhlmannH. In vitro imaging of angiogenesis using embryonic stem cell-derived endothelial cells. Stem Cells Dev. 2011[published ahead of print April 27, doi:10.1089/scd.2010.0587].
31.
RaimondiFCrivaroVCapassoL. Unconjugated bilirubin modulates the intestinal epithelial barrier function in a human-derived in vitro model. Pediatr Res. 2006;60(1):30–33.
32.
McCallICBetanzosAWeberDANavaPMillerGWParkosCA. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization. Toxicol Appl Pharmacol. 2009;241(1):61–70.
33.
BritoMABrondinoCDMouraJJGBritesD. Effects of bilirubin molecular species on membrane dynamic properties of human erythrocyte membranes: a spin label electron paramagnetic resonance spectroscopy study. Arch Biochem Biophys. 2001;387(1):57–65.
34.
RodriguesCMPSoláSBritoMABritesDMouraJJG. Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria. J Hepatol. 2002;36(3):335–341.
35.
PagèsGPouysségurJ. Transcriptional regulation of the vascular endothelial growth factor gene—a concert of activating factors. Cardiovasc Res. 2005;65(3):564–573.
36.
BritesDFernandesAFalcãoASGordoACSilvaRFMBritoMA. Biological risks for neurological abnormalities associated with hyperbilirubinemia. J Perinatol. 2009;29(suppl 1):S8–S13.
37.
HachiyaYHayashiM. Bilirubin encephalopathy: a study of neuronal subpopulations and neurodegenerative mechanisms in 12 autopsy cases. Brain Dev. 2008;30(4):269–278.
38.
WatchkoJF. Kernicterus and the molecular mechanisms of bilirubin-induced CNS injury in newborns. Neuromolecular Med. 2006;8(4):513–529.
39.
DaoodMTsaiCAhdab-BarmadaMWatchkoJF. ABC transporter (P-gp/ABCB1, MRP1/ABCC1, BCRP/ABCG2) expression in the developing human CNS. Neuropediatrics. 2008;39(4):211–218.
40.
NagSManiasJLStewartDJ. Pathology and new players in the pathogenesis of brain edema. Acta Neuropathol. 2009;118(2):197–217.
41.
RoscoeWAWelshMECarterDEKarlikSJ. VEGF and angiogenesis in acute and chronic MOG((35-55)) peptide induced EAE. J Neuroimmunol. 2009;209(1-2):6–15.
42.
Martich-KrissVKolliasSSBallWS Jr. MR findings in kernicterus. AJNR Am J Neuroradiol. 1995;16(4suppl):819–821.
43.
LoftspringMCJohnsonHLFengRJohnsonAJClarkJF. Unconjugated bilirubin contributes to early inflammation and edema after intracerebral hemorrhage. J Cereb Blood Flow Metab. 2011;31(4):1133–1142.
44.
CorichLArandaACarrassaLBellarosaCOstrowJDTiribelliC. The cytotoxic effect of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells is modulated by the expression level of MRP1 but not MDR1. Biochem J. 2009;417(1):305–312.
45.
GazzinSBerengenoALStrazielleNModulation of Mrp1 (ABCc1) and Pgp (ABCb1) by bilirubin at the blood-CSF and blood-brain barriers in the Gunn rat. PLoS One2011;6(1):e16165.
46.
ConleeJWShapiroSM. Development of cerebellar hypoplasia in jaundiced Gunn rats: a quantitative light microscopic analysis. Acta Neuropathol. 1997;93(5):450–460.
