Reduced Myelination and Increased Glia Reactivity Resulting from Severe Neonatal Hyperbilirubinemia.
ABSTRACT: Bilirubin-induced neurologic dysfunction (BIND) and kernicterus has been used to describe moderate to severe neurologic dysfunction observed in children exposed to excessive levels of total serum bilirubin (TSB) during the neonatal period. Here we use a new mouse model that targets deletion of the Ugt1 locus and the Ugt1a1 gene in liver to promote hyperbilirubinemia-induced seizures and central nervous system toxicity. The accumulation of TSB in these mice leads to diffuse yellow coloration of brain tissue and a marked cerebellar hypoplasia that we characterize as kernicterus. Histologic studies of brain tissue demonstrate that the onset of severe neonatal hyperbilirubinemia, characterized by seizures, leads to alterations in myelination and glia reactivity. Kernicterus presents as axonopathy with myelination deficits at different brain regions, including pons, medulla oblongata, and cerebellum. The excessive accumulation of TSB in the early neonatal period (5 days after birth) promotes activation of the myelin basic protein (Mbp) gene with an accelerated loss of MBP that correlates with a lack of myelin sheath formation. These changes were accompanied by increased astroglial and microglial reactivity, possibly as a response to myelination injury. Interestingly, cerebellum was the area most affected, with greater myelination impairment and glia burden, and showing a marked loss of Purkinje cells and reduced arborization of the remaining ones. Thus, kernicterus in this model displays not only axonal damage but also myelination deficits and glial activation in different brain regions that are usually related to the neurologic sequelae observed after severe hyperbilirubinemia.
Project description:Importance:Neonatal hyperbilirubinemia can cause lifelong neurodevelopmental impairment (kernicterus) even in high-resource settings. A better understanding of the incidence and processes leading to kernicterus may help in the design of preventive measures. Objectives:To determine incidence rates of hazardous hyperbilirubinemia and kernicterus among near-term to term newborns and to evaluate health care professional adherence to best practices. Design, Setting, and Participants:This population-based nationwide cohort study used prospectively collected data on the highest serum bilirubin level for all infants born alive at 35 weeks' gestation or longer and admitted to neonatal care at all 46 delivery and 37 neonatal units in Sweden from 2008 to 2016. Medical records for newborns with hazardous hyperbilirubinemia were evaluated for best neonatal practices and for a diagnosis of kernicterus up to 2 years of age. Data analyses were performed between September 2017 and February 2018. Exposures:Extreme (serum bilirubin levels, 25.0-29.9 mg/dL [425-509 μmol/L]) and hazardous (serum bilirubin levels, ≥30.0 mg/dL [≥510 μmol/L]) neonatal hyperbilirubinemia. Main Outcomes and Measures:The primary outcome was kernicterus, defined as hazardous neonatal hyperbilirubinemia followed by cerebral palsy, sensorineural hearing loss, gaze paralysis, or neurodevelopmental retardation. Secondary outcomes were health care professional adherence to national guidelines using a predefined protocol with 10 key performance indicators for diagnosis and treatment as well as assessment of whether bilirubin-associated brain damage might have been avoidable. Results:Among 992 378 live-born infants (958 051 term births and 34 327 near-term births), 494 (320 boys; mean [SD] birth weight, 3505  g) developed extreme hyperbilirubinemia (50 per 100 000 infants), 6.8 per 100 000 infants developed hazardous hyperbilirubinemia, and 1.3 per 100 000 infants developed kernicterus. Among 13 children developing kernicterus, brain injury was assessed as potentially avoidable for 11 children based on the presence of 1 or several of the following possible causes: untimely or lack of predischarge bilirubin screening (n = 6), misinterpretation of bilirubin values (n = 2), untimely or delayed initiation of treatment with intensive phototherapy (n = 1), untimely or no treatment with exchange transfusion (n = 6), or lack of repeated exchange transfusions despite indication (n = 1). Conclusions and Relevance:Hazardous hyperbilirubinemia in near-term or term newborns still occurs in Sweden and was associated with disabling brain damage in 13 per million births. For most of these cases, health care professional noncompliance with best practices was identified, suggesting that a substantial proportion of these cases might have been avoided.
Project description:Neurotoxic bilirubin is solely conjugated by UDP-glucuronosyltransferase (UGT) 1A1. Due to an inadequate function of UGT1A1, human neonates develop mild to severe physiological hyperbilirubinemia. Accumulation of bilirubin in the brain leads to the onset of irreversible brain damage called kernicterus. Breastfeeding is one of the most significant factors that increase the risk of developing kernicterus in infants. Why does the most natural way of feeding increase the risk of brain damage or even death? This question leads to the hypothesis that breast milk-induced neonatal hyperbilirubinemia might bring certain benefits to the body. One of the barriers to answering the above question is the lack of animal models that display mild to severe neonatal hyperbilirubinemia. A mouse model that develops neonatal hyperbilirubinemia was previously developed by a knockout of the Ugt1 locus. Deletion of Ugt1a1 results in neonatal lethality from bilirubin neurotoxicity. Bilirubin is the end product of heme catabolism in which heme oxygenase-I is largely involved. When zinc protoporphyrin, an inhibitor of heme oxygenase I, was administered to newborn Ugt1-/- mice, serum bilirubin levels dropped dramatically, rescuing the mice from bilirubin-induced neonatal lethality. Zinc protoporphyrin-treated Ugt1-/- mice developed normally as adults capable of reproducing, but their newborns showed even more severe hyperbilirubinemia. Microarray analysis of the hyperbilirubinemic livers indicated that a number of genes associated with nucleotide, transport, and immune response were significantly down-regulated in a serum bilirubin level-dependent manner. Conclusion: Our study provides an opportunity to advance the development of effective therapeutics to effectively and rapidly prevent bilirubin-induced toxicity. Neonatal hyperbilirubinemia has various impacts on the body that could be driven by the antioxidant property of bilirubin.
Project description:Crigler-Najjar syndrome type I (CN-I, MIM #218800) is a rare and severe autosomal disorder. It is caused by deficiency of the liver enzyme responsible for bilirubin elimination, the uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1; EC 126.96.36.199). Biologically, the disease manifests itself with severe and persistent unconjugated hyperbilirubinemia. Kernicterus is a well-known complication of severe unconjugated hyperbilirubinemia in infants and young children, especially in patients with CN-I.Few articles have shown the efficiency of plasmapheresis for extreme hyperbilirubinemia.In this report, we describe the efficiency of plasmapheresis for a rapid control of acute and severe unconjugated hyperbilirubinemia in a 6-year-old CN-I patient who had previously developed kernicterus in the neonatal period. In spite of intensification of phototherapy, the patient developed severe hyperbilirubinemia (up to 830 ?mol/l, with bilirubin/albumin ratio at 1.2). With two plasmapheresis procedures, bilirubin serum concentration decreased to 420 ?mol/ and bilirubin/albumin ratio to 0.55. Following the acute episode of very severe unconjugated hyperbilirubinemia, the child recovered and neurological examination was unchanged, thus suggesting that plasmapheresis possibly prevented further worsening of kernicterus.
Project description:Unconjugated bilirubin is considered a potent antioxidant when present at moderate levels. However, at high concentrations, it produces severe neurological damage and death associated with kernicterus due to oxidative stress and other mechanisms. While it is widely recognized that oxidative stress by different toxic insults results in severe damage to cellular macromolecules, especially to DNA, no data are available either on DNA damage in the brain triggered by hyperbilirubinemia during the neonatal period or on the activation of DNA repair mechanisms. Here, using a mouse model of neonatal hyperbilirubinemia, we demonstrated that DNA damage occurs <i>in vivo</i> in the cerebellum, the brain region most affected by bilirubin toxicity. We studied the mechanisms associated with potential toxic action of bilirubin on DNA in <i>in vitro</i> models, which showed significant increases in DNA damage when neuronal and nonneuronal cells were treated with 140?nM of free bilirubin (Bf), as determined by <i>?</i>H2AX Western blot and immunofluorescence analyses. Cotreatment of cells with N-acetyl-cysteine, a potent oxidative-stress inhibitor, prevented DNA damage by bilirubin, supporting the concept that DNA damage was caused by bilirubin-induced oxidative stress. Bilirubin treatment also activated the main DNA repair pathways through homologous recombination (HR) and nonhomologous end joining (NHEJ), which may be adaptive responses to repair bilirubin-induced DNA damage. Since DNA damage may be another important factor contributing to neuronal death and bilirubin encephalopathy, these results contribute to the understanding of the mechanisms associated with bilirubin toxicity and may be of relevance in neonates affected with severe hyperbilirubinemia.
Project description:<h4>Objectives</h4>This study was intended to explore the etiology and risk factors of severe neonatal hyperbilirubinemia and to analyze the adverse events associated with ECT (Exchange Transfusion), as well as to identify the factors related to the poor prognosis.<h4>Methods</h4>All of the full-term neonates who had undergone ECT for hyperbilirubinemia at Children's Hospital of Chongqing Medical University from January 2001 to December 2011 were enrolled in this study. General demographic characteristics, comorbidities, pre- and post-exchange TSB(Total Serum Bilirubin) levels, duration and frequency of ECT, and clinical outcomes were recorded and analyzed anonymously.<h4>Results</h4>Of 614 total infants, 368 patients (59.9%) with ABO incompatibility were identified, of whom 197 (53.5%) developed acute bilirubin encephalopathy (ABE) and 16 (4.3%) suffered a poor prognosis. The etiology was unidentified in 103 patients (16.8%), of whom 62 (60.1%) developed ABE and 9 (8.7%) had a poor prognosis. Identified adverse events secondary to ECT included thrombocytopenia (54.6%), hyperglycemia (42.8%), apnea (3.3%) and necrotizing enterocolitis (NEC) (1.3%). No ECT-related mortality was documented in this study.<h4>Conclusions</h4>The etiology, peak TSB level before ECT, and time of ECT had a significant impact on the outcome of severe neonatal hyperbilirubinemia. ABO incompatibility was the most common cause of extreme neonatal hyperbilirubinemia. Pathological weight loss could be involved in the development of extreme hyperbilirubinemia with an unidentified cause.
Project description:The recognition, follow-up, and early treatment of neonatal jaundice has become more difficult, since the earlier discharge of newborns from hospitals has become common practice. Since intrapartum hypoxic stress has been pointed as predisposing factor for the occurrence of hyperbilirubinemia risk, we tested the association with the cord blood acid-base index tests.A cohort of healthy term and near-term newborns underwent umbilical cord hemogasanalysis at birth and capillary heel total serum bilirubin (TSB) pre-discharge, scheduled at 36 h of life, to define the risk of significant hyperbilirubinemia, defined as >9 mg/dL TSB level, ≥ 75th percentile on nomogram of Bhutani et al.It was found that among 537 studied neonates, 133 (24.8%) had pre-discharge TSB levels of >9 mg/dL. When the cord blood gas analysis index tests were compared, their acidemia levels were significantly higher than those of neonates with normal TSB levels: HCO3- (20.71 ± 2.37 vs. 21.29 ± 2.25 mEq/L, p < 0.010), base deficit (-3.52 ± 3.188 vs. -2.68 ± 3.266 mEq/L, p < 0.010), and lactacidemia (3.84 ± 1.864 vs. 3.39 ± 1.737 mEq/L, p < 0.012), respectively. However, logistic regression analysis showed that base deficit was the strongest index of the pre-discharge hyperbilirubinemia risk (OR, 95% CI 0.593; 0.411-0.856), and the hyperbilirubinemia risk increased by 40% with the decrease of 1 mEq/L of base deficit.Umbilical cord blood acidemia and lactacidemia are significant indexes of adaptive mechanisms at birth. The base deficit provides the strongest association with future development of high bilirubin on an hour specific bilirubin nomogram generating risk stratification score in term and near-term neonates.
Project description:Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced neurological damage and eventually death by kernicterus. Bilirubin neurotoxicity is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, due to bilirubin accumulation in the brain. Despite the abundant literature documenting the in vitro and in vivo toxic effects of bilirubin, it is unclear which molecular and cellular events actually characterize bilirubin-induced neurodegeneration in vivo.We used a mouse model of neonatal hyperbilirubinemia to temporally and spatially define the response of the developing cerebellum to the bilirubin insult.We showed that the exposure of developing cerebellum to sustained bilirubin levels induces the activation of oxidative stress, ER stress and inflammatory markers at the early stages of the disease onset. In particular, we identified TNF? and NFK? as key mediators of bilirubin-induced inflammatory response. Moreover, we reported that M1 type microglia is increasingly activated during disease progression. Failure to counteract this overwhelming stress condition resulted in the induction of the apoptotic pathway and the generation of the glial scar. Finally, bilirubin induced the autophagy pathway in the stages preceding death of the animals.This study demonstrates that inflammation is a key contributor to bilirubin damage that cooperates with ER stress in the onset of neurotoxicity. Pharmacological modulation of the inflammatory pathway may be a potential intervention target to ameliorate neonatal lethality in Ugt1 -/- mice.
Project description:Therapies to prevent severe neonatal unconjugated hyperbilirubinemia and kernicterus are phototherapy and, in unresponsive cases, exchange transfusion, which has significant morbidity and mortality risks. Neurotoxicity is caused by the fraction of unconjugated bilirubin not bound to albumin (free bilirubin, Bf). Human serum albumin (HSA) administration was suggested to increase plasma bilirubin-binding capacity. However, its clinical use is infrequent due to difficulties to address its potential preventive and curative benefits, and to the absence of reliable markers to monitor bilirubin neurotoxicity risk. We used a genetic mouse model of unconjugated hyperbilirubinemia showing severe neurological impairment and neonatal lethality. We treated mutant pups with repeated HSA administration since birth, without phototherapy application. Daily intraperitoneal HSA administration completely rescued neurological damage and lethality, depending on dosage and administration frequency. Albumin infusion increased plasma bilirubin-binding capacity, mobilizing bilirubin from tissues to plasma. This resulted in reduced plasma Bf, forebrain and cerebellum bilirubin levels. We showed that, in our experimental model, Bf is the best marker to determine the risk of developing neurological damage. These results support the potential use of albumin administration in severe acute hyperbilirubinemia conditions to prevent or treat bilirubin neurotoxicity in situations in which exchange transfusion may be required.
Project description:Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.
Project description:Jaundice may cause auditory toxicity (auditory neuropathy and hearing loss). However, total serum bilirubin (TSB) does not discriminate neonates at risk for auditory toxicity. We compared TSB, bilirubin:albumin molar ratio (BAMR), and unbound bilirubin for their association with auditory toxicity in neonates with severe jaundice (TSB ?342?mol/L, or that met exchange transfusion).Neonates greater or equal to 34 weeks gestational age with severe jaundice during the first 2 postnatal weeks were eligible for prospective cohort study, unless they had craniofacial malformations, chromosomal disorders, toxoplasmosis, other infections, rubella, cytomegalovirus, herpes simplex infections, surgery, or family history of congenital deafness.Twenty-eight out of 100 neonates (mean gestational age 37.4wks; 59 males, 41 females) had auditory toxicity. Peak unbound bilirubin, but not peak TSB and BAMR, was associated with auditory toxicity (p<0.05) in neonates with severe (TSB <427.5?mol/L) and extreme hyperbilirubinemia (TSB ?427.5?mol/L). Area under the receiver operating characteristic curve for unbound bilirubin (0.78) was significantly greater (p=0.03) than TSB (0.54) among neonates with severe but not extreme hyperbilirubinemia.Unbound bilirubin is more strongly associated with auditory toxicity than TSB and/or BAMR in greater or equal to 34 weeks gestational age neonates with severe jaundice. Unbound bilirubin is a better predictor than TSB in neonates with severe hyperbilirubinemia.