Bilirubin-induced Neurotoxicity Research Articles

Bilirubin-Induced Transcriptomic Imprinting in Neonatal Hyperbilirubinemia.

Recent findings indicated aberrant epigenetic control of the central nervous system (CNS) development in hyperbilirubinemic Gunn rats as an additional cause of cerebellar hypoplasia, the landmark of bilirubin neurotoxicity in rodents. Because the symptoms in severely hyperbilirubinemic human neonates suggest other regions as privileged targets of bilirubin neurotoxicity, we expanded the study of the potential impact of bilirubin on the control of postnatal brain development to regions correlating with human symptoms. Histology, transcriptomic, gene correlation, and behavioral studies were performed. The histology revealed widespread perturbation 9 days after birth, restoring in adulthood. At the genetic level, regional differences were noticed. Bilirubin affected synaptogenesis, repair, differentiation, energy, extracellular matrix development, etc., with transient alterations in the hippocampus (memory, learning, and cognition) and inferior colliculi (auditory functions) but permanent changes in the parietal cortex. Behavioral tests confirmed the presence of a permanent motor disability. The data correlate well both with the clinic description of neonatal bilirubin-induced neurotoxicity, as well as with the neurologic syndromes reported in adults that suffered neonatal hyperbilirubinemia. The results pave the way for better deciphering the neurotoxic features of bilirubin and evaluating deeply the efficacy of new therapeutic approaches against the acute and long-lasting sequels of bilirubin neurotoxicity.

Microglial Priming in Bilirubin-Induced Neurotoxicity.

Neuroinflammation is a major contributor to bilirubin-induced neurotoxicity, which results in severe neurological deficits. Microglia are the primary immune cells in the brain, with M1 microglia promoting inflammatory injury and M2 microglia inhibiting neuroinflammation. Controlling microglial inflammation could be a promising therapeutic strategy for reducing bilirubin-induced neurotoxicity. Primary microglial cultures were prepared from 1-3-day-old rats. In the early stages of bilirubin treatment, pro-/anti-inflammatory (M1/M2) microglia mixed polarization was observed. In the late stages, bilirubin persistence induced dominant proinflammatory microglia, forming an inflammatory microenvironment and inducing iNOS expression as well as the release of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Simultaneously, nuclear factor-kappa B (NF-κB) was activated and translocated into the nucleus, upregulating inflammatory target genes. As well known, neuroinflammation can have an effect on N-methyl-D-aspartate receptor (NMDAR) expression or function, which is linked to cognition. Treatment with bilirubin-treated microglia-conditioned medium did affect the expression of IL-1β, NMDA receptor subunit 2A (NR2A), and NMDA receptor subunit 2B (NR2B) in neurons. However, VX-765 effectively reduces the levels of proinflammatory cytokines TNF-α, IL-6, and IL-1β, as well as the expressions of CD86, and increases the expressions of anti-inflammatory related Arg-1. A timely reduction in proinflammatory microglia could protect against bilirubin-induced neurotoxicity.

Effects of lipid emulsions on unbound bilirubin and response to phototherapy in preterm infants

Objective Bilirubin-induced neurotoxicity is mediated by the fraction of total serum bilirubin (TSB) not bound to albumin (Bf). Unbound free fatty acids (FFAu) generated from lipid emulsions compete with bilirubin for albumin binding, increasing Bf. Soy-based (IL) and soy-MCT-olive-fish oil-based (SMOF) lipid emulsions contain different fatty acids with distinct albumin binding affinities. IL increases Bf in preterm infants, but the effects of SMOF on Bf are not known. Our objective was to compare changes in TSB, Bf, FFAu, and response to phototherapy in preterm infants receiving SMOF and IL. We hypothesized that SMOF would be associated with lower Bf and better response to phototherapy than IL. Methods Very preterm and low birth weight infants (<1500 g, <32 weeks) were infused with IL (n = 20) or SMOF (n = 20) as prescribed by providers. Phototherapy was prescribed using the standard care practice. FFAu profiles and levels, TSB, and Bf were measured on 0, 1, 2, and 3 g/kg/day of lipid infusion and at the initiation and termination of phototherapy. TSB was analyzed in the clinical laboratory using the diazo technique. FFAu and Bf were measured using fluorescent probes. Results Escalating doses of IL and SMOF increased FFAu levels and Bf, but not TSB. Phototherapy did not significantly decrease Bf for infants receiving either lipid. IL-treated infants had higher levels of unbound linoleic acid, and SMOF-treated infants had higher unbound arachidonic, oleic, and docosahexaenoic acids. Conclusions IL and SMOF both increase Bf similarly, and phototherapy does not significantly affect Bf for infants receiving them.

Chemoprevention of bilirubin encephalopathy with a nanoceutical agent.

Targeted rapid degradation of bilirubin has the potential to thwart incipient bilirubin encephalopathy. We investigated a novel spinel-structured citrate-functionalized trimanganese tetroxide nanoparticle (C-Mn3O4 NP, the nanodrug) to degrade both systemic and neural bilirubin loads. Severe neonatal unconjugated hyperbilirubinemia (SNH) was induced in neonatal C57BL/6j mice model with phenylhydrazine(PHz) intoxication. Efficiency of the nanodrug on both in vivo bilirubin degradation and amelioration of bilirubin encephalopathy and associated neurobehavioral sequelae were evaluated. Single oral dose (0.25 mg kg-1 bodyweight) of the nanodrug reduced both total serum bilirubin (TSB) and unconjugated bilirubin (UCB) in SNH rodents. Significant (p < 0.0001) UCB and TSB-degradation rates were reported within 4-8 h at 1.84 ± 0.26 and 2.19 ± 0.31 mg dL-1 h-1, respectively. Neural bilirubin load was decreased by 5.6 nmol g-1 (p = 0.0002) along with improved measures of neurobehavior, neuromotor movements, learning, and memory. Histopathological studies confirm that the nanodrug prevented neural cell reduction in Purkinje and substantia nigra regions, eosinophilic neurons, spongiosis, and cell shrinkage in SNH brain parenchyma. Brain oxidative status was maintained in nanodrug-treated SNH cohort. Pharmacokinetic data corroborated the bilirubin degradation rate with plasma nanodrug concentrations. This study demonstrates the in vivo capacity of this novel nanodrug to reduce systemic and neural bilirubin load and reverse bilirubin-induced neurotoxicity. Further compilation of a drug-safety-dossier is warranted to translate this novel therapeutic chemopreventive approach to clinical settings. None of the current pharmacotherapeutics treat severe neonatal hyperbilirubinemia (SNH) to prevent risks of neurotoxicity. In this preclinical study, a newly investigated nano-formulation, citrate-functionalized Mn3O4 nanoparticles (C-Mn3O4 NPs), exhibits bilirubin reduction properties in rodents. Chemopreventive properties of this nano-formulation demonstrate an efficacious, efficient agent that appears to be safe in these early studies. Translation of C-Mn3O4 NPs to prospective preclinical and clinical trials in appropriate in vivo models should be explored as a potential novel pharmacotherapy for SNH.

Activin A is Not a Reliable Prognostic Biomarker For Bilirubin Induced Neurotoxicity in Neonates

Activin A is Not a Reliable Prognostic Biomarker For Bilirubin Induced Neurotoxicity in Neonates

Bilirubin-induced neurotoxicity and visuocortical dysfunction

Bilirubin-induced neurotoxicity and visuocortical dysfunction

HP01: Usefulness of auditory brainstem evoked response in the assessment of bilirubin-induced auditory toxicity in infants with jaundice: A case-control study

To assess ABR alterations due to hyperbilirubinemia in jaundiced infants compared to a control group. Background: Bilirubin-induced neurotoxicity has predilection to specific areas of the brain including the auditory pathway structures. These latter are assessed by auditory brainstem evoked response (ABR). Methods: This was a retrospective study including two groups: jaundiced infants (G1) and age-matched (p>0.05) infants without jaundice (G2). All infants underwent ABR on each ear to screen deafness. Responses obtained at 90 dB were studied. Responses of G2 were normal. The Mann-Whitney U test was used to compare the responses between the two groups. Results: Twenty infants (40 ears) were included with a mean age of 33.5±13 months. G1 and G2 included 18 and 20 ears, respectively. Wave-I latency was significantly more prolonged in G1 (1.71±0.2 ms) compared to G2 (1.56±0.1 ms) with p=0.02. There was no difference in I-III interpeak interval (IPL). The III-V IPL was significantly increased in G1 (2.04±0.1 ms) compared to G2 (1.92±0.1 ms) with p=0.04. I-V IPL was more prolonged in G1. Discussion: Both peripheral and central auditory pathways may be altered in infants with jaundice. Bilirubin-induced toxicity may affect the inner part of the ear. The central dysfunction may occur at the brainstem level between the bulbo-protuberant junction and the inferior colliculus. This may be due to the hyperbilirubinemia-induced changes in the central synaptic transmission. Conclusion: Hyperbilirubinemia-induced auditory toxicity can occur at different levels of the auditory pathway. Therefore, there is a need for an early ABR assessment in jaundiced infants.

Animal Kernicterus Models: Progress and Challenges

Kernicterus is a leading cause of neonatal death throughout the world, especially in low-middle-income countries. It is developed by an unconjugated hyperbilirubinemia in the blood and brain tissue, triggering pathological processes that spawn neurotoxicity and neurodegeneration. However, the biological mechanism (s) of bilirubin-induced neurotoxicity and Kernicterus development remain to be well elucidated. Likewise, a practical therapeutic approach for human Kernicterus has yet to be found. Undoubtedly, animal models of Kernicterus can be helpful in the identification of underlying biological processes of hyperbilirubinemia evolution to Kernicterus, as well as the evaluation of various treatments efficacy in preclinical studies. More importantly, establishing an animal model that can mimic the Kernicterus and its behavioral, neuro-histological, and hematological manifestations is a severe priority in preclinical studies. So far, several Kernicterus animal models have been established that could partially mimic one or more clinical and paraclinical signs of human Kernicterus. The present study aimed to review all methods modeling Kernicterus with a focus on their potentials and shortcomings and subsequently provide the optimal methods for an ideal Kernicterus animal model.

Minocycline protects neurons against glial cells-mediated bilirubin neurotoxicity

Unconjugated bilirubin, the end product of heme catabolism and antioxidant, induced brain damage in human neonates is a well-recognized clinical syndrome. However, the cellular and molecular mechanisms underlying bilirubin neurotoxicity remain unclear. To characterize the sequence of events leading to bilirubin-induced neurotoxicity, we investigated whether bilirubin-induced glial activation was involved in bilirubin neurotoxicity by exposing co-cultured rat glial cells and cerebellar granule neurons (CGN) to bilirubin. We found that bilirubin could markedly induce the expression of TNF-α and iNOS in glial cells, and even at low concentrations, the co-culture of glial cells with neurons significantly enhances neurotoxicity of bilirubin. Pretreatment of the co-cultured cells with minocycline protected CGN from glia-mediated bilirubin neurotoxicity and inhibited overexpression of TNF-α and iNOS in glia. Furthermore, we found that high doses of bilirubin were able to induce glial injury, and minocycline attenuated bilirubin-induced glial cell death. Our data suggest that glial cells play an important role in brain damage caused by bilirubin, and minocycline blocks bilirubin-induced encephalopathy possibly by directly and indirectly inhibiting neuronal death pathways.

Experimental models assessing bilirubin neurotoxicity.

The molecular and cellular events leading to bilirubin-induced neurotoxicity, the mechanisms regulating liver and intestine expression in neonates, and alternative pathways of bilirubin catabolism remain incompletely defined. To answer these questions, researchers have developed a number of model systems to closely recapitulate the main characteristics of the disease, ranging from tissue cultures to engineered mouse models. In the present review we describe in vitro, ex vivo, and in vivo models developed to study bilirubin metabolism and neurotoxicity, with a special focus on the use of engineered animal models. In addition, we discussed the most recent studies related to potential therapeutic approaches to treat neonatal hyperbilirubinemia, ranging from anti-inflammatory drugs, activation of nuclear receptor pathways, blockade of bilirubin catabolism, and stimulation of alternative bilirubin-disposal pathways.

Bilirubin-induced neurotoxic and ototoxic effects in rat cochlear and vestibular organotypic cultures

Exposure to high levels of bilirubin in hyperbilirubinemia patients and animal models can result in sensorineural deafness. However, the mechanisms underlying bilirubin-induced damage to the inner ear, including the cochlear and vestibular organs, remain unknown. The present analyses of cochlear and vestibular organotypic cultures obtained from postnatal day 3 rats exposed to bilirubin at varying concentrations (0, 10, 50, 100, or 250 μM) for 24 h revealed that auditory nerve fibers (ANFs) and vestibular nerve endings were destroyed even at low doses (10 and 50 μM). Additionally, as the bilirubin dose increased, spiral ganglion neurons (SGNs) and vestibular ganglion neurons (VGNs) exhibited gradual shrinkage in conjunction with nuclei condensation or fragmentation in a dose-dependent manner. The loss of cochlear and vestibular hair cells (HCs) was only evident in explants treated with the highest concentration of bilirubin (250 μM), and bilirubin-induced major apoptosis most likely occurred via the extrinsic apoptotic pathway. Thus, the present results indicate that inner ear neurons and fibers were more sensitive to, and exhibited more severe damage following, bilirubin-induced neurotoxicity than sensory HCs, which illustrates the underlying causes of auditory neuropathy and vestibulopathy in hyperbilirubinemia patients.

High unbound bilirubin for age: a neurotoxin with major effects on the developing brain.

Neonatal hyperbilirubinemia is one of the most frequent diagnoses made in neonates. A high level of unconjugated bilirubin that is unbound to albumin is neurotoxic when the level exceeds age-specific thresholds or at lower levels in neonates with neurotoxic risk factors. Lower range of unbound bilirubin results in apoptosis, while moderate-to-high levels result in neuronal necrosis. Basal ganglia and various brain stem nuclei are more susceptible to bilirubin toxicity. Proposed mechanisms of bilirubin-induced neurotoxicity include excessive release of glutamate, mitochondrial energy failure, release of proinflammatory cytokines, and increased intracellular calcium concentration. These mechanisms are similar to the events that occur following hypoxic-ischemic insult in neonates. Severe hyperbilirubinemia in term neonates has been shown to be associated with increased risk for autism spectrum disorders. The neuropathological finding of bilirubin-induced neurotoxicity also includes cerebellar injury with a decreased number of Purkinje cells, and disruption of multisensory feedback loop between cerebellum and cortical neurons which may explain the clinical characteristics of autism spectrum disorders. Severe hyperbilirubinemia occurs more frequently in infants from low- and middle-income countries (LMIC). Simple devices to measure bilirubin, and timely treatment are essential to reduce neurotoxicity, and improve outcomes for thousands of neonates around the world.

Developmental influence of unconjugated hyperbilirubinemia and neurobehavioral disorders.

Bilirubin-induced brain injury in the neonatal period has detrimental effects on neurodevelopment that persist into childhood and adulthood, contributing to childhood developmental disorders. Unconjugated bilirubin is a potent antioxidant that may be useful for protecting against oxidative injuries, but it becomes a potent neurotoxin once it crosses the blood brain barrier. Because bilirubin toxicity involves a myriad of pathological mechanisms, can damage most types of brain cells, and affects brain circuits or loops that influence cognition, learning, behavior, sensory, and language, the clinical effects of bilirubin-induced neurotoxicity are likely to be manifold. One possible effect that several experts have identified is bilirubin-induced neurological dysfunction (subtle kernicterus). However, the underlying biological mechanisms or pathways by which subtle kernicterus could lead to developmental disorders has not been elucidated previously. Our aim in this review is to describe a spectrum of developmental disorders that may reflect subtle kernicterus and outline plausible biological mechanisms for this possible association. We review existing evidence that support or refute the association between unconjugated hyperbilirubinemia and developmental disorders, and limitations associated with these studies.

Histone acetylation as a new mechanism for bilirubin-induced encephalopathy in the Gunn rat

Bilirubin neurotoxicity has been studied for decades and has been shown to affect various mechanisms via significant modulation of gene expression. This suggests that vital regulatory mechanisms of gene expression, such as epigenetic mechanisms, could play a role in bilirubin neurotoxicity. Histone acetylation has recently received attention in the CNS due to its role in gene modulation for numerous biological processes, such as synaptic plasticity, learning, memory, development and differentiation. Aberrant epigenetic regulation of gene expression in psychiatric and neurodegenerative disorders has also been described. In this work, we followed the levels of histone 3 lysine 14 acetylation (H3K14Ac) in the cerebellum (Cll) of the developing (2, 9, 17 days after the birth) and adult Gunn rat, the natural model for neonatal hyperbilirubinemia and kernicterus. We observed an age-specific alteration of the H3K14Ac in the hyperbilirubinemic animals. The GeneOntology analysis of the H3K14Ac linked chromatin revealed that almost 45% of H3K14Ac ChiP-Seq TSS-promoter genes were involved in CNS development including maturation and differentiation, morphogenesis, dendritogenesis, and migration. These data suggest that the hallmark Cll hypoplasia in the Gunn rat occurs also via epigenetically controlled mechanisms during the maturation of this brain structure, unraveling a novel aspect of the bilirubin-induced neurotoxicity.

Role of caspase-1 activation in bilirubin-induced injury in cultured primary rat hippocampal neurons

To investigate the role of caspase-1 activation in bilirubin-induced neuronal injury and the protective effect of VX-765 against bilirubin-induced neurotoxicity in cultured primary rat hippocampal neurons. Cultured primary rat hippocampal neurons were exposed to DMSO (control group), 50 µmol/L bilirubin, or 50 µmol/L bilirubin 1 h after 50 µmol/L VX-765 treatment. The expressions of NLRP3 and caspase-1 in the neurons were detected by Western blotting, and the relative cell survival and death rates were assessed with a modified MTT assay, lactate dehydrogenase assay and Typan blue staining. Interleukin-18 (IL-18) concentration in the culture supernatant was measured using enzyme-linked immunosorbent assay (ELISA). In cultured primary rat hippocampal neurons, bilirubin exposure for 3 and 6 h caused significant increases in the expressions of NLRP3 and activated caspase-1 compared with those in the control group (P<0.05). Pretreatment of the cells with VX-765 obviously suppressed bilirubin-induced activation of caspase-1 (P<0.05). The relative survival rate of the neurons was (84.02∓2.31)% in VX-765 intervention group, significantly higher than that in bilirubin group (P<0.05) but lower than that in the control group (P<0.05); LDH release rate in VX-765 intervention group was (10.78∓1.58)%, significantly lower than that in bilirubin group (P<0.05) but higher than that in the control group (P<0.05). The cell death rate in VX-765 intervention group was (5.58∓1.23)%, significantly lower than that in bilirubin group (P<0.05) but higher than that in the control group (P<0.05). In cultured primary rat hippocampal neurons, caspase-1 activation plays a role in bilirubin-induced neurotoxicity, and VX-765 treatment provides protection against bilirubin-induced neuronal injury by inhibiting caspase-1 activation.

The Neuroprotective Effects of Hypothermia on Bilirubin-Induced Neurotoxicity in vitro

Background: In high-risk newborns indirect hyperbilirubinemia can lead to acute bilirubin encephalopathy and kernicterus. Despite the current therapeutic modalities, preventing or reversing the neurotoxicity cannot be achieved in all infants. Objective: To investigate the neuroprotective effects of hypothermia on bilirubin-induced toxicity in primary mouse neuronal cell cultures. Methods: Hippocampal cell cultures, isolated from newborn mouse brains, were incubated with unconjugated bilirubin (UCB) at 3 days in vitro (DIV) and immediately exposed to varying degrees of hypothermia. Neuronal viability and mitochondrial health were compared between the normothermia (37°C), mild (34°C), moderate (32°C) and severe (29°C) hypothermia groups. Confocal microscopy and fluorescent dyes (propidium iodide and JC-1) were used for cell evaluation. To determine the late effects of hypothermia, the cultures were also examined at 7 DIV after returning to normothermic conditions. Results: Induction of any degree of hypothermia increased the neuronal survival after 24 h of UCB treatment. Neuronal death rate and mitochondrial membrane potential loss were lowest in the neurons exposed to moderate hypothermia. We also observed that mild to moderate hypothermia had late protective effects on neuronal cell viability, whereas deep hypothermia did not improve neuronal survival. Conclusions: We conclude that hypothermia reduces the cell death induced by bilirubin toxicity in neuronal cells. Although moderate hypothermia has a better outcome than mild hypothermia, deep hypothermia as low as 29°C has adverse effects on neuronal cell viability.

Attenuation of neuro-inflammation improves survival and neurodegeneration in a mouse model of severe neonatal hyperbilirubinemia

All pre-term newborns and a high proportion of term newborns develop neonatal jaundice. Neonatal jaundice is usually a benign condition and self-resolves within few days after birth. However, a combination of unfavorable complications may lead to acute hyperbilirubinemia. Excessive hyperbilirubinemia may be toxic for the developing nervous system leading to severe neurological damage and death by kernicterus. Survivors show irreversible neurological deficits such as motor, sensitive and cognitive abnormalities. Current therapies rely on the use of phototherapy and, in unresponsive cases, exchange transfusion, which is performed only in specialized centers.During bilirubin-induced neurotoxicity different molecular pathways are activated, ranging from oxidative stress to endoplasmic reticulum (ER) stress response and inflammation, but the contribution of each pathway in the development of the disease still requires further investigation.Thus, to increase our understanding of the pathophysiology of bilirubin neurotoxicity, encephalopathy and kernicterus, we pharmacologically modulated neurodegeneration and neuroinflammation in a lethal mouse model of neonatal hyperbilirubinemia. Treatment of mutant mice with minocycline, a second-generation tetracycline with anti-inflammatory and neuroprotective properties, resulted in a dose-dependent rescue of lethality, due to reduction of neurodegeneration and neuroinflammation, without affecting plasma bilirubin levels. In particular, rescued mice showed normal motor-coordination capabilities and behavior, as determined by the accelerating rotarod and open field tests, respectively. From the molecular point of view, rescued mice showed a dose-dependent reduction in apoptosis of cerebellar neurons and improvement of dendritic arborization of Purkinje cells. Moreover, we observed a decrease of bilirubin-induced M1 microglia activation at the sites of damage with a reduction in oxidative and ER stress markers in these cells.Collectively, these data indicate that neurodegeneration and neuro-inflammation are key factors of bilirubin-induced neonatal lethality and neuro-behavioral abnormalities.We propose that the application of pharmacological treatments having anti-inflammatory and neuroprotective effects, to be used in combination with the current treatments, may significantly improve the management of acute neonatal hyperbilirubinemia, protecting from bilirubin-induced neurological damage and death.

Unconjugated bilirubin induces pyroptosis in cultured rat cortical astrocytes

BackgroundBilirubin-induced neurological dysfunction (BIND), a severe complication of extreme neonatal hyperbilirubinemia, could develop into permanent neurodevelopmental impairments. Several studies have demonstrated that inflammation and nerve cell death play important roles in bilirubin-induced neurotoxicity; however, the underlying mechanism remains unidentified.MethodsThe present study was intended to investigate whether pyroptosis, a highly inflammatory form of programmed cell death, participated in the bilirubin-mediated toxicity on cultured rat cortical astrocytes. Further, VX-765, a potent and selective competitive drug, was used to inhibit the activation of caspase-1. The effects of VX-765 on astrocytes treated with bilirubin, including the cell viability, morphological changes of the cell membrane and nucleus, and the production of pro-inflammation cytokines, were observed.ResultsStimulation of the astrocytes with unconjugated bilirubin (UCB) at the conditions mimicking those of jaundiced newborns significantly increased the activation of caspase-1. Further, caspase-1 activation was inhibited by treatment with VX-765. Compared with UCB-treated astrocytes, the relative cell viability of VX-765-pretreated astrocytes was improved; meanwhile, the formation of plasma membrane pores was prevented, as measured by lactate dehydrogenase release, trypan blue staining, and ethidium bromide (EtBr) uptake. Moreover, DNA fragmentation was partly attenuated and the release of IL-1β and IL-18 was apparently decreased.ConclusionPyroptosis is involved in the process of UCB-induced rat cortical astrocytes’ injury in vitro and may be the missing link of cell death and inflammatory response exacerbating UCB-related neurotoxicity. More importantly, the depression of caspase-1 activation, the core link of pyroptosis, attenuated UCB-induced cellular dysfunction and cytokine release, which might shed light on a new therapeutic approach to BIND.

Melatonin Promotes Brain-Derived Neurotrophic Factor (BDNF) Expression and Anti-Apoptotic Effects in Neonatal Hemolytic Hyperbilirubinemia via a Phospholipase (PLC)-Mediated Mechanism.

BackgroundMelatonin therapy shows positive effects on neuroprotective factor brain-derived neurotrophic factor (BDNF) expression and neuronal apoptosis in neonatal hemolytic hyperbilirubinemia. We hypothesized that melatonin promotes BDNF expression and anti-apoptotic effects in neonatal hemolytic hyperbilirubinemia through a phospholipase (PLC)-mediated mechanism.Material/MethodsA phenylhydrazine hydrochloride (PHZ)-induced neonatal hemolytic hyperbilirubinemia model was constructed in neonatal rats. Four experimental groups – a control group (n=30), a PHZ group (n=30), a PHZ + melatonin group (n=30), and a PHZ + melatonin+U73122 (a PLC inhibitor) group (n=30) – were constructed. Trunk blood was assayed for serum hemoglobin, hematocrit, total and direct bilirubin, BDNF, S100B, and tau protein levels. Brain tissue levels of neuronal apoptosis, BDNF expression, PLC activity, IP3 content, phospho- and total Ca2+/calmodulin-dependent protein kinase type IV (CaMKIV) expression, and phospho- and total cAMP response element binding protein (CREB) expression were also assayed.ResultsPHZ-induced hemolytic hyperbilirubinemia was validated by significantly decreased serum hemoglobin and hematocrit as well as significantly increased total and direct serum bilirubin (p<0.05). Neonatal bilirubin-induced neurotoxicity was validated by significantly decreased serum BDNF, brain BDNF, and serum S100B, along with significantly increased serum tau protein (p<0.05). PHZ-induced hemolytic hyperbilirubinemia significantly decreased serum BDNF, brain BDNF, and PLC/IP3/Ca2+ pathway activation while increasing neuronal apoptosis levels (p<0.05), all of which were partially rescued by melatonin therapy (p<0.05). Pre-treatment with the PLC inhibitor U73122 largely abolished the positive effects of melatonin on PLC/IP3/Ca2+ pathway activation, downstream BDNF levels, and neuronal apoptosis (p<0.05).ConclusionsPromotion of BDNF expression and anti-apoptotic effects in neonatal hemolytic hyperbilirubinemia by melatonin largely operates via a PLC-mediated mechanism.

Bilirubin Albumin Binding and Unbound Unconjugated Hyperbilirubinemia in Premature Infants

To evaluate the associations between unbound bilirubin (UB) and total serum bilirubin (TSB), bilirubin:albumin molar ratio (BAMR), and bilirubin albumin binding affinity (Ka) as a function of gestational age (GA) in infants born at 24-33 weeks GA. In a prospective observational study, TSB and UB were measured twice daily at least 8 hours apart during the first postnatal week. Serum albumin was measured to calculate BAMR on each day. The highest UB on each day, corresponding TSB, and serum albumin were used to calculate the Ka on each day. For the 166 infants studied, peak UB significantly correlated with concomitant Ka (r = -0.44, P = .001) but not with concomitant TSB or BAMR after adjusting for GA. On multiple regression analyses, there was a significant association of concomitant Ka (-0.06, 95% CI -0.08 to -0.04, P = .0001), but not concomitant TSB or BAMR with peak UB after controlling for GA, birth weight, race, and sex. GA group was a significant effect modifier for the association between Ka and peak UB (0.03, 95% CI 0.02-0.04, P < .001). Interaction analyses showed the association between concomitant Ka and peak UB was significant for the 24-30 weeks GA group infants, but not for the 301/7-33 weeks GA group infants. Peak UB was primarily associated with a decrease in binding affinity in infants ≤30 weeks GA. Interventions aimed at improving binding affinity may be important in decreasing the risk of bilirubin-induced neurotoxicity.