Diagnostics Value of Quantitative Magnetic Resonance Imaging (MRI) in Neonatal Acute Bilirubin Encephalopathy.

Background: This study was designed to investigate the diagnostic value of relative signal intensity of globus pallidus on T1-weighted magnetic resonance imaging (MRI) in neonatal acute bilirubin encephalopathy (ABE). Methods: Participants who were recruited in hospital from April 2019 to May 2020 were grouped into mildly increased total serum bilirubin (TSB) group (n = 30), severely increased TSB group (n = 25), or extremely increased TSB group (n = 10) based on the total serum bilirubin level. Bilirubin-induced neurologic dysfunction scale score was used to determine if participants had acute bilirubin encephalopathy. All neonates underwent conventional brain MRI and the relative signal intensity of globus pallidus was measured on T1-weighted images. The diagnostic value of these 3 indices was assessed by receiver operating characteristic curve analysis. Results: There was a significant correlation between relative signal intensity of globus pallidus and total serum bilirubin level in neonates with hyperbilirubinemia (r = 0.551, P < .001). Relative signal intensity of globus pallidus in the extremely increased TSB group was significantly higher than that in severely increased TSB, mildly increased TSB, and healthy control groups. Relative signal intensity of globus pallidus in the acute bilirubin encephalopathy group was significantly higher than that in the non-acute bilirubin encephalopathy group (P < .01). The area under the receiver operating characteristic curve of the relative signal intensity of globus pallidus was 0.765 (P < .01), with sensitivity of 0.655 and specificity of 0.861. The area under the curve of the total serum bilirubin and visual inspection of globus pallidus signal was 0.621 and 0.579, respectively. The area under the curve of relative signal intensity was significantly greater than that of total serum bilirubin and visual inspection (P = .04 for both). Conclusion: Relative signal intensity of globus pallidus, which is an objective assessment, has the potential to be used as a diagnostic tool for acute bilirubin encephalopathy.

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.

Magnetic Resonance Imaging Findings in Preterm Infants With Bilirubin Encephalopathy Beyond Three Years Corrected Age.

BACKGROUND: Magnetic resonance imaging (MRI) abnormalities in preterm infants with bilirubin encephalopathy (BE) become less clear as the infants age. We assessed MRI findings in children with preterm BE older than 36 months corrected age (CA). METHODS: In a previous questionnaire survey, hospitals were asked to provide head MRI data of patients older than 36 months CA. MRI findings were reviewed by three pediatric neurology specialists and classified as no abnormalities, partial globus pallidus (GP) lesions, or diffuse GP lesions. RESULTS: In total, 33 MRI scans were available from 28 patients. The median gestational age and birth weight were 26 weeks and 824 g, respectively. The prevalence of MRI abnormalities was 100% in patients at 37 to 48 months CA, 71% in those at 49 to 60 months CA, 50% in those at 61 to 72 months CA, 67% in those at 73 to 84 months CA, and 38% in those at 85 months CA or older. Partial GP lesions were more common than diffuse GP lesions at all ages. No significant differences in sex, gestational age, birth weight, or gross motor function impairment were observed among lesion groups. CONCLUSIONS: GP lesions were detected on MRI in most children with preterm BE when studied after 36 months CA, although MRI abnormalities became less apparent along with age. Partial GP lesions may be a characteristic of older children with preterm BE.

Neonatal acute liver failure with pulmonary yellow hyaline membrane and kernicterus

Background Neonatal acute liver failure (NALF) is a rare and life-threatening condition. It causes bilirubin to accumulate to a dangerous level in the body, causing permanent damage to vital organs such as the brain and lungs. In many cases, the etiology of NALF remains unknown. Case presentation We described a case of an 8-day-old baby girl who presented with poor oral intake, lethargy, and jaundice. Her clinical condition rapidly deteriorated with progression to multi-organ failure, and despite intensive resuscitation efforts, she expired. At autopsy, the most significant findings were liver necrosis, yellow hyaline membrane deposition in the lungs, and bilirubin deposition in the brain (kernicterus). Conclusions NALF is a rare and potentially fatal condition necessitating prompt recognition and disease-specific treatment approaches. Toxic accumulation of bilirubin in the lungs can lead to hypoxia and precipitate further ischemic injury to the liver.

Kernicterus with abnormal high-signal changes bilaterally in the globus pallidus: A case report.

Kernicterus is a relatively rare consequence of hyperbilirubinemia. There is an important role for MRI imaging for this entity in the appropriate clinical context as there are distinct signal changes in the globus pallidus. A case report and image findings are presented

A new rat model of neonatal bilirubin encephalopathy (kernicterus).

INTRODUCTION: Hemolytic kernicterus, an indirect bilirubin-induced brain dysfunction, is associated with hyper-bilirubinemia in mammalian neonates. In this study, a new model of kernicterus has been developed using intra-peritoneal injections of phenyl hydrazine and subcutaneous injections of sulfisoxazole. These drugs can potentially induce kernicterus in neonatal through changes in hemolysis and hypo-albumin. METHODS: For this purpose, 7-day-old male Wistar rats (n=72; mean weight 11±1g) were used. The animals have been divided into six different groups which received the drugs alone and their combination, and the drugs' solvents and their combination. Biochemical parameters, brain iron and bilirubin, behavioural performance, auditory function and apoptosis were measured using auto-analyser instruments; atomic absorption spectroscopy, Sawasaki, footprint, auditory brainstem response (ABR) and TUNEL test, respectively. RESULT: The drug-injected groups showed a significant reduction in serum haematocrit and an increase in the concentration of brain bilirubin, total and indirect bilirubin as well as TUNEL positive cells in basal ganglia. In addition, the obtained results showed that there was a significant increase in behavioural disturbance and auditory dysfunction in the group injected with the combination of two drugs. CONCLUSION: This kernicterus-induced rat model could perfectly mimic the common conditions of the hyperbilirubinemia in human neonates. This study offers an easy technique to develop more stable models for follow-up studies.

Bilirubin Encephalopathy in a Domestic Shorthair Cat With Increased Osmotic Fragility and Cholangiohepatitis.

A 7-month-old female domestic shorthair cat was diagnosed with chronic regenerative hemolytic anemia characterized by increased osmotic fragility of unknown etiology. At 13 months of age, the cat was evaluated for acute collapse. The cat was icteric with severe hyperbilirubinemia but no hematocrit changes. Severe obtundation and lateral recumbency progressed to tetraparesis and loss of proprioception in all 4 limbs, and a cerebellar or brainstem lesion was suspected. Postmortem examination revealed suppurative cholangiohepatitis and acute neuronal necrosis in the nuclei of the brainstem and cerebellum, consistent with bilirubin encephalopathy. This is the first known occurrence of cholangiohepatitis and bilirubin encephalopathy in an adult cat with chronic hemolytic anemia. Although rare, bilirubin encephalopathy should be considered a possible sequela to hyperbilirubinemia in adult patients. It remains unknown whether increased osmotic fragility was related to the cholangiohepatopathy.

Magnetic resonance imaging of bilirubin encephalopathy: current limitations and future promise.

Infants with chronic bilirubin encephalopathy often demonstrate abnormal bilateral, symmetric, high-signal intensity on T2-weighted magnetic resonance imaging of the globus pallidus and subthalamic nucleus, consistent with the neuropathology of kernicterus. Early magnetic resonance imaging of at-risk infants, while frequently showing increased T1-signal in these regions, may give false-positive findings due to the presence of myelin in these structures. Advanced magnetic resonance imaging including diffusion-weighted imaging, magnetic resonance spectroscopy, and diffusion tensor imaging with tractography may shed new insights into the pathogenesis of bilirubin-induced brain injury and the neural basis of long-term disability in infants and children with chronic bilirubin encephalopathy.

The enigma of low bilirubin kernicterus in premature infants: why does it still occur, and is it preventable?

Low bilirubin kernicterus in preterm neonates, though rare, remains an unpredictable and refractory form of brain injury. Hypoalbuminemia, co-morbid CNS insult(s), infection, and inflammation are contributing causes that, in many cases, appear to interact in potentiating bilirubin neurotoxicity. Despite compulsive attention to serum bilirubin levels, and clinical and laboratory indices of neurotoxicity risk, low bilirubin kernicterus continues to be seen in contemporary NICUs. While efforts to refine and improve current treatment guidelines are certainly needed, such revision(s) will also have to take into account the risks and benefits of any intervention, including phototherapy.

Subtle bilirubin-induced neurodevelopmental dysfunction (BIND) in the term and late preterm infant: does it exist?

Subtle bilirubin-induced neurological dysfunction (BIND) is defined as disturbances in sensory and sensorimotor integration, central auditory processing, coordination, and muscle tone in the absence of the classical findings of kernicterus. This review is restricted to the (sensori)motor signs of BIND associated with unconjugated hyperbilirubinemia in term and late preterm neonates. The diagnosis of BIND at follow-up requires validated, age-specific techniques that are designed to identify these disturbances in infancy and later childhood. The (sensori)motor signs of BIND are compatible with the pathological substrate of unconjugated hyperbilirubinemia and its known effects on the brain.

A novel newborn rat kernicterus model created by injecting a bilirubin solution into the cisterna magna.

BACKGROUND: Kernicterus still occurs around the world; however, the mechanism of bilirubin neurotoxicity remains unclear, and effective treatment strategies are lacking. To solve these problems, several kernicterus (or acute bilirubin encephalopathy) animal models have been established, but these models are difficult and expensive. Therefore, the present study was performed to establish a novel kernicterus model that is simple and affordable by injecting unconjugated bilirubin solution into the cisterna magna (CM) of ordinary newborn Sprague-Dawley (SD) rats. METHODS: On postnatal day 5, SD rat pups were randomly divided into bilirubin and control groups. Then, either bilirubin solution or ddH2O (pH = 8.5) was injected into the CM at 10 µg/g (bodyweight). For model characterization, neurobehavioral outcomes were observed, mortality was calculated, and bodyweight was recorded after bilirubin injection and weaning. Apoptosis in the hippocampus was detected by H&E staining, TUNEL, flow cytometry and Western blotting. When the rats were 28 days old, learning and memory ability were evaluated using the Morris water maze test. RESULTS: The bilirubin-treated rats showed apparently abnormal neurological manifestations, such as clenched fists, opisthotonos and torsion spasms. Bodyweight gain in the bilirubin-treated rats was significantly lower than that in the controls (P<0.001). The early and late mortality of the bilirubin-treated rats were both dramatically higher than those of the controls (P = 0.004 and 0.017, respectively). Apoptosis and necrosis in the hippocampal nerve cells in the bilirubin-treated rats were observed. The bilirubin-treated rats performed worse than the controls on the Morris water maze test. CONCLUSION: By injecting bilirubin into the CM, we successfully created a new kernicterus model using ordinary SD rats; the model mimics both the acute clinical manifestations and the chronic sequelae. In particular, CM injection is easy to perform; thus, more stable models for follow-up study are available.

Developmental onset of bilirubin-induced neurotoxicity involves Toll-like receptor 2-dependent signaling in humanized UDP-glucuronosyltransferase1 mice.

Biological and signaling events that connect developmentally induced hyperbilirubinemia to bilirubin-induced neurological dysfunction (BIND) and CNS toxicity in humans are poorly understood. In mammals, UDP-glucuronosyltransferase 1A1 (UGT1A1) is the sole enzyme responsible for bilirubin glucuronidation, a rate-limiting step necessary for bilirubin metabolism and clearance. Humanized mice that express the entire UGT1 locus (hUGT1) and the UGT1A1 gene, develop neonatal hyperbilirubinemia, with 8-10% of hUGT1 mice succumbing to CNS damage, a phenotype that is presented by uncontrollable seizures. We demonstrate that neuroinflammation and reactive gliosis are prominent features of bilirubin brain toxicity, and a disturbed redox status resulting from activation of NADPH oxidase is an important contributing mechanism found in BIND. Using knock-out mice and primary brain cells, we connect a key pattern recognition receptor, Toll-like receptor 2 (TLR2), to hyperbilirubinemia-induced signaling. We illustrate a requirement for TLR2 signaling in regulating gliosis, proinflammatory mediators, and oxidative stress when neonatal mice encounter severe hyperbilirubinemia. TLR2-mediated gliosis strongly correlates with pronounced neuroinflammation in the CNS with up-regulation of TNFα, IL-1ß, and IL-6, creating a pro-inflammatory CNS environment. Gene expression and immunohistochemistry staining show that hUGT1/Tlr2(-/-) mice fail to activate glial cells, proinflammatory cytokines, and stress response genes. In addition, bilirubin-induced apoptosis was significantly enhanced by blocking TLR2 signaling indicating its anti-apoptotic property. Consequently, a higher neonatal death rate (57.1%) in hUGT1/Tlr2(-/-) mice was observed when compared with hUGT1 mice (8.7%). These results suggest that TLR2 signaling and microglia neuroinflammation are linked to a repair and/or protection mode against BIND.

MRI findings in a dog with kernicterus.

A severe increase in total bilirubin coincided with a decline in neurologic status to comatose in a 9 yr old spayed female mixed-breed dog being treated for immune-mediated hemolytic anemia. MRI of the brain was performed to investigate potential causes for the neurologic signs. MRI revealed bilaterally symmetrical hyperintensities within the caudate nuclei, globus pallidus, thalamus, deep cerebellar nuclei, and cortical gray matter on T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequences, which coincided with areas of bilirubin deposition and neuronal necrosis (kernicterus) identified on necropsy examination. This is the second case report of an adult dog exhibiting kernicterus, and the first report to document MRI findings associated with that condition. Kernicterus is an uncommonly reported complication of hyperbilirubinemia in dogs, but is potentially underreported due to difficulties in recognizing subtle lesions and distinguishing kernicterus from other potential causes of neurologic abnormalities with readily available antemortem tests. MRI may be helpful in supporting the diagnosis of kernicterus.

Cerebellar axon/myelin loss, angiogenic sprouting, and neuronal increase of vascular endothelial growth factor in a preterm infant with kernicterus.

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.

Bilirubin production and the risk of bilirubin neurotoxicity.

Neonatal jaundice usually occurs in the transitional period after birth, presenting as an elevation of circulating bilirubin. Bilirubin neurotoxicity can occur if the levels of bilirubin become excessive (hyperbilirubinemia). This pathologic phenotype of newborn jaundice can develop because of excessive bilirubin production or impaired conjugation, with the risk for developing bilirubin-induced neurologic dysfunction, depending on the degree of the resultant bilirubin load. The plasma bilirubin level thus can be used to assess an infant's risk for developing bilirubin neurotoxicity relative to an infant's age in hours. Because all infants have an impaired conjugation ability, infants at greatest risk are those who have increased bilirubin production rates, because of hemolysis, for example. Therefore, developing potential preventive strategies as well as noninvasive technologies to treat and to identify infants with increased bilirubin production rates, respectively, are tantamount to reducing the incidence of bilirubin-induced neurologic dysfunction.

Elevated levels of bilirubin and long-term exposure impair human brain microvascular endothelial cell integrity.

The pathogenesis of encephalopathy by unconjugated bilirubin (UCB) seems to involve the passage of high levels of the pigment across the blood-brain barrier (BBB) and the consequent damage of neuronal cells. However, it remains to be clarified if and how the disruption of BBB occurs by UCB. We used confluent monolayers of human brain microvascular endothelial cells (HBMEC) to explore the sequence of events produced by UCB. A cell line and primary cultures of HBMEC were exposed to 50 or 100 µM UCB, in the presence of 100 µM human serum albumin, to mimic moderate and severe jaundice, for 1-72 h. UCB caused loss of cell viability in a concentration-dependent manner. UCB inhibited the secretion of interleukin-6, interleukin-8, monocyte chemoattractant protein-1 and vascular endothelial growth factor at early time points, but enhanced their secretion at later time points. Upregulation of mRNA expression, particularly by 100 µM UCB, preceded cytokine secretion. Other early events include the disruption of glutathione homeostasis and the increase in endothelial nitric oxide synthase expression followed by nitrite production. Prolonged exposure to UCB upregulated the expression of ß-catenin and caveolin-1. In conclusion, elevated concentrations of UCB affect the integrity of HBMEC monolayers mediated by oxidative stress and cytokine release. UCB also induced increased expression of caveolin-1, which has been associated with BBB breakdown, and ß-catenin, probably as an attempt to circumvent that impairment. These findings provide a basis for target-directed therapy against brain endothelial injury caused by UCB.

Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation.

Microglia constitute the brain's immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target.

Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus.

High levels of unconjugated bilirubin (UCB) in newborn children is associated with a reduction in hepatic UDP glucuronosyltransferase (UGT) 1A1 activity that can lead to CNS toxicity, brain damage, and even death. Little is known regarding those events that lead to UCB accumulation in brain tissue, and therefore, we sought to duplicate this condition in mice. The human UGT1 locus, encoding all 9-UGT1A genes including UGT1A1, was expressed in Ugt1(-/-) mice. Because the most common clinical condition associated with jaundice in adults is Gilbert's syndrome, which is characterized by an allelic polymorphism in the UGT1A1 promoter, hyperbilirubinemia was monitored in humanized UGT1 mice that expressed either the Gilbert's UGT1A1*28 allele [Tg(UGT1(A1*28))Ugt1(-/-) mice] or the normal UGT1A1*1 allele [Tg(UGT1(A1*1))Ugt1(-/-) mice]. Adult Tg(UGT1(A1*28))Ugt1(-/-) mice expressed elevated levels of total bilirubin (TB) compared with Tg(UGT1(A1*1))Ugt1(-/-) mice, confirming that the promoter polymorphism associated with the UGT1A1*28 allele contributes to hyperbilirubinemia in mice. However, TB accumulated to near toxic levels during neonatal development, a finding that is independent of the Gilbert's UGT1A1*28 promoter polymorphism. Whereas serum TB levels eventually returned to adult levels, TB clearance in neonatal mice was not associated with hepatic UGT1A1 expression. In approximately 10% of the humanized UGT1 mice, peak TB levels culminated in seizures followed by death. UCB deposition in brain tissue and the ensuing seizures were associated with developmental milestones and can be prevented by enhancing regulation of the UGT1A1 gene in neonatal mice.

Neurodevelopmental outcome of acute bilirubin encephalopathy.

The aim of the study was to determine the neurodevelopmental outcome of acute bilirubin encephalopathy (ABE) in children who underwent double volume exchange transfusion (DVET). The 25 referred newborns of ≥ 35 weeks gestation with total serum bilirubin >20 mg dl(-1) and signs of ABE were enrolled and followed up at 3, 6, 9 and 12 months. Denver Development Screening Test (DDST), Neurological examination along with MRI at discharge and brain stem evoked response audiometry (BERA) at 3 months were done. Abnormal neurodevelopment was defined as either (i) cerebral palsy or (ii) abnormal DDST or (iii) abnormal BERA. The mean bilirubin at admission was 37 mg dl(-1). MRI and BERA were abnormal in 61% and 76%. At 1 year, DDST and neurological abnormality were seen in 60% and 27% and 80% had combined abnormal neurodevelopment. MRI had no relation (P = 0.183) but abnormal BERA had a significant association (P = 0.004) with abnormal outcome. Intermediate and advanced stages of ABE associated with significant adverse outcome in spite of DVET.