The Mechanism Of Hyperbilirubinemia Concurrent With Acidosis Leading To Neurotoxicity

PURPOSE: To explore the evidence of blood bilirubin and other indicators and evidence of brain damage in clinical data of newborns with hyperbilirubinemia and acidosis;establish an animal model of hyperbilirubinemia with acidosis and explore bilirubin sensitivity to acids;Mechanisms for potentiation of Acid-Sensing Ion Channels(ASICs)by bilirubin;to clarify the behavioral manifestations of central nervous system injury in model animals,to reveal the mechanism of brain damage exacerbated by hyperbilirubinemia with acidosis.METHODS: All children in ICU who had undergone cerebrospinal fluid examination as the basic object(897 cases)were collected according to the inclusion criteria in Shanghai Children’s Hospital from June 2014 to June 2017.The collected data mainly included: blood direct bilirubin concentration,blood total bilirubin concentration,blood p H,cerebrospinal fluid biochemical examination.According to the diagnostic criteria,they were divided into simple hyperbilirubinemia group,simple acidosis group and hyperbilirubinemia concurrent with acidosis group.Wildtype and ASIC1 a knockout mice from P5-12 were used,brain slices containing medial vestibular nucleus(MVN)were used in experiments such as Calcein-PI apoptosis staining and LDH analysis.Intracellular calcium was detected by calcium imaging.ASIC1a/2a transfected CHO cells were used in electrophysiology and cell death analysis.Animal models of hyperbilirubinemia and acidosis were established by intraperitoneal injection of bilirubin and hypoxia culture.The animal models were used for water maze,fear memory,ABR test,rod test and gait analysis and other behavioral tests.All data were analyzed using statistical methods such as paired and grouped Student t-test,normal distribution test,and spearman correlation analysis.RESULTS: Analysis of clinical data shows that in children with hyperbilirubinemia and acidosis,the level of cerebrospinal fluid LDH is significantly higher than that in the other two groups.Cerebrospinal fluid LDH is directly related to blood bilirubin and blood p H.Micromolar concentration of bilirubin promoted the amplitude of ASICs in MVN neurons.In the current-clamp recordings,bilirubin increased the frequency of action potential firings induced by ASICs when exposed extracellular solution at p H 7.0.calcium imaging shows that bilirubin exposure at p H 7.0 increased [Ca2+]i.viability analysis in ASIC1a/2a transfected CHO cells and Calcein-PI cell death staining results show that bilirubin significantly decreased the cell viability and increased the mortality of MVN neurons in a mild acidic environment.Animal behavior experiments show that: the animals with hyperbilirubinemia combined with acidosis show a decline in cognitive and spatial memory and learning ability;advanced motor balance function was significantly decreased.CONCLUSION: Our findings show that bilirubin promoted ASICs activity,exacerbated neuronal excitotoxicity,and caused neuronal death.Combined with the behavioral results in model animals,it implies that the neonates concurrent hyperbilirubinemia with acidosis are more prone to brain damage.