| Neonatal hypoxic-ischemic encephalopathy(HIE)is the neonatal brain injury caused by perinatal asphyxia,leading to cerebral palsy,long-term neurological sequelae or neonatal death.Multiple mechanisms have been invoked to explain the development of brain injury caused by hypoxia,including reducing cerebral blood flow,energy metabolism changes in brain tissues,inflammatory regulation factors,stimulation of reactive oxygen species(ROS),glutamate accumulation in the synaptic cleft,high concentrations of intracellular calcium and brain cell apoptosis.Currently,the early diagnosis of neonatal HIE in the clinic depends on observing clinical symptoms and signs using a combination of Sarnat scoring,computer tomography(CT),magnetic resonance imaging(MRI),ultrasound and electroencephalogram(EEG).However,these examinations have different limitations and effectiveness.Biomarkers in the blood circulation are bio-chemical factors released by specific tissues or organs.After the onset of neonatal HIE,the injured brain tissues release the special tissue components or products into blood or cerebrospinal fluid(CSF).Therefore,the accurate detection of biomarkers in blood or CSF in neonatal HIE is important and will allow early interventions to reduce neonatal mortality,morbidity and degree of disability.To date,potential biomarkers have been identified in neonates with HIE.These biomarkers were obtained from CSF,serum,or urine,including S100β,neuron specific enolase(NSE),glial fibrillary acidic protein(GFAP),brain-derived neurotrophic factor(BDNF),spectrin breakdown products(SBDPs),ubiquitin C-terminal hydrolase L1(UCHL1),Tau and p NF-H,being helpful to determine the time of brain injury occurrence and to predict the long-term prognosis.A large number of studies have focused on the function of S100β、NSE、GFAP、BDNF.A few studies about the role of UCH-L1,SBDPs and Tau in brain injury were reported and mostly focused on traumatic brain injury.Thus,this study aimed to explore the function of these biomarkers in the diagnosis of neonatal HIE.Ubiquitin carboxyl-terminal esterase L1(UCHL1),belongs to cysteine proteinase and is a cytoplasmic enzyme selectively expressed in neurons and nervous tissue.UCHL1 was recently identified as a biomarker of neuronal injury.Document data has suggested that high level of UCHL1 in cerebrospinal fluid and blood was closely associated with neuronal injury and the destruction of blood brain barrier.UCHL1 was found to be upregulated in several neurogenic disease,such as aneurysmal subarachnoid hemorrhage,traumatic brain injury,stroke,hypoxic encephalopathy and neonatal HIE.However,it is still poorly understood about the mechanism of UCHL1 upregulation and its relationship with HIE-induced neuronal apoptosis.SBDPs are the breakdown products of αII Spectrin.αII Spectrin is primarily found in neurons and is abundant in axons and presynaptic terminals.It is processed into SBDPs of 145,150,and 120 k Da.SBDP150 and SBDP145 are produced by cleavage with calpain and SBDP120 is produced by proteolytic cleavage with caspase-3.Calpain and caspase-3 are primarily associated with necrosis and apoptosis in the process of cerebral ischemia and brain injury.Therefore,SBDPs might be clinically useful biomarkers in patients with brain injury.Tau protein is a microtubule-associated protein that is necessary for cytoskeleton structure and axonal transport.Tau protein is found primarily in axons and peripheral nervous system.In healthy adult or newborns,the level of Tau protein is commonly very low in cerebrospinal fluid or serum.While Tau protein could be released by axon or cell in patients with nerve injury,traumatic brain injury or certain diseases of the central nervous system,causing the increased level in cerebrospinal fluid or serum.This suggested that elevated tau protein levels in cerebrospinal fluid or serum can indicate neuronal injury.It has been shown that extracellular Tau protein levels were significantly upregulated in immature brain after subjecting hypoxia ischemia.In this study,serum UCHL1 level in newborns with HIE was detected by using a sandwich enzyme-linked immunosorbent assay(ELISA).The expression of UCHL1 was inhibited by si RNA in neuronal cell AGE1.HN to detect the function of UCHL1 on cell apoptosis.In addition,the mechanism of UCHL1 in hypoxia-induced neuronal apoptosis was explored.To evaluate the use of SBDPs and Tau protein levels for diagnosis and a mild hypothermia therapy for treatment of neonatal HIE,the serum level of SBDPs and Tau in newborns with different degree of HIE were detected.Finally,we explored the application of amplitude integrated electroencephalography(a EEG)for the early diagnosis and long-term prognosis of newborns with HIE.This study aimed to search the serum biomarkers for the early diagnosis of neonatal HIE and provided theoretical foundation for the prediction and clinical intervention therapy of HIE.Part Ⅰ HIF1α and HIF2α mediated UCHL1 upregulation in hypoxia-induced neuronal injury following neuronal hypoxic ischemic encephalopathyObjectives:To explore the function of UCHL1 on the hypoxia-induced neuronal apoptosis and its regulation mediated by HIF1α and HIF2α.Methods:1.Total twenty cases of neonates with HIE and corresponding normal neonates who were treated in the neonatal department were analyzed in this study.Blood samples from these subjects were subjected to serum preparation for the measurement of UCHL1 levels by using a sandwich enzyme-linked immunosorbent assay(ELISA).2.AGE1.HN cells were propagated in the maintenance medium(1:1 Dulbecco’s modified Eagle’s medium: Ham’s F-12 mix)supplemented with 5% fetal bovine serum,1% antibiotic-antimycotic,10 mg/m L human transferring and 30 mmol/L sodium selenite at 37°C with normoxia(20% O2).For hypoxia treatment,cells were maintained in hypoxia(1% O2)at 37°C for indicated time intervals.3.Small interference RNAs(si RNAs)were used here for knockdown experiments.Three UCHL1 si RNAs against UCHL1 m RNA and those specific si RNAs for HIF1α and HIF2α m RNA were all designed and synthesized by Ribobio(Guangzhou,China),as well as scrambled negative control si RNA(si-NC).4.AGE1.HN cells were pre-incubated and transfected with si RNAs by using Lipofectamine 2000 according to the manufacturer’s instructions.5.Annexin V/PI Apoptosis Detection Kit was used to measure the percentage of apoptotic cells of AGE1.HN cell treated by 1 ng/ml,5 ng/ml,10 ng/ml of UCHL1 for 48 h.6.Cell Counting Kit(CCK)-8 assay was performed to detect the cell proliferation of AGE1.HN cell transfected with si UCHL1-1,si UCHL1-2 and si UCHL1-3.7.Quantitative real-time PCR(q RT-PCR)was performed to examine the m RNA level of UCHL1 in AGE1.HN cell cultured under hypoxia.8.Western blot was used to detect the protein level of UCHL1 in AGE1.HN cells cultured under hypoxia and transfected with si HIF1α or si HIF2α.9.Chromatin immunoprecipitation(Ch IP)assay was performed to verify the binding of HIF1α or HIF2α protein to the promoter of UCHL1 by using a Chromatin Immunoprecipitation Kit.Results:1.Individual UCHL1 concentrations were plotted,with a mean value at 3.3 ng/ml or 2.0 ng/ml for HIE or healthy subjects respectively.Further data analysis showed that serum UCHL1 levels were significantly elevated in neonates with HIE as compared with healthy ones(P<0.05).2.After the incubation with UCHL1 for 48 h,the percentage of AGE1.HN apoptotic cells increased in a dosage dependent manner,with a maximum of 5-fold increase after treated with 10 ng/ml UCHL1(P<0.05).3.Compared to normal cells,better cell growth of AGE1.HN was observed in cells with downregulated UCHL1 expression that obtained by the transfection of UCHL1 si RNAs.4.AGE1.HN cells were cultured under hypoxia condition for 12 h,24 h or 48 h,and the results of q RT-PCR and Western blot showed that UCHL1 expression was upregulated.The m RNA level of UCHL1 in AGE1.HN cells cultured under hypoxia for 48 h was increased by 1.9-fold.5.The upregulated UCHL1 expression induced by hypoxia was counteracted in HIFα inhibiting cells by pretreated with YC-1 or transfected with si HIF1α or si HIF2α.6.The percentage of apoptotic cells was strongly increased by hypoxia treatment,which could be attenuated in both HIFα inhibiting cells and UCHL1 silencing cells.7.The results of Ch IP assay showed that HIF1α and HIF2α could bind to the UCHL1 promoter in hypoxia-treated cells,but not in normoxia incubated cells.Quantitative real time PCR analysis demonstrated that HIF1α or HIF2α was significantly enriched on the proximal UCHL1 promoter with putative HRE sites,compared to a distal region of the promoter without HRE in AGE1.HN cellsConclusions:UCHL1 was involved in the cell apoptosis of hypoxic-ischemic encephalopathy.In addition,the transcription level of UCHL1 was promoted by the binding between HIF with the HRE sites within UCHL1 promoter.Part Ⅱ The expression of SBDPs and Tau proteins in Neonatal Hypoxic Ischemic EncephalopathyObjectives:To detect the expression of SBDPs and Tau protein in newborns with various degrees of HIE and treated by different treatments to evaluate their values for HIE diagnosis and prognosis.Methods:1.A total of 132 patients with neonatal HIE were continuously selected.There were 30 cases with mild symptoms,52 cases with moderate symptoms and 50 cases with severe symptoms.During the same period,30 infants admitted to this hospital for neonatal pneumonia were enrolled for the control group.2.Newborns treated with regular therapy included those in the control and mild symptoms groups and 30 infants from the moderate symptoms and 30 infants from the severe symptoms groups.On the 1st,3rd,5th and 7th days after birth,blood samples were collected from each patient and preserved.The expression levels of SBDPs and Tau protein were measured by sandwich-ELISA.3.The other 42 patients,22 from the moderate symptoms and 20 from the severe symptoms groups,were treated by mild hypothermia therapy.On the 1st,3rd,5th and 7th days after birth,blood samples were collected and saved for detection of expression levels of SBDPs and Tau protein by sandwich-ELISA.4.The clinical conditions after 28 days,3,6 and 12 months were evaluated.Comprehensive physical(weight,body length,head circumference,etc.)and neurological development examinations were performed.On the 28 th day after birth,the NBNA scoring was determined,and the total points for a perfect score were set at 40.Any score lower than 35 points indicated brain injury,a score between 35 and 37 points indicating suspected brain injury and a score of 37 or more points indicated normal development.The DQ values were obtained at 3,6 and 12 months after birth using the Gesell development scale.The test included motor ability,adaptability,language,and social contact.The results of each test were presented as DQ values,in which 55 to 75 points indicated mild intelligence disability,40 to 54 points indicated moderate intelligence disability and points less than 40 indicated severe intelligence disability.Results:1.At each time point,the plasma levels of SBDPs and Tau protein increased with the augmentation of HIE severity and decreased with the prolongation in treatment duration in each group with statistically significant differences between the averages in all groups(P<0.05).For groups with moderate and severe symptoms treated by mild hypothermia therapy,the serum levels of SBDPs and Tau protein at any point in time were significantly lower than those of groups treated by regular therapy(P<0.05).2.During the follow-up period,the NBNA score decreased with the increase of HIE severity.For groups with moderate and severe symptoms treated by mild hypothermia therapy,the NBNA score was significantly better than that of groups treated by regular therapy(P<0.05).3.At each time point,DQ was worse with the augmentation of HIE severity and improved with the prolongation in treatment duration in each group,with statistically significant differences between the averages of different groups(P<0.05).For groups with moderate and severe symptoms treated by mild hypothermia therapy,DQ at any point in time was significantly better than that of groups treated by regular therapy(P<0.05).4.The area under the curve(AUC)of ROC for HIE diagnosis in accordance with the level of SBDPs was 0.934(P=0.0001).And the sensitivity and specificity of diagnosis reached 84.1 and 87.2% respectively,when the critical point was 1.57 ng/ml.The AUC of ROC for HIE diagnosis according with the level of Tau was 0.938(P=0.0001).The sensitivity and specificity of diagnosis reached 79.1 and 96.3% respectively,when the critical point was 4.72 pg/ml.Conclusions:1.SBDPs and Tau protein were involved in the brain damage process of HIE.The serum levels of SBDPs and Tau protein were increased with the increasing severity of HIE injury.2.The expression of SBDPs and Tau protein were decreased in HIE infants treated by mild hypothermia therapy.The infants with the same degree of HIE and lower expression of SBDPs and Tau protein showed better long-term follow-up prognosis.Part Ⅲ The application of a EEG for the early diagnosis and long-term prognosis of newborns with hypoxic ischemic encephalopathy Objectives: To study the application of amplitude integrated electroencephalography(a EEG)for the early diagnosis and long-term prognosis of newborns with hypoxic ischemic encephalopathy.Methods:1.A total of 132 cases with HIE were as the observation group.During the same period,30 infants admitted to this hospital for neonatal pneumonia were enrolled for the control group.Two rows were received a EEG diagnosis.The image of a EEG was analyzed by professional.2.Observation index:(1)Continuity: Based on the a EEG background activity,there are two types of a EEG,including continuous EEG and discontinuous EEG.Continuous EEG showed continuous activity with a minimum voltage of(5)7-10 u V and a maximum voltage of 10-25(50)u V.Discontinuous EEG showed discontinuous activity with a minimum amplitude less than 5 u V and a maximum amplitude of more than 10 u V.(2)Sleep wake cycling(SWC): EEG was showed a regular sine wave with the cycling more than 20 min.The broadband represents sleeping period and the narrow band represents the waking period.(3)Minimum voltage: namely brain activities at the lowest level.(4)Maximum voltage: namely brain activities at the highest level.(5)The abnormal degree of a EEG: continuous normal voltage represents normal a EEG waveform.Discontinuous voltage or continuous normal voltage combining with single convulsion represents mild abnormal a EEG.Severe abnormal a EEG is characterized by the other type of background activity,recurrent convulsion or status epilepticus.Results:1.The continuous EEG of HIE group accounted for 34.09%(45/132),and the mature SWC accounted for 27.27%(36/132),which were significantly lower than the control group with 100%(30/30)and 100%(30/30),respectively.These data indicated that the EEG type of HIE group was mainly discontinuous EEG and the SWC type was mainly immature SWC.2.The maximum voltage of the HIE group was 56.52 ± 19.29 u V,which was significantly higher than the control group with 37.61 ± 2.53 u V;the minimum voltage was 4.28 ± 1.38 u V,which was significantly lower than the control group of 7.61 ± 0.86 u V.Differences were statistically significant(all P<0.05).These data indicated that the maximum voltage of the HIE group was increased,while the minimum voltage was decreased.3.According to the clinical grading,among the 132 HIE cases 30 cases of children showed mild HIE,52 cases of children showed moderate HIE and 50 cases of children showed severe HIE.Based on the results of a EEG,36 cases showed normal,50 cases of children showed mild HIE and 46 cases of children showed severe HIE.The results of a EEG of 30 cases in control group showed normal.Spearman correlation analysis found that a EEG monitoring results with clinical grading of HIE had positive correlation.4.According to the inspection results before discharge of the 132 cases,we found that 74 cases showed normal a EEG activity,40 cases showed mild abnormality and 18 cases showed severe abnormality.When the infants were followed up for 12 months,the DQ values of 11 cases were less than 55 among the 132 cases,accounting for 8.33%.Conclusion:a EEG,as a simple,effective diagnosis and continuous monitoring way,can reflect the severity of HIE and long-term prognosis and is worth for promoting the use in clinic. |
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