Study On Early Predictor Of Neurobehavioral Outcomes In Neonates With Hypoxic-Ischemic Encephalopathy And Preliminary Exploration On Therapeutic Effect Of Different Doses Of Inhaled Nitric Oxide

Introduction Hypoxic ischemic encephalopathy(HIE)is the major cause of death and disability among full-term newborns,which imposing heavy stress on individuals,families and society.There are several reasons why the incidence of neurological sequelae has not improved significantly even with the current application of mild hypothermia based on Grade A evidence-based medical evidence.How to identify the occurrence of neonatal HIE early,judge the severity of HIE,predict the prognosis,give effective treatment early,and focus on follow-up intervention are important measures to improve the prognosis of children with HIE.The exploration of the relationship between early nerve reflex,brain imaging changes and synaptic damage in HIE children and their neurodevelopmental level,as well as the effective intervention measures will help clinicians to better manage neonates with HIE.In this study,amplitude integrated electroencephalogram(aEEG),serum neurone specific enolase(NSE)and magnetic resonance imaging(MRI)were used alone and in combination to predict the long-term neurobehavioral function of neonates with HIE.To further investigate the effects of hypoxic ischemic brain injury(HIBI)on nerve reflex and percentage of residual brain volume(RBV%)in neonatal SD rats.And to investigate the relationship between early nerve reflex,RBV%,synaptic gap width and learning and memory function after HIBI;the therapeutic effect of inhaled nitric oxide(iNO)at different doses was further discussed.Materials and Methods From June 2013 to December 2020,neonates with HIE who were born and treated in the third affiliated hospital of Anhui Medical University were continuously included in this prospective cohort study,and were classified into mild,moderate and severe by Sarnat grading criteria.All children were undergone aEEG monitoring within 6 hours,serum NSE levels were detected on days 1(NSE1d)and3(NSE3d),and routine head MRI was performed on days 4 to 7 after birth.Long-term systematic neurobehavioral follow-up was conducted for these children,the children were assessed and classified at 18 months by neurological examination and the Bayley Scales of Infant development II(BSID II).To evaluate the prognostic accuracy of aEEG,serum NSE,brain MRI and their combination.The sensitivity,specificity,positive predictive value(PPV)and negative predictive value(PPV)of aEEG,serum NSE and head MRI were calculated to predict neurological prognosis.A neonatal SD rat model of HIBI was established,cliff turning reflex and negative geotaxis reflex were evaluated,RBV% was measured,the latency time and swimming distance of Morris water maze were calculated,and cell damage,synaptic vesicles and macula densa in hippocampus CA1 on the side of common carotid artery ligation(left side)were evaluated,and the width of synaptic gap was measured.Then different doses of iNO(5ppm and 20ppm)were given to HIBI neonatal rats.Morris water maze test,synaptic gap width and cell apoptosis in left hippocampal CA1 region were evaluated in all surviving rats in control group,model group and different iNO dose treatment groups.Results(1)Among the 52 children with HIE,1 did not meet the inclusion criteria and 1died.Finally,50 children with HIE met the inclusion criteria and completed the entire study.According to Bayley II score,the children were divided into three groups:normal group(32 cases),critical state group(5 cases)and neurodevelopmental retardation group(13 cases)respectively.There were significant differences in aEEG monitoring among the three groups within 6h after birth(P < 0.001).The serum NSE3 d level of the three groups was significantly different,and the normal group was lower than the critical state group(P < 0.01)and the developmental retardation group(P <0.001).There were significant differences in head MRI findings 4 to 7 days after birth among the three groups(P < 0.001).AEEG showed high sensitivity but poor specificity,serum NSE3 d had higher sensitivity and specificity,and brain MRI had better sensitivity and specificity.The combination of the three variables(abnormal aEEG,high NSE level,and abnormal MRI)had a sensitivity of 100%,specificity of97.70%,PPV of 98.25%,and NPV of 99.98%.(2)The mortality rate in the model group was 15%,higher than that in the control group(0%)(P < 0.001).The growth rate of in model group was significantly lower than that in control group(P = 0.001).At Postnatal 14 days(P14),the cliff turning and negative geotaxis reflex of rats in the model group were significantly delayed compared with those in the control group(all P < 0.05).The left side of the brain in the model group showed significant injury on MRI postnatal 10 days(P10),and the mean RBV%was significantly lower than that of the control group(P < 0.001).Morris water maze experiment showed that the latency time and swimming distance of the model group were significantly longer than those of the control group(all P < 0.001).The degeneration and necrosis of the cells in the left hippocampal CA1 region of the model group were observed under light microscope,and the presynaptic membrane vesicles were decreased,the posterior synaptic dense plaques were thinner under electron microscope,and the synaptic gap was wider than that in the control group(P < 0.001).Linear regression analysis showed that latency time and swimming distance were negatively correlated with the RBV% of P10(all P < 0.01),significantly positively correlated with cliff turning reflex and negative geotaxis reflex at P14(all P < 0.01),and significantly positively correlated with the width of synaptic gap(all P < 0.01).(3)The mortality rate of model group was higher than that of HIBI+5ppm iNO group and HIBI+20ppm iNO group,but there was no statistical significance(all P > 0.05).The growth rate of model group was significantly lower than that of HIBI+5ppm iNO group and HIBI+20ppm iNO group(all P < 0.05).The results of Morris water maze experiment showed that the latency time and swimming distance of the four groups were significantly different(all P < 0.001).The synaptic gap width in the left hippocampal CA1 region of the four groups was significantly different(P = 0.000),and the model group was significantly larger than that of HIBI+5ppm iNO group(P = 0.002)and HIBI+20ppm iNO group(P = 0.000),and HIBI+5ppm iNO group was significantly larger than HIBI+20ppm iNO group(P = 0.000).TUNEL method was used to detect apoptotic cells in CA1 region of the left hippocampus of rats.The apoptotic cell count in the model group was higher than that in HIBI+5ppm iNO group and HIBI+20ppm iNO group(all P < 0.01),and the apoptotic cell count in HIBI+5ppmiNO group was higher than that in HIBI+20ppmiNO group(P = 0.000).Conclusion(1)aEEG within 6 hours,NSE at 3 days,and head MRI at 4 to 7 days after birth can predict the neurological outcome of HIE neonates,and the combination of them can improve the ability to predict the long-term neurobehavioral outcome.(2)The neonatal rats with HIBI showed abnormal nerve reflexes at a later stage,structural damage of brain tissue,learning and memory dysfunction and microscopic cytological damage.(3)The long term learning and memory function of HIBI neonatal rats were correlated with the nerve reflexes at a later stage,the percentage of remaining brain capacity and the width of synaptic gap.(4)Treatment with 5ppm iNO and 20 ppm iNO can reduce synaptic damage and cell apoptosis,improve growth and development,and improve long-term learning and memory ability of neoantal male rats with HIBI,and the efficacy of 20 ppm iNO is more obvious than that of 5ppm iNO.The First Part Early predictors of neurobehavioral outcomes of neonatal hypoxic-ischemic encephalopathyIntroduction Hypoxic-ischemic encephalopathy(HIE)can lead to death,and leave a variety of neurobehavioral sequelae,such as retardation of mental development and psychomotor development.In this study,the long-term neurobehavioral development of children with HIE was systematically followed up over a long period of time,and the predictive value of aEEG,serum NSE,and conventional head MRI alone and in combination was also discussed.Materials and Methods Term neonates with HIE were enrolled in the study.AEEG within 6 hours after birth,serum NSE(NSE1d)level on day 1 and NSE(NSE3d)level on day 3 were monitored,and routine head MRI examination was performed on day 4to 7 after birth,and long-term systemic neurobehavioral function follow-up was performed.Bayley Infant Development Scale II(BSID II)and neurological examination were performed at 18 months.Results(1)Among the 52 children with full-term HIE,1 died and 1 did not meet the inclusion criteria.Finally,50 children with HIE completed the study and were divided into normal(32 cases,64.0%),critical state(5 cases,10.0%)and developmental retardation(13 cases,26.0%)groups according to Bayley II score.(2)aEEG was abnormal in 42 cases(84.0%),normal in 8 cases(16.0%),and 6 cases with recurrent convulsion,which was significantly associated with poor prognosis(P <0.001).(3)The serum NSE3d level in developmental retardation patients(44.60±9.01 μg/L)was significantly higher than that in critical state patients(39.82±5.92 μg/L,P < 0.001)and normal patients(20.52±6.42 μg/L,P < 0.001).(4)The head conventinal MRI of 24 patients(48%)was normal,7 patients(14%)grade1,6 patients(12%)grade 2,6 patients(12%)grade 3,and 7 patients(14%)grade 4,the MRI findings were statistically significant in the three groups at 4 to 7 days after birth.(P < 0.001).(5)aEEG showed high sensitivity(100%)but poor specificity(25.00%)in predicting neurodevelopmental outcomes at 18 months.Serum NSE3 d had high sensitivity(100%)and specificity(87.50%).Brain conventional MRI showed good sensitivity(94.44%)and specificity(71.88%)at 4 to 7 days after birth.The combination of three variables(abnormal aEEG,high serum NSE3 d and abnormal MRI)had a sensitivity of 100%,specificity of 97.70%,PPV of 98.25%,and NPV of 99.98%.Conclusion The combination of aEEG monitoring within 6 hours,serum NSE3 d level and head MRI results at 4 to 7 days after birth has a good predictive value for the prognosis of neonates with HIE.The Second Part Study on the correlation between nerve function development and brain imaging and synaptic damage in hippocampal CA1 region of HIBI in neonatal SD ratsObjective To investigate the nerve reflexes,changes of brain structure on MRI,Morris water maze test and synaptic damage in hippocampal CA1 region of rats after HIBI,and the relationships between them applying neonatal SD rats with hypoxic ischemic brain injury(HIBI).Methods A total of 40 neonatal SD rats at 7 days after birth were randomly divided into model group(n=20)and control group(n=20).In the model group,HIBI model was made by modified Rice-Vannucci method,cliff turning reflex and negative geotaxis reflex at P9,P11 and P14 were evaluated,Brain MRI was performed on Postnatal 10 days(P10),and the percentage of residual brain volume(RBV%)was measured and calculated.Morris water maze test was performed one month after birth,and then brain histopathology was performed.The latency time and swimming distance of Morris water maze were calculated,and the cell damage in CA1 region of hippocampus was detected,and synaptic vesicles,macula densa and the width of synaptic gap were evaluated on the side(left side)of common carotid artery ligation,and the relationship between them was evaluated.Results(1)Three rats in the model group died during or after hypoxia,and the mortality rate was 15% in the model group,while no rats in the control group died(P <0.001).The growth rate in the model group was significantly lower than that in the control group(P = 0.001).(2)Rats in the model group showed significantly slower in cliff turning reflex and negative geotaxis reflex than those in the control group at 14 days after birth(all P <0.05).(3)The left side of the brain in the model group showed significant structural damage on MRI at P10,and the mean RBV% was significantly less than that in the control group(P < 0.001).(4)Morris water maze traces showed that the latency time and swimming distance of the model group were significantly longer than those of the control group(all P <0.001).(5)The degeneration and necrosis of cells was observed in the left hippocampus CA1 region of model group under light microscope.Presynaptic membrane vesicles were significantly reduced,postsynaptic membrane dense spots were thinner,and and synaptic gap was significantly wider in the model group compared with the control group under electron microscope(P < 0.001).The latency time and swimming distance were negatively correlated with RBV% at P10(all P < 0.01),positively correlated with cliff turning reflex and negative geotaxis reflex at P14(all P < 0.01)and with synaptic cleft width(all P < 0.01).Conclusion HIBI neonatal rats showed abnormal nerve reflexes later in life,structural damage of brain tissue,learning and memory dysfunction and synaptic damage in hippocampal CA1 region,and the learning and memory function was correlated with nerve reflexes,percentage of residual brain volume and synaptic damage.The Third Part Effects of different doses of iNO treatment on learning and memory function and cell damage in hippocampal CA1 area in neonatal male SD rats with HIBIObjective To evaluate the long-term improvement of learning and memory function,synaptic damage and apoptosis of brain cells in hippocampal CA1 area after treatment,which applying different doses(5ppm and 20ppm)of inhaled nitric oxide(iNO)to treat neonatal SD rats with hypoxic ischemic brain injury(HIBI)Methods A total of 80 neonatal SD male rats postnatal 7 days(P7)were divided into control group,HIBI model group(i.e model group),HIBI+5ppm iNO group and HIBI+20ppm iNO group with 20 rats in each group according to random number table method.The control group did not do any treatment such as neck skin incision;model group production is the same as the second part;HIBI+5ppm iNO group and HIBI+20ppm iNO group were put into iNO boxes with concentrations of 5ppm and20 ppm immediately after the production of HIBI model.The Morris water maze test was performed on all surviving rats 1 month after birth,and synaptic damage and cell apoptosis in hippocampal CA1 region of the common carotid artery ligation side(left side)were detected.Results(1)The mortality rate of the model group(15%)was higher than that of the HIBI+5ppm iNO group(10%)and HIBI+20ppm iNO group(5%),but the differences were not statistically significant(all P > 0.05).(2)The growth rate of rats in the four groups was significantly different(P = 0.001),and the model group was significantly lower than that of the HIBI+5ppm iNO group(P= 0.040)and HIBI+20ppm iNO group(P = 0.001).There was no significant difference between HIBI+5ppm iNO group and HIBI+20ppm iNO group(P = 0.205).(3)The results of Morris water maze experiment showed that there were statistically significant differences in latency time and swimming distance among the four groups of rats(all P < 0.001),while there were no statistically significant differences in latency time and swimming distance between the model group and the HIBI+5ppm iNO group(all P > 0.05).However,they were all longer than those in HIBI+20ppm iNO group(all P < 0.01).The latency time and swimming distance of HIBI+5ppm iNO group were longer than those of HIBI+20ppm iNO group(all P < 0.01).(4)The synaptic gap width in the left hippocampal CA1 region of the four groups was significantly different(P = 0.000),and the model group was significantly larger than that in HIBI+5ppm iNO group(P = 0.002)and HIBI+20ppm iNO group(P = 0.000).HIBI+5ppm iNO group was significantly larger than HIBI+20ppm iNO group(P =0.000).(5)TUNEL method was used to detect apoptotic cells in CA1 region of the left hippocampus of rats.The apoptotic cell count in model group was morer than that in HIBI+5ppm iNO group and HIBI+20ppm iNO group(all P < 0.01).The apoptotic cell count in HIBI+5ppm iNO group was more than that in HIBI+20ppm iNO group(P =0.000).Conclusion 5ppm iNO and 20 ppm iNO can reduce synaptic damage,reduce brain cell apoptosis,and improve growth development and long-term learning and memory ability of neonatal male SD rats with HIBI,and the efficacy of 20 ppm iNO is more significant than that of 5ppm iNO.