| Part I Evaluation of hypoxic-ischemic encephalopathy in neonatal rats by ultrasound techniqueObjective Neonatal hypoxic-ischemic encephalopathy(NHIE)is a newborn brain injury caused by asphyxia in the perinatal period,including prenatal fetal distress and postnatal hypoxia,which is a main reason of newborn death and infant neurological function sequelae formation.The etiology of NHIE is very complex and the extent of the lesion is difficult to identify in time.Therefore,timely diagnosis of NHIE is required to help the early recovery of children,in order to improve long-term prognosis and reduce disability.In this study,we established the NHIE rat model.The ultrasonographic changes of the lateral ventricle and brain parenchyma were observed by two-dimensional ultrasonography(2DUS).The middle cerebral artery(MCA)Blood flow parameters were monitored by Doppler ultrasonography in the whole process of cerebral ischemia and hypoxia.To evaluate NHIE model brain injury,acoustic radiation force impulse imaging(ARFI)technique was applied.The brain tissue pathological changes were compared,and the neurobehavioral examination wereconducted to investigate the relationship between the ultrasonographic parameters and the histopathological changes and neurobehavioral changes of NHIE models in neonatal rats.Method1.Animal model establishment: a total of 108 Wistar rats of Guangxi Medical University experimental animal center were randomly divided into control group and two model groups.Control group: a total of 36 rats were sutured the wound directly without ligation of right common carotid artery.Ischemia group: a total of 36 rats were directly ligatured the right common carotid artery with 5.0 silk,then we sutured the wound.Asphyxia group: a total of 36 rats were ligatured the right common carotid artery with 5.0 silk,and after we sutured the wound and the rats recovered for 2 hours,we put the rats in a cabin was filled with 8% gas mixture of oxygen and nitrogen,the oxygen concentration was monitored by oxygen analyzer,hypoxia duration was last for 2 hours.2.Ultrasonic examination of animal models: Three groups of NHIE model rats were followed up for 3,12,24,48 and 72 hours after operation.The head of the rat was fixed on the ultrasound examination console,the probe was placed in the head metacarpal midline.And 2DUS was used observe the lateral ventricle and brain parenchymal lesions.The maximum systolic velocity(Vs),diastolic velocity(Vd)and resistance index(RI)at the beginning of the bilateral MCA was measured by color Doppler flow imaging(CDFI)and pulsed wave Doppler(PW).The shear wave velocity(SWV)was measured by acoustic radiation force impulse imaging(ARFI)technique.3.Histopathological examination of animal models: a total of 12 rats in each group were randomly selected for brain tissue histopathological specimens establishment and pathological changes observation.The edema range,nervecells,glial cell status,and the bleeding location and extent of brain tissue were observed by HE staining.4.Neurobehavioral observation of animal models: The rest rats in each group were kept up for 30 days of neurobehavioral observation.Results1.Animal model making success rate: In ischemia group,there were 4 deaths in the modeling process,the mortality rate was 11.11%(4/36),modeling success rate was 88.89%(32/36);in asphyxia group,there were 6 deaths in the modeling process,the mortality rate was 16.67%(6/36),in which 3 died during suffocation,2 died within 12 h to 24 h after suffocation,1 died within 48 h to 72 h after suffocation,modeling success rate was 83.33%(30/36).In control group,modeling success rate was 100.00%(36/36).2.Animal model PW results: There was no significant difference in preoperative Vs,Vd and RI among the three groups(P>0.05).For ischemia group,the preoperative Vs of right MCA was 10.62±1.28,compared to 3h Vs 6.80±1.62,the difference was statistically significant(P<0.05),compared with each previous time point,the changes of Vs value in 12 h,24 h and 48 h were not significant(P>0.05),and Vs of 72 h was 6.15±1.51,which decreased from 48 h Vs,the difference was statistically significant(P<0.05);the preoperative Vd of right MCA was 3.17±0.69,compared to 3h Vd was 3.00±0.46,the difference was statistically significant(P<0.05),the 12 h Vd raised to 3.44±0.65,compared to 3h Vd,the difference was statistically significant(P<0.05),compared with each previous time point,the changes of Vd in 24 h,48h and 72 h were not significant(P>0.05);the preoperative RI of right MCA was 0.69±0.05,which decreased to 0.52±0.07 in postoperative 12 h,the difference was statistically significant(P<0.05),compared with each previous time point,the changes of RIin 24 h,48h were not significant(P>0.05),RI value was 0.48±0.10 in postoperative 72 h,compared to each previous time point,the difference was statistically significant(P<0.05).For hypoxia-ischemia group,preoperative Vs of right MCA was 10.77±1.30,compared to 12 h Vs 5.97±0.60,the difference was statistically significant(P<0.05),compared with each previous time point,the changes of Vs value in 24 h,48 h and 72 h were not significant(>0.05);the preoperative Vd of right MCA was 3.2±0.77,raised to 4.20±0.97 in 3 h postoperative,the difference was statistically significant(P<0.05),it dropped back to 3.31±0.75(P<0.05)in 12 h postoperative,compared with 3h,the change was significant(P<0.05),compared with each previous time point,the changes of Vd in 24 h,48h and 72 h were not significant(P>0.05);the preoperative RI value of right MCA was 0.71±0.03,compared with 0.40±0.05 in 3h postoperative,the difference was statistically significant(P<0.05),12 h RI value was 0.45±0.64,compared with 3h RI value,the difference was significant(P<0.05),compared with each previous time point,the changes of RI in 24 h,48h were not significant(P>0.05),72 h RI value was 0.47±0.06,compared with48 h RI value 0.42±0.08,the difference was significant(P<0.05);to sum up,compared with ischemia group,the postoperative RI value in each time point was all significantly lower(P<0.05).3.Pre and postoperative SWV values of Animal model brain tissues: For control group,there was no significant difference in the SWV results preoperative and each time point postoperative(P>0.05).For ischemia group,the preoperative SWV value was 0.86±0.22,compared with 3h postoperative value 1.34±0.12,the difference was statistically significant(P<0.05),compared with each previous time point,the changes of SWV value in 12 h and 24 h were not significant(P>0.05),48 h SWV value was 1.41±0.12,compared with 24 h value,the difference was significant(P<0.05),the changes between 48 h and 72 h value was not significant(P>0.05).For hypoxia-ischemia group,the preoperative SWV value was 0.83±0.20,compared with 3h postoperative value 1.61±0.08,the difference was statistically significant(P<0.05),the changes between 3h and12 h were not significant(P>0.05),compared with each previous time point,the changes of the value in 24 h and 48 h were all significant(P<0.05),72 h value was decreased to 1.64±0.18,compared with 48 h value 1.76±0.15,the difference was significant(P<0.05).Each postoperative time point SWV value of hypoxia-ischemia group was higher than ischemia group,the difference was statistically significant(P<0.05).4.HE staining pathological changes of animal model brain tissues: For control group,hierarchical structure of the brain tissue was clear;the boundaries of cortex and medulla were distinct;neuronal cell nucleuses were visible with clear nucleoli,abundant cytoplasm and Nissl's Body.For ischemia group,hierarchical structure of the brain tissue was still clear;the boundaries of cortex and medulla could be identified;neuronal cell nucleuses were visible with occasional karyopyknosis.The perivascular cells were shown with edema.For hypoxia-ischemia group,hierarchical structure of the brain tissue was less distinct;the boundaries of cortex and medulla were unclear;neuronal cell nucleuses were visible with occasional karyopyknosis,unclear nucleuses and less Nissl's Body;clear nucleoli,abundant cytoplasm and Nissl's Body;accumulation of a large number of red blood cells were visible in small blood vessels.5.Neurological test results of animal models: compared with control group,the neurological results were significantly lower in the ischemia group and asphyxia group(P<0.05).For open field test,the horizontal score and vertical score ofischemic group and hypoxia-ischemia group were lower(horizontal score:ischemic group 49.81± 8.00,hypoxia-ischemia group 32.00± 5.29,control group63.30±11.60,(P<0.05);vertical score: ischemic group 9.75±2.08,hypoxia-ischemia group 7.21±1.67,control group 13.50±3.0,(P<0.05).For suspention test,the residence time of the rats in the ischemia group and the hypoxic-ischemia group was significantly lower than that in the control group(ischemic group 1.38±0.62 min,hypoxia-ischemia group 0.79±0.70 min,control group 2.05±0.76 min).For slope test,the time for the head of the rat turned from downwards 45 degrees to upwards into >135 degrees was significantly slower in ischemia group and hypoxia-ischemia group(ischemia group 4.00±0.73 sec,hypoxia-ischemia group 7.79±1.63 sec,control group 2.80±0.83 sec),the difference was significant(P<0.05).For capture experiment,scores in ischemia group and hypoxia-ischemia group were significantly higher(ischemia group3.81±0.75,hypoxia-ischemia group 4.85±0.75,control group 2.36±0.93),the difference was significant(P<0.05).Overall,neurological score: sham operation group> ischemic group> hypoxia ischemia group,the difference was statistically significant(P<0.05).Conclusion1.Application of newborn rats with unilateral common carotid artery ligation combined with the placement in hypoxia box to make NHIE model is a reliable method for human NHIE simulation.2.Changes of hemodynamic parameters in the brain can reflect the early lesions of NHIE,and corresponds to the degree of disease,NHIE can lead to changes in brain tissue structure,the number of neurons decreased,nerve cell swelling and deformation,bleeding and other pathological changes.3.ARFI technique can quantitatively reflect the hardness of NHIE brain tissue,can accurately assess the degree of brain injury,its simple operation,high safety factor,the impact on the organization is small,can become early clinical evaluation of neonatal NHE degree of new indicators.4.In this study,the neonatal rat model can be observed with NHIE-induced neurobehavioral adverse consequences,assess the extent of injury by neurobehavioral experiments can provide basic data for clinical brain injury data.Part II Clinical study of neonatal hypoxic-ischemic encephalopathy by ultrasonography and Gesell Development Diagnosis Scale assessmentObjective NHIE may lead to an increase in infant mortality and the incidence of long-term adverse disease.Severe NHIE can cause severe neurological sequelae and long-term adverse effects on children,including mental and motor development damage,cortical blindness,neurological hearing loss,mental disability,cerebral palsy,epilepsy and cerebral palsy,and even death.ARFI technology obtains information related to tissue elastic deformation characteristics,can fully display organization texture,hardness,specific elasticity value,shape ratio,area ratio,etc.,similar to the organization's physical palpation check,can give the specific tissue hardness measurement,with a broad clinical prospects,and can be applied to almost all organs of the human body.In this study,we used Doppler ultrasonography to detect MCA blood flow indexparameters and ARFI technique to evaluate the early brain injury of HIE in neonates,with follow-ups.The results were compared with Gesell Development Diagnosis Scale(GDDS)results,to provide basic research and clinical exploration of the theoretical basis for the early diagnosis of NHIE and predict long-term prognosis.Methods1.A total of 204 children with clinically diagnosed NHIE were enrolled in this study.The time of examination was 3 days after birth.Normal control group was 200 cases.2.Observation indicators: The echo status of bilateral ventricles and brain parenchyma was observed by 2DUS.MCA was applied by color Doppler ultrasound,and PW technique was used to measure Vs,Vd and RI parameters.Fourteen days and 1 month after,patients were asked for ultrasound re-examination.The left and right parietal white matter area,brain falculus and bilateral thalamic SWV values were obtained using ARFI technique.Patients were asked to receive the first GDDS test in 4 weeks to 8 weeks after birth,and also receive GDDS reviews in 10 months and 20 months of age,development quotient(DQ)was recorded for intelligence test scores.Results1.2DUS examination results: mild group showed echo enhancement limited to the lateral ventricle side of the brain parenchymal,compared with the choroid plexus the echo was slightly lower,14 days after,the echo recovered.Including28 cases ependymal hemorrhage,and 15 cases intraventricular hemorrhage.Moderate to severe group showed diffuse echo of brain parenchyma increased,equal or higher than the choroid plexus,14 days after,72 cases showed normal echo,24 cases showed ventricles expaned,and 6 cases showed brainparenchymal exploration and no-echo capsule formation.Including subventricular hemorrhage in 34 cases,intraventricular hemorrhage in 20 cases,and 4 cases of cerebral parenchymal hemorrhage.2.Results of CDFI and PW: The blood flow parameters of MCA were measured within 3 days after birth.For term patients,Vs in mild group and term moderate to severe group were lower than those in control group,the difference was significant(P<0.05);Vd had a downward trend in mild group and moderate to severe group compared with the control group,but the difference was not significant(P>0.05);the changes of RI in the three groups had no significant difference(P>0.05).For preterm patients,Vs in mild group and moderate to severe group were lower than control group,the difference was statistically significant(P<0.05);Vd in mild group and moderate to severe group lower than control group,the difference was significant(P<0.05);there was no significant difference in RI between three groups(P>0.05).A scatter plot showed that RI of moderate and severe term children was concentrated in the range of <0.65 and>0.75,RI of moderate and severe preterm children was concentrated in the range of <0.60 and> 0.80.Fourteen days and 1 month after,most of the patients showed hemodynamics parameters improved,that was,Vs raised,and Vd raised.A scatter plot showed that RI distribution of 14 days in moderate to severe term children was concentrated in the range of 0.70 to 0.80,and RI distribution of 14 days in moderate to severe preterm children was concentrated in the range of0.60 to 0.80;for 1 month,RI distribution in moderate to severe term children was concentrated in the range of 0.65 to 0.75,that in moderate to severe preterm children was concentrated in the range of 0.65 to 0.80.3.ARFI results: Due to fewer children received ARFI examination(A total of137 cases,including 40 cases in mild group,42 cases in moderate to severegroup and 55 cases in control group),therefore,we merged the data to analyze.The 3 days SWV results showed that the difference of SWV value of left and right cranial parietal lobe and thalamus in each group was not significant(P>0.05).Compared between groups,SWV values of parietal lobe and thalamus in moderate to severe group were higher than those in mild group and control group,the difference was significant(P<0.05).the SWV value of thalamus in mild group was higher than control group,the difference was significant(P<0.05).Among them,there were 72 cases of NHIE patients(34 cases in mild group,38 cases in moderate to severe group)returned to hospital in 14 days and1 month after the first examination.The results showed that the SWV values of parietal lobe in mild group and moderate to severe group decreased in both reviews,the difference was significant(P<0.05).The SWV changes of thalamus in both reviews were not significant(P>0.05).The SWV changes of brain sickle in mild group and moderate to severe group was not significant compared with the first check,the difference was not significant(P>0.05).4.GDDS and neurological outcome in NHIE children: After primary ultrasound examination,1 case died in preterm mild group due to the merger of severe pneumonia and sepsis,1 case died in term infants with moderate to severe NHIE due to the merger of renal insufficiency,2 cases died in preterm moderate to severe NHIE group,the difference of mortality rate was not statistically significant(P>0.05).After 10 months and 20 months of development and rehabilitation,the GDDS score were increased,the difference was statistically significant(P<0.05)There was no significant difference in the incidence of neurological sequelae in each group(P>0.05).DQ of 20 months showed that mild group of term infants> mild group of premature infants> moderate to severe children of term infants> moderate to severe of premature infants,thedifference was statistically significant(P<0.05).Conclusion1.CDFI and PW hemodynamic parameters can reflect the degree of NHIE lesions,which has important clinical significance in early clinical prediction of neonatal brain damage and assessment of prognosis.2.Ultrasound ARFI technique used SWV to quantitatively assess the degree of brain injury in NHIE and can provide new parameter information for the evaluation of NHIE brain injury.However,a better elastic imaging technology for the diagnosis of NHIE remains to be further studied.3.The Gesell Development Diagnostic Scale allows early assessment of the level of neurological development in children,which can provide important clinical data for early clinical diagnosis and early intervention. |
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