| 1Introduction Neonatal hypoxic-ischemia(HI)remains an important cause of acute mortality and chronic morbidity in infants and children.The neurologic consequences of injury include mental retardation,epilepsy,cerebral palsy,and blindness.It has been estimated that HI injury occurs in approximately 2-4 out of every 1000 full-term births in developed countries and is 20 to 30 times higher in developing countries.Despite the advances in neonatal healthcare,the increased understating of the pathophysiology of hypoxic-ischemic brain injury,and the introduction of therapeutic hypothermia as standard care for moderate to severe birth asphyxia,HI continues to lead to significant long-term neurodisabilities or mortality.Birth asphyxia causes an annual estimate of one million or 23%of all neonatal deaths worldwide.It is now well established that HI brain injury is a syndrome that evolves over days,even weeks.Over the few last years,evidence has accumulated pointing to a key role for connexin43(Cx43)signaling in neurophysiology.Cx43 forms gap junctions(GJ)and hemichannels(HC),which mediate intercellular and extracellular communication,respectively.In the central nervous system,Cx43 is expressed in developing neurons,activated microglia,pericytes and endothelial cells of blood vessels,but it is predominantly and abundantly found in astrocytes.These negative results can be explained in part by a neuron-centered approach,which could lead to overlooking the participation of other cell types and pathogenic mechanisms.Alternatively,cell types that could play a relevant role in neuronal survival are glia and particularly astrocytes,because they play major roles both in physiologic and pathologic states.Astrocytes are highly interconnected via gap junctions that allow for spatial buffering of extracellular K+,H+,and glutamate,among others.Astrocytes express connexin43 and hemichannel in the exploration of the diseases of the nervous system get more and more,for us to study the pathogenesis of hypoxic ischemia brain damage and treatment brings new view direction.In the brain,astrocytes represent the cellular population that expresses the highest amount of connexins(Cxs).Membrane topology of a generic connexin with four transmembrane domains,two extracellular loops and three cytoplasmic portions.Six connexins oligomerize into connexons that can be either homomeric,if they contain one connexin type,or heteromeric,if multiple connexins are assembled.Two connexons interact and align to form complete intercellular channels that can be homotypic,heterotypic,or heteromeric.Connexin channels cluster in specialized domains of the plasma membrane called gap junctions.Connexins are transmembrane proteins that form two different pathways for intercellular communication.One pathway involves the gap junction channels(GJCs)located between adjacent cells allowing direct cell-to-cell transfer of ions and small molecules.The other pathway is through hemichannels(HCls,half GJCs)located at unopposed cellular surface that permit the exchange between intra-and extracellular compartments,including release of autocrine/paracrine signaling molecules(e.g.,ATP,NAD1,and glutamate)to the extracellular milieu.At present,we have found that CX family has at least 21 members in mammals,according to different molecular weight named CX,according to the degree of gene sequence homology and intracytoplasmic circular structure domain length can be divided into different classes,namely,alpha,beta,and gamma.Cx43 belongs to connexin alpha,43 kd molecular mass.Cx43 expressed strongest in brain tissue,astrocytes,activated microglia,neurons in the development stag,vascular smooth muscle and endothelial cells can also express Cx43.For years it has been assumed that the hemichannel is half of a gap junction and cannot function independently.In recent years,the presence of hemichannels on the cell surface has been documented with several experimental approaches.Freeze-fracture replicas of the plasma membrane of Xenopus oocytes expressing Cx50 exhibit a new population of intramembrane particles(-9 nm in diameter)associated with a whole-cell current.Using an anti-Cx26 antibody that reacts with a region of the C-terminus of Cx26 and immunofluorescence,Cx26 has been localized at the membrane of the dendritic tips of horizontal cells of a teleost fish where gap junctions are absent,suggesting that Cx26 might form hemichannels.Moreover,in human polymorphonuclear cells treated with proinflammatory agents,but not in resting cells,Cx43 hemichannels have been detected by immunofluorescence using an antibody directed to a region of extracellular loop 1 of Cx43.The relative levels of Cx43 hemichannels located in the surface of osteocytic MLO-Y4 and NRK-cells have been measured by Western blot analysis of biotinylated cell surface proteins.Usually,most of the unapposed/non-junctional hemichannels are closed but a fraction of Cx43 HCs can be open under resting conditions and have physiological roles while they become more active in pathological situations.Accordingly,astrocytic HCs seem predominantly active in pathological conditions,e.g.,ischemia and inflammation,where they are proposed to be involved in damage-associated adenosine triphosphate(ATP)release,disturbed transmembrane ion fluxes and loss of essential metabolites.Connexins regulating channel functions,are essential for metabolism and dynamic interaction,the interaction is that astrocytes build with their own and their interface between neurons and vasculature.Indeed,transgenic animals in which the two major astroglial Cxs,i.e.,Cx43 and Cx30,have been deleted,exhibit impaired potassium clearance,synaptic transmission and plasticity,a dysmyelinating phenotype and a loss in blood-brain barrier integrity.In stroke,a possible role of astrocytes contributing to the amplification of damage has been previously proposed.In fact,they are the principal defense against excitotoxicity by active clearance of glutamate.However,energy failure could reverse transport and subsequently result in down-regulation of the glutamate transporter GLT-1,which exacerbates excitotoxicity.Under tissue stress,astrocytes could spread apoptotic signals through GJCs and HCs.In stress induced by metabolic inhibitors,the enhanced connexin HC activity of astroglia accelerates cell death.In a model of trauma in vivo,increased Cx43 immunore-activity is observed in the ipsilateral hippocampus 24-72 h post injury,and phosphorylation of Cx43 is detected already at 1 h post-trauma.In hippocampal slices,the connexin-based channel blockers,carbenoxolone and octanol,significantly reduced cell death post trauma.Similar results were obtained using antisense oligonucleotides for Cx43,and in brain slices from Cx43 KO mice.Studies on spinal cord injury suggest that Cx43 HC was involved in early injury.Some scholars found a spatial distribution of increased HC activity:cells at-17 mm away from the scratch gained Cx43 HC-mediated communication without changes in GJC-dependent coupling.This increase in HC activity was inhibited by connexin HC blockers,was prevented by blocking P2 purinergic receptors,and did not occur in astrocytes from Cx43 KO mice.The increased HC activity was associated with an increment of apoptotic cells at 24 h after scratch,which was abolished by inhibition of connexin HC.As the structural basis of blood-cerebrospinal fluid barrier(BCB),epithelial cells in the choroid plexus(CP)are targets for lead(Pb).Recently,hemichannels of Connexin 43(Cx43),the most ubiquitously expressed gap junction proteins in the CP,were found to be important pathways for many substances.This study was designed to investigate the roles of Cx43 in Pb uptake in the epithelial cells.Activation of Cx43 hemichannels by reduced serum conditions caused an increase of Pb concentrations.Cx43-induced Pb up-take was attenuated after blockage of Cx43 hemichannels with its inhibitor,carbenoxolone.Additionally,down-regulation of Cx43 protein levels by Pb exposure paralleled cellular Pb concentrations in the time study.Concomitantly,expressions of phosphor-Src and phosphor-Erk were both significantly increased by Pb.These data establish that Pb can accumulate in the BCB and validate the role of Cx43 hemichannel in Pb uptake and its regulations through Erk phosphorylation.Apoptosis is a prominent form of neuronal death in the neonatal hypoxia/ischemia model.In cerebral ischemia,the caspase cascade is mediated specially by caspase-3,classified as an executioner subclass member.Activated directly bycaspase-3 stimulates the mitochondrial path way causing apoptosis,and it is considered a biomarker of neuronal apoptosis and of brain lesion levels.Active caspase-3 is the protein that executes programmed cell death and removes unnecessary neurons.In the progression of HI injury,apoptosis closely follows necrosis which happens due to energy failure.Apoptosis is triggered by a cascade of proapoptotic initiator and effector caspase activity,caspase 3 in particular.Pharmacological inhibitors of apoptosis can reduce damage and improve sensorimotor function in neonatal models of brain injury.2 Objectives(1)To determine whether Cx43 and hemichannels are expressed in immature brain tissue in hypoxic ischemia model of neonatal rats;(2)Dynamic observation in vivo after hypoxia ischemia oxygen and glucose deprivation of Cx43,hemichannel and caspase 3,and the change of astrocytes activity,to seek the pathogenesis and treatment of HIBI provide new theoretical basis.3 Methods3.1 subjects(1)A total of 80 Wistar rats were born at the age of 7 day,a weight of 12 to 17g,with an average weight(14 ± 2.8)g,and the rats were male and female,provided by the experimental animal center in Shandong University.(2)Human cerebral cortex astrocytes:purchased from the United States ScienCell research laboratory.3.2 Research object grouping and model preparation(1)animal experiment grouping80 healthy newborn Wistar rats,only 7 days of age,were randomly divided into 5 groups,namely the control group(Sham),1 h after hypoxic ischemia group(Tlh group),12 h after hypoxic ischemia group(group T12h),24 h after hypoxic ischemia group(group T24h),48 h after hypoxic ischemia group(group T 48 h).(2)Neonatal hypoxic-ischemia encephalopathy(HIE)rat model establishmentA model of neonatal HIE was developed at postnatal-day 7(P7).Under anesthesia using lidocaine local anethesia,the left common carotid artery was isolated,double ligated,and cut.After recovery for 2 hours with their mothers,the pups were exposed to 8%oxygen with an ambient temperature 35? for 3 hours and then returned to their mothers.The pups were conditioned at 23±1? and a constant humidity of 60±10%in a room(12 h/12 h light/dark cycle).They had free access to food and water until they were killed.The same mother littermates as age-matched controls underwent neither surgical procedure nor hypoxic exposure and were killed at postnatal-day 9(P9).3.3 Hematoxylin-Eosin dyeingThe control group,1 h after hypoxia ischemia group,12 h after hypoxia ischemia group,24 h after hypoxia ischemia group and 48 h after hypoxia ischemia group were given hematoxylin-eosin dye.3.4 Immunofluorescent detectionThe expression of Cx43 + GFAP,HCl+GFAP in 1 h after hypoxia ischemia group was detected by immunofluorescence double labeling method?3.5 immunofluorescence histochemistryThe expression of Cx43,Cx43-hemichannel and Caspase-3 in the groups of rats were detected by immunofluorescence histological method.3.6 Western blot analysis of brain tissueThe protein content of Cx43,Cx43-hemichannel and Caspase-3 in the groups of rats were determined by Western blot for the determination of sham operation group and hypoxic ischemia groups.3.7 Cell culture,retroviral constructs and transfectionHuman cerebral cortex astrocyte cell line purchased from ScienCell Research Laboratories,were transduced with a Cx43-specific shRNA construct(Sequence GGTGTGGCTGTCAGTGCTC)or a retroviral empty vector.The GP2-293 packaging cell line(Clontech)was transfected by calcium phosphate precipitation with retroviral plasmid containing the Cx43 shRNA insert or empty vector.In this study,astrocytes cell line and astrocytes transfected with Cx43 shRNA were used to detect the roles of connexin 43 and its hemichannel in astrocytic death and apoptosis.Astrocytes transfected with an empty vector were used as a control to compare with Cx43 shRNA.3.8 Oxygen-glucose deprivation techniques(OGD)Cell cultures were subjected to the oxygen-glucose deprivation astrocytes media(previously bubbled with 5%C02/95%N2 for 30 min).Hypoxia was induced by placing the cultures inside of an incubator chamber and removing the air with a C02/N2(5,95%)flow for 15 min,after which the chamber was closed for 3 hours as previously described.Then,recovery was allowed in normal media(MEM with 5 mM glucose and 10%FBS)at 37?in a 5%C02/95%air atmosphere at nearly 100%relative humidity with different time points when appropriate(1h,12h,24h and 48h).3.9 Cell death quantification-MTT assayThe MTT reduction assay was used as an index of cell viability and was performed according to the manufacturer's instructions.Briefly,astrocytes were cultured in 96-well plates at a density of 5000 cells/well with culture medium for 24 hours to let the cells attach stably.After the treatment,0.4 mg/ml MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]was added into the culture medium.After 3 hours,the MTT solution was aspirated and dimethylsulfoxide(0.3mL/well)was added.Optical density of the supernatant was read at 570 nm using a microplate spectrophotometer.Absorbances were normalized to the untreated control cultures which represented 100%viability:%Viability = Mean Absorbance of Sample/Mean Absorbance of Control x 1003.10 Western blot analysis of astrocytesWestern blot was used to detect the protein of astrocytes,Psup astrocytes and shRNA astrocytes in the control group and OGD group in 1 hour,12 hours,24 hours and 48 hours.4 Results4.1 General conditionCompared with mice in the Sham group,mice in 1 hours after hypoxia ischemia group(Tlh),12 hours after hypoxia ischemia group(T12h),24 hours after hypoxia ischemia group(T24h)and 48 hours after hypoxia ischemia(T48h)were no statistical difference in weight(P>0.05).4.2 Successful mark of modelThe mice in hypoxic-ischemia groups were treated with hypoxia after ligation of the carotid artery.Hypoxia about 10 min,all the mice were fidgety,activity increased;Hypoxia 15-20 min,we could see the mice mouth and nose obviously cyanosis,breathing difficulty,breathing rate increase fast;Hypoxia 20-30min,the mice appeared to be unstable,waddling,crawling and dragging.During 35-60 min,the activities of mice were significantly reduced.After hypoxia 1 h,most of the mice showed lethargy and irritability,convulsions,shrieks,and even death.4.3 General view of brain tissueThe mice in Sham group and hypoxic-ischemic groups were given intraperitoneal injection of chloral hydrochloride and then put to death.The brains in Sham group were symmetrical with no abnormal changes.The brains in groups of different time points after hypoxic ischemia were edema,volume increased,enlarged left brain volume than on the right side.The brains in 48 hours after hypoxic ischemia group were found visible necrosis and liquiation on the left side.4.4 Brain tissue stainingBrain tissue staining showed that the neurons in the Sham group were normal;but neurons in different time points of hypoxia ischemia groups were swollen,arranged disordered,and even decreased in number of cells.At the same time glial cells were found increased,arranged disorders,and white matter tissues around the ventricle were loose.4.5 Expression of Cx43 + GFAP,HCl + GFAP in astrocytes in rat brainTo confirm that Cx43 and HCl can be expressed in the astrocytes with GFAP-an astrocytic marker,double immunostaining of Cx43 + GFAP and HCl + GFAP was performed.The results showed Cx43 and HCl expressed in the astrocytes which GFAP was detected in the same time.There appears to be markedly lower level of labeling for HC,compared to Cx43.4.6 Expression of Cx43,HCl and Casp3 in rat brainTo detect the expression of Cx43,HCl and Casp3 in rats' brain,immunostaining was undertaken following HI with appropriate time points.Compared with mice in the Sham group,the results showed that the expression of Cx43,HCl and Casp3 significantly increased after hypoxia ischemia in different groups of SVZ.The expression of Cx43,HCl and Casp3 in rats' brain hypoxia ischemia after 24 hours,48 hours expression were significantly increased and with significant difference(P<0.05).4.7 Western blot was used to detect the expression of Cx43,HCl,Casp3 after hypoxic ischemic injuryWestern blot was performed to further confirm above results.The elevated expression of Cx43,HCl and Casp3 from brain tissue in SVZ appeared after 1 hours of HI.Compared with control group,the mice expressed Cx43,HCl,Casp3 after hypoxic ischemic 24 hours,48 hours were significantly increased and with significant difference(P<0.05).4.8 Effect of transfection with a retroviral empty vector and null Cx43 gene in astrocytesWestern blot was performed to confirm the effects of the transfection.The results showed there was expression of Cx43 and HCl in both astrocytic cell line and Psup astrocytes,but not in shRNA astrocytes after the transfection.4.9 Viability of Psup and shRNA astrocytes after OGDTo investigate the viability of astrocytes after OGD,the MTT assay was performed.The cells that were not treated with OGD were in the control group,and the results showed that the activity of the cells increased;but the results showed there were significant decreases of viability for astrocytes cell line(P<0.05)and Psup astrocytes(P<0.05)on 24 hours after OGD.In contrast,there were no changes of the viability for shRNA astrocytes after OGD(P>0.05).4.10 OGD increases the expression of Cx43,HC1 and Casp3 in cell line and Psup astrocytes but not in shRNA cellsWestern blot was processed to detect the expression of astrocytes after OGD insult.The results showed the elevated expression of Cx43,HCl and Casp3 from cell line(P<0.05)and Psup astrocytes(P<0.05)on 24,48 hours after OGD.There were no changes of the expression of Cx43,HCl and Casp3 for shRNA cells after OGD(P>0.05).5 Conclusions(1)This study shows that connexin43 and its hemichannels significantly mediated with Casp3 in brain tissues and astrocytes.They may play the important roles in HI-induced astrotic death.They transform each other in all process of the cell death.(2)Cx43 and its hemichannels may become the signals to predict HI injury.The blockage or inhibiters of connexin43 and its hemichannels may contribute to neuroprotection for the clinic.1 BackgroundAutism spectrum disorders(ASD)are neurodevelopmental disorders in early childhood,characterized by a combination of social deficits,communication impairment and a variety of obstacles in behavior repetitions,and accompanied by a certain degree of mental retardation.ASD are persistent disabling neurodevelopmental disorders clinically evident from early childhood.In 2010,there were an estimated a prevalence of 7.6 per 1000 or one in 132 persons.However,the prevalence in Chinese children is unknown.In recent years,there was a number of reports on the prevalence of autism in asmall area.In 2013,Autism research report was analyzed on mainland China,Taiwan and Hongkong in recent years,someone pointed out the prevalence of autism from 2.8/ten thousand to 29.5/ten thousand in recent years.Many factors such as environment,genetic factor,inflammation and so on have been implicated in the etiology of autism.However,the pathogenesis of ASD is unclear.At present,many studies support that genetic factors are the important pathogenic factors of autism.The researchers found that De Novo gene mutations in autism patients were far higher than the general population.The study of twins and their families showed that genetic factors of ASD accounted for more than 90%,which had familial aggregation.Several dozens of ASD susceptibility genes had been identified in the past decade,collectively accounting for 10-20%of ASD cases,which suggested that we need to further identify the susceptibility genes of ASD,but also need to consider other factors.Many studies had shown that environmental factors,such as air pollutants,maternal exposed during pregnancy etc may promote the occurrence of autism.Environmental factors were likely to increase the incidence of ASD by amplifying genetic susceptibility to this adverse effect.Heavy metals,pesticides,infections,parental age and other environmental factors are also considered to be a possible cause of ASD.Neuroimaging studies by means of magnetic resonance imaging(MRI)had provided important insights into the neurobiological basis for autism.Children with autism had early brain overgrowth and brain slow growth later which were found by magnetic resonance imaging and head circumference measurement.Epigenetic regulation of neuronal structure and function was important for the development of the nervous system,alteration or destruction of this regulation may lead to many neurological diseases,such as ASD.Some scholars had suggested that immune factors were involved in ASD,especially the expression of cytokines in the brain and cerebrospinal fluid(CSF)related molecular immune abnormalities and then this was a permanent existence of early development of the brain immune disorder.One possibility was maternal infection that was a known risk factor for autism and activation of the immune response.Glial fibrillary acidic protein(GFAP)is the hallmark intermediate filament protein in astrocytes,a main type of glial cells in the central nervous system(CNS).GFAP had been studied in many neurological diseases,such as neuromyelitis optica attacks,stroke,traumatic brain injury,glioblastoma and Parkinson's disease.Further,Schiff et al.suggested that GFAP could provide clinically valuable information for the prognosis of TBI and stroke,but it was still at an early stage of development as a biomarker.Larsson et al.found serum GFAP increased in patients with poor outcome,but did not show sufficient sensitivity to predict neurological outcome after cardiac arrest.GFAP in ASD research reports are relatively fewer.GFAP immunoreactivity levels in the anterior cingulate cortex were significantly increased in some patients with ASD.Animal experiments demonstrated that GFAP immunoreactivity was increased in the CA3 specific region of hippocampus.In recent years,homocysteine is considered as a new risk factor for cardiovascular disease,which is closely related to thrombosis,vascular endothelial injury,inflammatory reaction,lipid metabolism and oxidative stress.Studies have shown that hyperhomocysteinemia is an independent risk factor for ischemic stroke.Some studies had shown that plasma and serum homocysteine levels were increased in ASD patients.The level of urine homocysteine in patients with ASD was also increased,which was directly associated with the severity of social communication deficits.However,some studies suggested that there was some controversy about the low level of plasma homocysteine in patients with ASD.Most children with ASD have different degrees of mental retardation.Studies had shown that the mortality rate of ASD patients was 2.8 times higher than that of the same age group of non autistic people?So far,there is still lack of specific therapy for ASD,which causes great pain and social burden for patients,families and society.Therefore,more mechanistic studies are needs to further explain the pathogenesis of ASD,so that we could prevent ASD earlier,reduce morbidity and it is great significant.2 Objectives2.1 To investigate the possible risk factors of ASD children based on the above-mentioned possible causes of ASD,to understand the high risk factors of ASD children in our city,and to explore the relationship between autism and children's general condition,birth,family factors and mother's aspects of pregnancy factors.2.2 To investigate the potential role of GFAP and HCY in ASD children by comparing the levels of GFAP,HC Y and CRP in serum of ASD and control group,and to determine the early diagnostic value and clinical significance of GFAP.3 Methods3.1Access to literature,design questionnairesBased on a large number of literatures at home and abroad,the possible influencing factors of ASD children were analyzed,and the questionnaire of this study was designed.The contents mainly included four parts:(1)children's general situation;(2)children's birth;(3)children's parents and families(4)mother's aspects during pregnancy.3.2 Determine the object of studyAn observational study was conducted over the period from January 2015 to December 2015 in Yantaishan Hospital.A total of one hundred and fifty 2-6 years old Chinese children(75 confirmed autism cases and 75 their age-gender matched typical development children)participated in this study.ASD were diagnosed as autistic disorder according to Diagnostic and Statistical Manual of Mental Disorders,4th Edition,and excluded all children with another axis I psychiatric disorder or having another chronic medical comorbid condition.All enrolled children with ASD were newly diagnosed,drug-naive,and exhibited symptoms were within the typical triad of autistic traits:communication impairment,social deficits,and ritualistic interests.The typical development children from a kindergarten near the hospital were assigned to the normal control group.In order to exclude the possibility that the controls could have any sub-clinical autistic features,all control subjects were also clinically examined by the pediatricians.Children with controls were excluded from the study if they receiving treatment for any reason and if endocrinological disease,mental retardation,communication disorder,psychotic disorder,attention deficit hyperactivity disorder and learning disorders seen in the children or their family members.3.3 Sign informed consentThe protocol and informed consent for this study were reviewed and approved by the Institutional Review Board at Yantaishan Hospital.The details of the study were explained to the parents of the participating children,and written informed consent was obtained from the parents.3.4 Issue questionnairesQuestionnaires were issued on ASD children and parents of normal children who matched the same age and same sex,and accessed to basic data.3.5 Conduct the Children's Autism Rating ScaleThe severity of autism symptoms was measured using the Chinese version of the Children's Autism Rating Scale(CARS).All children included in the study were evaluated for ASD severity.3.6 Test CRPEvery children had a routine CRP test,using the automated blood analyzer.3.7 GFAP detectionFasting blood samples were detected by GFAP,5 ml of venous blood was collected and serum was collected.After centrifugation,the serum was transferred to a centrifuge tube and stored at-80? for measurement.The serum GFAP was measured using a commercial ELISA kit by a blinded coworker.3.8 Determination of HCY Fasting blood samples were tested for HCY at the same time.After centrifugation,serum was transferred to a centrifuge tube and stored at-80? for measurement.Serum level of homocysteine(HCY)was tested using an enzyme cycling method.4 Results4.1 General Information ComparisonASD group and control group were all Han nationality,two groups of gender,age matched,including 59 boys and 16 girls,male to female ratio of 3.7:1,mean age was 3.75 years(SD:1.21),according to 1:1 case match.There was no significant difference between ASD group and control group(P>0.05).CARS score was 41.7 ±6.7 in the ASD group and 21.7 ±4.4 in the control group.There was significant difference between the two groups,(P<0.05).4.2 Univariate analysis of questionnairesThe results of single factor analysis showed that there was significant difference between ASD group and control group in the following aspects:contact time of electronic products,father's type of work,father's education,household income,whether there was a man of few words in the family,whether mother was exposed to electromagnetic radiation during pregnancy,maternal mood during pregnancy.4.3 Multivariate regression analysis of conditional questionnaireThe regression results of multivariate analysis showed that contact time of electronic products,household income,whether there was a man of few words in the family had effect on ASD.From the(Hazard Ratio)estimation point of view,household income more,contact the electronic products of the late time,man of few words less in the family were less risk of ASD.4.4 Correlative Analysis of the Influencing Factors of the Questionnaire and the Severity of ASDCorrelation analysis of influencing factors and severity of ASD showed contact time of electronic products differed significantly in different degrees of children with ASD.4.5 Determination of serum GFAP,HCY and CRPThe results on the serum GFAP levels in both autistic children and controls indicated that the mean serum GFAP level was significantly(p<0.001)higher in autistic children as compared to controls(1.71 ±0.53ng/ml vs.0.99±0.25ng/mll).Serum HCY and CRP were higher in autistic children compared to controls(P<0.01).4.6 Correlation between GFAP and CARS score,CRP,HCYLevels of GFAP increased with increasing severity of ASD as defined by the CARS score.There was a significant positive association between serum GFAP levels and CARS scores(r[Pearson]=0.390,P=0.001).GFAP and CRP levels were correlated(r ?0.205,P = 0.011).Serum GFAP levels were not correlated with gender,age,BMI and HCY levels(P>0.05).4.7 Conditional multivariate logistic regression analysisThe level of serum GFAP was related to the time of exposure to electronic products and the history of mothers during pregnancy(P<0.05).4.8 Determine the best diagnostic threshold for GFAPBased on the Receiver operating characteristic(ROC)curve,the optimal cut-off value of serum GFAP levels as an indicator for auxiliary diagnosis of autism was projected to be 1.28ng/ml which yielded a sensitivity of 77.3%and a specificity of 88.4%,the area under the curve was 0.895(95%CI,0.844—0.947).5 Conclusion5.1 In this study,we investigated the risk factors of children with local ASD in relation to the time of exposure to electronic products,family income and family.The more people in the family have fewer words,the lower the family income,the sooner the time of exposure to electronic products,the higher the risk of ASD.5.2 This study evaluated the serum levels of GFAP and HCY in ASD children and the correlation with the severity of ASD,suggesting that GFAP may be involved in the pathophysiology of children with autism and is a useful biomarker for autistic children's disease and can be used as an early detection assistant for ASD Diagnostic tools. |
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