Spatio-Temporal Expression Of Progranulin In Brains Of VPA-Induced ASD Rats And Its Effect On Neurodevelopment

Autism spectrum disorder(ASD)is a congenital developmental disorder of central nervous system,which is characterized by impaired social interaction,repetitive stereotyped behaviors,language difficulties,mental retardation and hypoesthesia or hyperesthesia.The prevalence of ASD has been rising gradually and it impacts one out of every 100 children born in China.ASD is now recognized as a common,lifelong neurodevelopmental disorder,which has become a urgent public health problem in recent years.The development of central nervous system during embryonic and postnatal periods is a vast array of biological spatiotemporal processes which is elaborately orchestrated and precisely regulated.Neurodevelopmental hypothesis suggests that genetic and environmental factors in the early development of individual growth lead to abnormal neuronal proliferation and differentiation and synaptic dysplasia,resulting in neurological dysfunction.A growing body of neuroimaging and neuropathology evidences have reported that there are neuroanatomicalabnormalities during early development of individuals with ASD.In ASD children aged 6 to 24 months over growth of specific brain regions was observed.However,in childhood or adolescence development of these regions slowed down and growth retardation occurred.These structural abnormalities may alter the local connectivity in specific regions of the brain,which affects social interaction,language,cognition and movement regulations.The exact reasons for these neurodevelopmental abnormalities are not yet known.progranulin(PGRN)is a kind of secretory growth factor with the activity of neural growth factor,which is widely involved in physiological and pathological reactions like growth and development,injury and repair,inflammation and metabolism regulation.It has been shown that PGRN can reduce neuronal apoptosis,promote the survival of neurons,and be involved in the formation and growth of synapses.Previous studies have confirmed that PGRN knockout mice presented neurological symptoms,such as motor dysfunction,spatial learning and memory impairment,anxiety and social behavior disorder.It have also been found that the concentration of PGRN was significantly reduced in serum samples of ASD children.Therefore,it is reasonable to suppose that abnormal pattern of PGRN expression during nervous system development in ASD children may alter the ordinary processes of neuronal apoptosis and synaptic development,resulting in ASD-associated neurologic impairments.However,no studies have reported the expression pattern of PGRN in the brains of ASD patients and its effect on nervous system development.Part one Comparison of VPA-induced autism spectrum disorder models in SD rats and C57 miceObjective: To compare the differences and similarities between SD rat and C57 mouse models of valproic acid(VPA)-induced ASD,and select an appropriate experimental animal model for the subsequent study.Methods:1.Animal modelVPA groups of pregnant SD rats or C57 mice were injected intraperitoneally 600 mg/kg of VPA on embryonic day 12.5(E12.5)of gestation,and control groups were administered by an equal amount of normal saline.2.GroupsVPA groups and control group of C57 mice : VPA-M group and CON-M group.VPA groups and control group of SD rats : VPA-R group and CON-R group.3.Detection indicators(1)The farrowing rate of pregnant rats or mice and 4-day survival rate,malformation of their offspring were detected.(2)The pups' physical development were continuously monitored by body weight,tail length,incisor eruption,eye opening,development of fur.(3)The pups' neurological function development were continuously monitored by surface righting reflex,cliff avoidance reflex,air reflex,crawling,bar holding test,pivoting,auditory startle.(4)Open-field test,three chamber sociability test,and self-grooming test were used to assess ASD-like neurobehaviors.Results:1.Compared with CON-M group,the farrowing rate of pregant VPA-M group was significantly lower,the external abnormality rate of pups was higher,and the 4-day survival rate of pups was reduced.There was no significant difference in the three indicators between the VPA-R group and the CON-R group.2.The VPA-M group showed significant growth retardation,such as lower body weight,delayed incisor eruption and eye opening.Although the weight of VPA-R group was also significantly lower than that of CON-R group,there was no significant delay in the incisor eruption and eye opening between the two groups.3.There were significantly differences in surface righting reflex,cliff avoidance reflex,crawling,bar holding test,auditory startle and air reflex between the VPA-R and CON-R groups.The VPA-M group was differentonly in pivoting,bar holding test and auditory startle from the CON-R group.4.Open-field test results showed that the vertical score of the VPA-M group was lower than that of CON-M group.Both the central grid score and the vertical score of VPA-R group were significantly lower than those of the CON-R group.5.Three chamber sociability test showed that in Stage ?,compared with the CON-R group,the VPA-R group spend less time to stay in stranger1 cage,to interact with stranger1 and the object,and more time to stay in object cage.There was no difference between the two groups of mice.In stage ?,compared with the CON-R group,the VPA-R group spend more time to stay in the case with familiar rat(stranger1),and less time to stay in the case with stranger rat(stranger2),to interact with stranger1 or stranger2.Compared with the CON-M group,the VPA-M group spent more time to stay in the stranger1 cage and to interact with stranger1,and less time to stay in the stranger2 cage.6.There was no difference in the cumulative self-gromming time between the VPA-M and CON-M groups.Compared with CON-R group,the gromming time of VPA-R group was markedly prolongedConclusion:Exposure to VPA in the second trimester of pregnancy,the offspring of both SD rats and C57 mice exhibited varying degrees of behavioral deficitsrelevant to ASD.However,growth retardation in nervous system and ASD-like neurobehavior of VPA-treated C57 mice offspring were mild.Moreover,the farrowing rate,external abnormality rate and 4-day survival rate of VPA-treated C57 mice were much higer than those of VPA-treated SD rats.Therefore,SD rats were superior to C57 mice in terms of model face validity,cost,and ethics in this VPA-induced ASD model.Part Two Spatiotemporal expression of PGRN in brains of VPA-induced ASD rats and its effect on neurodevelopmentObjective: This study using VPA-induced ASD rats model investigates spatiotemporal pattern of PGRN expression in brains and its effect on neuronal apoptosis and expressions of synapse-associated proteins to reveal that PGRN is involved in the phathogenesis of ASD and its underlying mechanism.Methods1.Animal model and groups:Pregnant SD rats were injected intraperitoneally 600 mg/kg of VPA on E12.5(VPA group),or administered by an equal amount of normal saline(CON group).2.Detection indicators(1)Western blot was used to detect the PGRN,PSD95 and SYN protein expressions in the frontal cortex,temporal cortex,hippocampus andcerebellum at postnatal(PN)7,14,35 and 72 d.(2)The PGRN,PSD95 and SYN protein expressions in the frontal cortex,hippocampus and cerebellum at PN7,14,35 and 72 d were detected by immunofluorescence.(3)Neuron apoptosis was tested in the frontal cortex,hippocampus and cerebellum at PN7,14,35 and 72 d by TUNEL stainingResults:1.PGRN expression of VPA group was upregulated in the frontal cortex at PN14 and 35 d and in the temporal cortex at PN7,14 and 35 d,and downregulated in the hippocampus at PN35 and 72 d and in the cerebellum at PN14 and 35 d.2.Compared with the CON group,neuron apoptosis index of VPA group was no significant difference in the frontal cortex at PN7,14 and 35 d while it was enhanced in the frontal cortex at PN72 d,in the hippocampus at PN35 and 72 d and in the cerebellum at PN14 and 35 d.3.PSD95 and SYN expressions of VPA group in the frontal cortex were significantly higher at PN14 and 35 d than those in the CON group,but significantly lower at PN72 d.In the temporal cortex of VPA group,PSD95 expression was higher than that of CON group at PN7,14 and 35 d and SYN expession was increased at PN35 and 72 d.In the hippocampus of VPA group,PSD95 and SYN expressions were lower than those of CON group at PN14,35 and 72 d.In the cerebellum of VPA group,PSD95 expression was downregulated at PN14 and 35 d while it was upregulated at PN72 d.SYN expession was decreased at PN14 and 35 d in the the cerebellum of VPA group.Conclusion:In the VPA-induced ASD rats,the abnormal expression of PGRN in critical brain regions at some critical periods and of the central nervous system development may disturb the normal spatio-temporal process of neuron apoptosis and synaptic development,which is likely to be associated with ASD-like neurological dysfunction induced by VPA exposure in pregnancy.