| Part one Establishment of the autistic animal modelObjectiveTo establish an animal model of autism induced by prenatal exposure to valproic acid.Methods:1.Reproductive age SD(Sprague-Dawley)male and female rats were caged for mating and randomly divided into two groups.SD rats were given a single intraperitoneal injection of 600 mg / kg VPA(concentration 250 mg / ml)or equal volume of physiological saline respectively at the 12.5th pregnancy day,and then the behavior test of 3-chamber social preference test,Open-field test and self-grooming test were performed.2.Groups:The male offspring of the SD rat that was received intraperitoneal injection of VPA or physiological saline were divided into VPA group and control group respectively.3.Detection indicator:3.1 Three chamber sociability test: Three chamber sociability test was used to assess social behavior of rats in two groups3.2 Open-field test: Open-field test was used to assess spontaneous locomotors activity and emotion of rats in two groups3.3 Self-grooming test: Self-grooming test was used to assess stereotyped behavior of rats in two groups Results:1.Compared with control group,the VPA rats have no significantly preference for strange rats and spend less time to interact with strange rats,and spend more time to interact with empty chamber in three chamber sociability test(p<0.05).2.Compared with Control group,the number of vertical movement and the exposure time of central area of VPA group are significantly decreased in open-field test(p<0.05).3.Compared with Control group,the cumulative self-gromming time of VPA group is markedly increased in self-grooming test(p<0.05).ConclusionThe animal model of autism was successfully established in the offspring of SD rat that was received intraperitoneal injection of sodium valproate at the 12.5th pregnancy day.Part Two Observation of the Early Development Characteristicsin VPA rats ObjectiveTo reveal the early development characteristics of physiological and central nervous system in VPA rats.Methods:1.Groups: Control group and VPA group(animal model of autism)2.Detection indicator:2.1 The early physiological development:The weight,body length,tail length and the appearance day of tooth eruption,eyes open,auricle separation,abdominal hair growth,body fluff growth,body white hair growth,ears open and testicular descent in two groups was observed.2.2 The early central nervous system development:2.2.1 Nervous reflex function were revealed by the appearance day of pivoting,surface righting reflex,negative geotaxis reflex,cliff aversion and air righting reflex.2.2.2 Motor coordination and muscular tension were observed and compared by the appearance day of crawl and front limb suspension,the retention time of front limb suspension,the score of swimming test,the distance of feet unfolded.2.2.3 Sensory reflex were observed and compared by appearance day of auditory startle,visual placing reflex,tail pinch reflex and the time of home cage emergence.2.3 Autistic-like behaviors:Three chamber social interaction test,open field test,self grooming test and Morris water maze were used to detect the autistic-like behaviors.Result:1.The early physiological development :There are no significant differences in weight and body length between two groups at PN28d(p>0.05).The appearance day of tooth eruption,eyes open,auricle separation,abdominal hair growth,body fluff growth,body white hair growth,ears open and testicular descent are same in both VPA group and Control group(p>0.05).But the tail of VPA rats is significant deformity and shorter than Control rats(p<0.01).2.The central nervous system development2.1 Nervous reflex function test shows that the positive criteria of surface righting reflex,Negative geotaxis reflex,cliff aversion and air righting reflex in VPA rats is latter than that in Control group(p<0.05),but the appearance day of pivoting is same in both VPA rats and Control rats(p>0.05).It shows that the development of nervous reflex in VPA rats was behind with the Control rats.2.2 Motor coordination and muscular tension test shows that VPA rats can persist less time in front limb suspension test than Control rats and show wider feet unfolded distance on PN15 d and PN30d(p<0.01),the appearance day of crawl is later than Control rats(p<0.01),at the same time,on PN8 d and PN10 d VPA rats get a lower score in swimming test than Control rats(p<0.01).Although there are no differences of swimming score and suspension time at PN6 d in VPA rats and Control rats(p>0.05),but the results still show that the muscular tension of VPA rats is decreased and the coordination ability is weak.2.3 Sensory reflex test shows that VPA rats represent more sensitive of auditory but dull vision compared with Control group(p<0.05).There are no differences in home cage emergence test and tail pinch reflex(p>0.05).It shows that VPA rats more sensitive of auditory but dull of vision,but no differences in smell and algesia.3.Autistic-like behaviors3.1 In the three chamber social interaction test,VPA rats spend less time with stranger1 and stranger zone 1 compared with Control rats(p<0.05),it prefer to stay with object or stay in center chamber(p<0.05),when object was replaced by stranger 2(p<0.05),VPA rats spend less time with stranger2 and stranger zone 2 compared with Control group(p<0.05),it prefer to stay in center chamber or stay with familiar(stranger1)(p<0.05).It shows that VPA rats have a significant social disabilities and novelty preference disfunction.3.2 In the OPT test,the VPA rats show less times of cross center grid and straight upward movements than Control rats.The results show that VPA rats have less independent exploratory behavior in the new environment,have weaken cognitive ability and adaptability than Control rats.There are no differences of defecation and urination in VPA and control group,it shows that all rats has no anxiety syndrome.3.3 In self-grooming test,VPA rats spend more time to groom than Control group,it shows that VPA rats have repetition behavior.3.4 In Morris water maze test,results of place navigation test shows that VPA rats have longer escape latency(p<0.05)and slower swimming speed(p<0.05).In the spatial probe test,VPA group shows less passing time,time ration in target quadrant and lower swimming speed than Control group.It shows that the spatial learning and memory ability in VPA rats is weaken than that in Control rats.Conclusion:The central nervous system early development in VPA rats is delay,and they show significant autistic-like behaviors.But there is no significant different in early physiological development in two groups?Part Three Exploration of GABAergic neurons expression in different brain regions in VPA rats ObjectiveTo investigate whether the nervous system dysplasia and autistic-like behaviors of VPA rats were related to the abnormal expression of GABAergic neurons in different brain regions at the different stages of development.Methods:1.Groups: Control group and VPA group(animal model of autism)2.Detection indicator:2.1 Western blot was used to detected the expression of GAD67 in prefrontal cortex(PFC),temporal cortex(TC),somatosensory cortex(SC),hippocampus(HC)and cerebellum cortex(CC)on PN7d?PN14d?PN28d?PN35d?PN45d and PN72 d.2.2 IHC was used to detected the expression of GAD67 in prefrontal cortex(PFC),temporal cortex(TC),somatosensory cortex(SC),hippocampus(HC)and cerebellum cortex(CC)on PN7d?PN35dand PN72 d.Result:1.The results of Western blot show that on PN7-72 d in VPA group,the expression of GAD67 in HC and CC is lower than that in Control group(p<0.05).On PN7-35 d,the expression of GAD67 in PFC,in both Control group and VPA group is same(p>0.05),but on PN45 d and PN72 d,in VPA group the expression of GAD67 in PFC is higher than that in Control group(p<0.05).On PN7-45 d the expression of GAD67 in TC in both Control group and VPA group is same(p>0.05),but on PN72 d,in VPA group the expression of GAD67 in TC is lower than that in Control group(p<0.05).The expression of GDA67 in SC is same compared with Control group on PN7-72 d.2.The results of ICH shows that at PN7 d and PN35 d,the expression of GAD67 in PFC,TC and SC in both Control group and VPA group is same(p>0.05).But in VPA group,the expression of GAD67 in HC and CC is lower than that in control group(p<0.05).At PN72 d,in VPA group,the expression of GAD67 in PFC in higher than that in control group but the expression of GAD67 in TC,HC,CC is lower than that in control group(p<0.05),at the same time,the expression of GDA67 in SC is same compared with control group(p>0.05).Conclusion:The abnormally expression of GABAergic neuronal of VPA rats in brain regions may be one of the important factors for the core symptoms of autism. |
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