The Effect Of Vitamin A Deficiency On The Motor Coordination Function And Its Possible Mechanism In A Rat Model Of Autism

Objectives: In this study,we built rat models with different vitamin A(VA)levelsbased on the valproic acid(VPA)-treated autism model to investigate the effect of gestational VA deficiency(VAD)onthe number and dendritic morphology of Purkinje cells of the cerebellum and motor coordination functionin offspring rats,and to explore the possible molecular mechanisminvolved.Methods:Establishment of a valproic acid-treated rat autism model:eight-week-old SD female rats were selected and mated with normal male rats.On gestational day 12.5,pregnant rats were intraperitoneally injected with 600 mg/kg VPAoran equivalent amount of normal saline randomly.The male offspring rats were divided into VPA group and CON group.Establishment of autism model with VAD: 3-week-old female SD rats were randomly divided into VA normal(VAN)group and VAD group,and were fed a VAN or VAD diet for 4 weeks,respectively.The tail vein blood of the rats was collected and serum VA concentrations were measured by HPLC.The female rats with standard VA levels(VAN?1.05?mol/L;VAS+VPA group.The rats in VAD+VPA group were always fed a VAD diet.The rats in the VAS+VPA groupwere transformed intoa VAN diet after birth,and VA was given intragastric supplement for 1 week from postnatal day(PND)1.The rats in each groupwere subjected to the pole test on PND28,the wire suspension test at PND35,the open-field test on PND42,the three-chamber test on PND43,and the rotarod test on PND46.After the behavioral tests,the offspring rats were sacrificed at the age of 8 weeks,and the serum and cerebellum tissues were collected.The serum VA concentration of the rats was determined by HPLC.Quantitative polymerase chain reaction(q PCR)was used to quantitatively analyze ROR? m RNA expression level in cerebellar tissue.The protein expression levels of RAR? and ROR? in cerebellar tissue were detected by Western blotting(WB).The dendritic morphology of Purkinje cells in the cerebellum were detected by Golgi staining.The distribution and expression of ROR? and Calbindin in the cerebellum were detected by immunofluorescence and laser-scanning confocal microscopy.Chromatin immunoprecipitation(Ch IP)assay was used to analyze the interaction between RAR? and ROR? gene.Dual-luciferase reporter assaywas used to VAD<0.70?mol/L)were selected to mated with normal male rats.On gestational day 12.5,all pregnant rats were intraperitoneally injected with600 mg/kg VPA.The male offspring of the pregnant rats in the VAN group were used as the VAN+VPA group and were fed a VAN diet all the time.In addition,the male offspring of the pregnant rats in the VAD groupwere randomly divided into two groups,the VAD+VPA group and the group(P<0.05),while the time spent on exploring in the central zone was significantly decreased compared with that in the CON group(P<0.05).In the three-chamber test,the VPA group spent significantly less time in the strange rat chamber(P<0.05)and more time in the object chamber(P<0.01)than the CON group.There was no significant difference in time that spent in the central chamber between VPA group and CON group(P>0.05).In the pole test,the VPA rats took longer to descend the pole than the CON rats(P< 0.05).In the wire suspension test,the time of falling from the wire of the rats was shorter in the VPA group than that in the CON group(P< 0.01).In the rotarod test,the time on the rod was decreased in the VPA group compared to the CON group(P< 0.01).Theimmunofluorescence results illustrated that the number of Purkinje cells in the VPA group was markedly decreased compared to that in the CON group in adult rats in 5 lobules(?????????).The most severely affected lobules were ??????and ?(P< 0.001).Golgi-Cox staining showed that the area of Purkinje cell dendritic arborization in the VPA rats was reduced compared with that in the CON rats on PND56(P<0.05).The number of intersections in the VPA group was decreased some distance(100–130 ?m)away from the soma compared with the CON group(P< 0.05).Compared with the CON group,the m RNA and protein verify the transcriptional regulatory activity and binding region of RAR? to ROR?.Results:(1)In the open field test,the time spent on self-grooming in the VPA group was significantly increased compared with that in the CON immunoreactivity in Purkinje cells.The colocalization of ROR? and Calbindin in Purkinje cells was observed using laser-scanning confocal microscopy.There was a visible reduction in ROR? and Calbindin expression levels as well as their coexpression in the VPA group compared to the CON group.(2)Compared with the VAN+VPA group,the serum VA concentrations in the VAD+VPA group were significantly decreased(all Ps <0.01)on PND1,PND7,PND14,PND28,PND42 and PND56.On PND1,the serum VA concentration of rats in the VAS+VPA group was not significantly different from that in the VAD+VPA group,but the serum VA concentrations in the VAS+VPA group were significantly decreased compared with VAN+VPA group(P<0.01).The serum VA concentrations in VAS+VPA group were significantly higher than those in VAD+VPA group at all time points from PND7(P<0.01),and were not different from those in the VAN+VPA group(P> 0.05).In the open field test,the time spent on self-grooming in the VAD+VPA group was significantly increased compared with that in the VAN+VPA group(P<0.01),and the time spent on exploring in the central zone was significantly less than that in the VAN+VPA group(P<0.001).Compared with the VAD+VPA group,the VAS+VPA group spent less time in self-grooming(P<0.01),and more time in exploring in the central zone(P<0.001).There was no significant difference in the time of self-grooming in the open field test and the time of expression levels of ROR? in the cerebellum of the VPA group were significantly reduced(P<0.05).Immunofluorescence showed strong ROR?exploring in the central zone between the VAS+VPA group and the VAN+VPA group(P> 0.05).In the three-chamber test,the VAD+VPA group spent significantly less time in the strange rat chamber(P<0.01)and more time in the object chamber(P<0.01)than the VAN+VPA group.The time that spent in the central chamber of the VAD+VPA group was not significantly different from that of the VAN+VPA group(P>0.05).Compared with VAD+VPA group,the VAS+VPA group spent significantly more time in the strange rat chamber(P<0.01),and significantly less time in the object chamber(P<0.01).Compared with the VAN+VPA group,the VAS+VPA group had no significant difference in the time spent in each chamber(P>0.05).In the pole test,the time to reach the bottom of the pole was significantly longer in the VAD + VPA group than in the VAN + VPA group(P< 0.05).The VAS + VPA group spent less time in reaching the bottom of the pole than the VAD+VPA group.There was no significant difference in descending timebetween VAS+VPA group and VAN+VPA group(P>0.05).In the wire suspension test,rats fell more quickly from the wire in the VAD + VPA group than in the VAN + VPA group(P< 0.001).In the wire suspension test,rats fell more quickly from the wire in the VAD + VPA group than in the VAN + VPA group(P<0.001).Significant improvement was detected in the VAS + VPA rats compared to the VAD + VPA rats(P< 0.001),and there was no significant difference between the VAN + VPA and VAS + VPA groups(P> 0.05).In the rotarod test,the time on the rod was significantly shorter in the VAD +VPA group than in the VAN + VPA group(P< 0.05).Thetime on the rod was significantly longer in the VAS + VPA group than in the VAD + VPA group(P< 0.05).Compared with the VAN+VPA group,the VAS+VPA group had no significant difference in the time spent on the rod(P>0.05).Theimmunofluorescence results illustrateda significantly fewer number of Purkinje cells in the VAD + VPA group than in the VAN +VPA group in some lobules,including lobules I(P< 0.01),?(P< 0.001)??(P< 0.05)??(P< 0.05)and ?(P< 0.01).In these lobules,the number of Purkinje cells in the VAS+VPA group was significantly increased compared with that in the VAD+VPA group.There was no significant difference in the number of Purkinje cells between the VAN + VPA and VAS + VPA groups(P> 0.05).Golgi-Cox staining showed that the total area of Purkinje cell dendritic arborization was remarkably reduced in the VAD + VPA group compared with the VAN + VPA group(P<0.05).Compared with the VAD + VPA group,a significant increase in the total area of Purkinje cell dendritic arborization was found in the VAS + VPA group(P< 0.05).Compared with VAN+VPA group,there was no significant difference in the total area of Purkinje cell dendritic arborization in VAS+VPA group(P>0.05).The number of intersections between dendrites and concentric circles of Purkinje neurons in VAD+VPA group was significantly less than that in VAN+VPA group and VAS+VPA group at the middle-to-distal segment(70–110 ?m from the soma)of the dendritic tree(P< 0.05).The protein levels of RAR? and ROR? in cerebella from the VAD+VPA group were significantly decreased compared withthe VAN+VPA group and the VAS+VPA group(P<0.05).Ch IP results illustrated that the binding of RAR? to the predicted binding-site of ROR?gene promoter region in cerebellawas enhanced.The binding was significantly decreased in VAD +VPA group compared with VAN+VPA group and VAS+VPA group(P< 0.05).Dual-luciferase reporter gene assay showed that RAR? has transcriptional activity on ROR?gene.Conclusions:(1)The VPA-treated autism rat model showed motor coordination abnormalities,Purkinje cell loss and impaired dendritic arborization of Purkinje cells.(2)Gestational VAD exacerbates motor incoordination and cerebellum impairment in autism model rats.VAS in the early postnatal period can effectively improve the motor incoordination and cerebellum impairment in an autism rat model with VAD.Increased motor dysfunction in ASD rats with VAD may be induced by lower expression of RAR? and RAR? regulation of the expression level of ROR? in the cerebellum.