Effect Of Form Deprivation Amblyopia On Retinal Cone Cells And Bipolar Cells In Tree Shrews

Objectives:Preliminary study on the conduction and transfection of recombinant adeno-associated virus with different promoter homotypes in the retina of normal tree shrews,to provide experimental evidence for the treatment of retinal diseases by gene vector transmission technology.Observation of the changes in the number and structure of cone cells and bipolar cells in the retina of form deprivation amblyopia tree shrews,and preliminary study on the plasticity of retina during the development of amblyopia,to provide theoretical basis for further understanding the pathogenesis of amblyopia.Methods:1.Tree Shrews:10 normal Tree Shrews at 50 days of birth.Two types of promoters with rAAV9 viruses,rAAV-mCAR-mCherry-WPREs-Pa and rAAV-Grm6-SV40-EYFP-WPRE-pA,were selected and injected into the subretinal space through the eye.The retina was taken 28 days after the virus was injected.Preparation of retina flattening and retinal frozen sections,Observation of transfection of retinal cone cells and bipolar cells by fluorescence microscope and confocal fluorescence microscope.2.A total of 36 cubs of Unopened eye tree shrews about 18 days old were selected,divided randomly into 6 groups,set up the form-deprived amblyopia model:Form deprivation amblyopia model groups:N1(suture right eye two month,n=6),N4(suture right eye one month,n=6);Form deprivation amblyopia recovery model groups:N2(after sutured right eye one month and open it,keep eyes open one month,n=6),N3(after sutured right eye one month and open it,suture left eye one month,n=6);Control groups:N5(normal binocular vision for two months,n=6),N6(normal binocular vision for one months,n=6).The retinal rAAV-mCAR-mCherry-WPREs-pA injection was performed and the retina samples were taken after the model was made.Observation of the number and structure of cone cells and bipolar cells by immunofluorescence staining.Results were analyzed through these softwares of IPP6.0 and ImageJ.Results:1.Expression of adeno-associated virus(rAAV)in the retina of normal tree shrews:After retinal injection,rAAV-mCAR-mCherry-WPREs-pA was successfully transfected and overexpressed in the retinal cones of tree shrews,and the expression of rAAV-Grm6-SV40-EYFP-WPRE-pA was low in the retinal bipolar cells of tree shrews.2.Changes of cone cells and bipolar cells:the number of cone cells of two groups of form-deprived amblyopia model group(N1,N4)was lower than that of control group(N5,N6)(P<0.01).With the prolongation of the time of the form-deprivation model group,the number of cone cells in group N1 was lower than that in group N4(p<0.01).In the two groups of form-deprived amblyopia recovery group,there was no obvious statistical difference in the number of cone cells in group N2 between group two and group N4(p>0.05).There was no statistically significant difference in the number of cone cells between N3 group and N4 group(p>0.05);The number of bipolar cells in the form deprivation amblyopia model group(N1,N4)was lower than that in the control(N5,N6)(P<0.01).With the prolongation of the time of the form-deprivation model group,the number of bipolar cells in group N1 was lower than that in group N4(p<0.01).In the two groups of form-deprived amblyopia recovery group,the number of bipolar cells in group N2 was significantly higher than that in group N4(p<0.05).The number of bipolar cells in group N3 was significantly higher than that in group N4(p<0.05).There was no statistically significant difference in the number of bipolar cells between N2 group and N3 group(p>0.05).The synaptic structure of retinal cones and bipolar cells did not change significantly.Conclusions:1.The rAAV9 virus vector containing different promoters,rAAV-mCAR-mCherry-WPREs-Pa and rAAV-Grm6-SV40-EYFP-WPRE-pA,was successfully used for the detection of retinal cone cells and bipolar cells in tree shrews.After subretinal injection of rAAV-mCAR-mCherry-WPREs-Pa,the transfection of most of the cone cells in tree shrews suggests that the cone cells can be transfected rAAV9 vector with mCAR promoter when gene therapy is involved in eye related diseases.2.In the process of form deprivation amblyopia,the number of cone cells and the number of bipolar cells in the retina decreased,and negatively correlated with the length of the covering time.3.In the process of form deprivation amblyopia,there is some plasticity in the bipolar cells of the retina.