The Role And Mechanism Of S100A4 On Ganglion Cells In A Mouse Model Of Retinal Ischemia-reperfusion Injury

Section one:Changes of retinal ganglion cells and calcium-binding protein S100A4 after retinal ischemia-reperfusion injury in miceObjective:To observe the density of retinal ganglion cells(RGCs)and the changes of S100A4 protein after ischemia-reperfusion(I/R)injury in mice.Determining whether the retinal I/R model was successful.Methods:27 adult C57BL/6J mice were randomly divided into the day 3 group,day 7 group and day 10 group after I/R.The right eyes were used as the I/R model and the left eyes were used as control.The I/R model establishment method is briefly described as follows:After C57 mice were intraperitoneally injected with sodium pentobarbital anesthesia and ocular surface anesthesia,the intraocular pressure of the mice was measured by a hand-held tonometer,and then dilated by the compound tropicamide.Under the operating microscope,the eyes of the mice were sterilized,and the right anterior chamber of the eye was inserted near the corneoscleral margin with a 30 G needle attached to a sterile saline bottle.Raise the bottle to a height of 150 cm for 60 minutes.The retina vessels were whitened by the ophthalmoscope.After the operation,tobramycin ophthalmic ointment was applied to the eyes to avoid infection.Only animals with intact cornea and lens were included in further studies.The mice were sacrificed at day 3,day 7 and day 10 after I/R,and retinas were collected for immunofluorescence staining of the whole retina.The RGCs were photographed by fluorescence microscope and the Image-J software was used to count the number of RGCs.Mouse retinal protein was collected 7 days after I/R,and the expression of S100A4 protein in mouse retina was detected by Western blot.The biomedical statistical analysis software GraphPad Prism 6.0 was used to analyze data.Results:The intraocular pressure of the mice before I/R was(8.3±1.5)mmHg,and it was(79.5±1.6)mmHg during I/R(P<0.001).The density of RGCs in the control group was(2403.9±384.5)/mm2,and the density of RGCs at day 3,day 7 and day 10 after I/R was(1132.7±113.8)/mm2,(636.9±135.7)/mm2 and(400.1±56.1)/mm2,respectively(F=68.83,P<0.0001).Western blot analysis showed that there was no significant difference in the expression of S100A4 protein in the retina of mice 7 days after I/R(P>0.05).Conclusion:We have successfully established retina I/R model in mice.The density of RGCs decreased after I/R injury,which was time-dependent.The subsequent experiments were carried out at day 7 after I/R.The expression level of ion-binding protein S100A4 did not change significantly at day 7 after retinal I/R injury.Section two:Construction of recombinant adenoassociated virus vector carrying S100A4 geneObjective:To design and construct a recombinant adeno-associated virus(rAAV)carrying the S100A4 gene.Methods:Construction of recombinant adeno-associated virus:using EF1α-DIOoChIEF-P2A-dTomato-WPRE-pA as a vector,digesting with Sali enzyme and ECoRI enzyme,followed by PCR amplification of the synthesized target gene EGFP-p2aS100A4 Carrier connection.Packaging and titer detection of virus:HEK 293 cells were cultured,plasmid DNA was extracted by plasmid extraction kit,and then transfected into plasmid to obtain recombinant adeno-associated virus,which was amplified and purified.Finally,the titer of the virus was determined by the endpoint dilution method for use.Results:The recombinant adeno-associated virus was constructed and confirmed by sequencing.The titer of rAAV-Eflα-s100a4-EGFP-WPRE titer was 3.34 x 1012 vg/ml,and the titer of rAAV-Eflα-EGFP-WPRE-Pa was 2.80 x 1012 vg/ml.Conclusion:A recombinant adeno-associated virus carrying the EGFP tag and capable of overexpressing the mouse S100A4 gene was successfully designed and constructed,which provided an effective tool to study the role and mechanism of S100A4 in retinal ischemia-reperfusion injury.Section three:Overexpressed S100A4 protect retinal ganglion cells in a mouse ischemia-reperfusion modelObjective:To overexpress S100A4 gene by intravitreal injection of rAAV-Eflas100a4-EGFP-WPR,and then to study the effect and possible mechanisms of S100A4 protein on retinal ganglion cells(RGCs)in a mouse ischemia-reperfusion(I/R)model.Methods:61 adult C57BL/6J mice were randomly divided into control group,I/R group(retinas were collected at day 7 after I/R),gene overexpression group(retinas were collected at 4 weeks after intravitreal injection of rAAV-Eflα-s100a4-EGFPWPRE),blank virus group(retinas were collected at 4 weeks after intravitreal injection of rAAV-Efla-EGFP-WPRE-Pa),gene overexpression+I/R group(retinas were collected at day 7 after I/R which was established at 4 weeks after intravitreal injection of rAAV-Eflα-s100a4-EGFP-WPRE)and blank virus vector+I/R group(retinas were collected at day 7 after I/R which was established at 4 weeks after intravitreal injection of rAAV-Efla-EGFP-WPRE-Pa).All mice were kept in the same environment.Control group:fast anterior chamber puncture with 30 G needle.4 weeks after injection of the virus into the vitreous cavity,the mice in the gene overexpression group and the blank virus group were taken and 10 eyes for each group.The retinal frozen sections were used to observe the efficiency of virus transfection.Mice in each group except the blank virus group were taken after I/R,and 6 eyes for each group were made into the whole retina.Immunofluorescence staining was performed to observe the changes in the density of retinal ganglion cells(RGCs).The retinal proteins of each group were taken,and the protein of S100A4 in the retina of control,gene overexpression group and blank virus group were detected by Western blot.Expression of Akt,P-Akt,Bcl-2 and Bax of the 5 groups were detected except the blank virus group.The data obtained was analyzed using biomedical statistical analysis software GraphPad Prism 6.0.Results:4 weeks after injection of rAAV-Efla s100a4-EGFP-WPRE in mice,retinal ganglion cell layer(GCL),inner plexiform layer(IPL)and inner nuclear layer(INL)showed strong green fluorescence and a small amount of weak fluorescence was observed in the outer plexiform layer(OPL)by confocal fluorescence microscopy,which indicated that rAAV-Efla-s100a4-EGFP-WPRE was successfully transfected into the retina.Western blot showed that the expression of S100A4 protein in the gene overexpressing group was significantly increased compared with the control group and the blank virus group(P<0.05),indicating that rAAV-Efla-s100a4-EGFP-WPRE overexpressed S100A4 protein on the retina successfully.Observation of retinal immunofluorescence staining revealed that the density of RGCs in the I/R group was significantly lower than that in the control group and the gene overexpression group at day 7 after I/R(P<0.0001).The density of RGCs in the gene overexpression+I/R model group was less than that in the control group,but was significant higher than that in I/R group(P<0.01)and the blank virus+I/R group(P<0.05).It is suggested that overexpression of S100A4 gene has protective effect on RGCs.Western blot showed that there was no significant change in the expression of Akt protein of the 5 groups(P>0.05).The expression of P-Akt and Bcl-2 in the retinas of the gene overexpression+I/R group were significantly higher than those in the I/R group and the blank virus+I/R group(P<0.05).The expression of Bax in the gene overexpression+I/R group was significantly lower than those in the I/R group and the blank virus+I/R group(P<0.05).The Bcl-2/Bax ratio of gene overexpression+I/R group was significantly higher than that in the I/R group and the blank virus+I/R group(P<0.01).Conclusion:rAAV-Efla-s100a4-EGFP-WPRE was efficiently and rapidly transfected into the inner layer of mice retinas and successfully increased the expression of S100A4 gene.Overexpression of S100A4 protein significantly increased the density of RGCs after I/R injury,which may be achieved by increasing the phosphorylation of Akt and the expression of Bcl-2 then inhibiting the expression of downstream Bax.