| ObjectiveTo construct a model of SD rat retinal hole induced by Nd.YAG laser,and to explore the significance of the expression of SOX2 and PRPH2 protein in the reconstruction of retinal structure and function during the healing process of the hole.Methods56 male SD rats were randomly divided into 7 groups with 8 rats in each group,and 1blank control group and 6 experimental groups were set.In the control group,the eyes were removed without any treatment,while in the experimental group,the retina hole model with diameter of 100μm was made by fundus Nd.YAG laser.The rats in six experimental groups were killed at 1 day,2 days,3 days,5 days,7 days and 10 days after operation respectively,and both eyes were removed.The healing of retinal holes was dynamically observed by SD-OCT scanning.Immunohistochemical staining was used to detect SOX2 protein and PRPH2 protein in retinal tissue.Western Blot and RT-PCR were used to detect the expression level of SOX2 protein and m RNA in retinal tissue,respectively.One-way ANOVA was used to compare the difference of SOX2 protein and m RNA expression among different groups of rats.Results1.SD-OCT scan results: Because of vitreous hemorrhage in the first 2 days after laser operation,OCT results are meaningless.On the 3rd day after Nd.YAG laser treatment,there is a liquid dark area at the retinal hole and a " bridge-shaped connection " is formed.On the5 th day after treatment,the retinal cleavage is filled by the "high reflection" agglomerate,and proliferative membrane appears in front of the retina.On the 7th day,the retinal cleft hole "high reflection " gradually disappears,and the structure of each layer is basically formed.On the 10 th day,the structure of each layer in the retinal hole is clear,and the retinal cleft holes are healing,and no obvious preretinal proliferative membrane is found.2.H&E staining results:In the Control group,under the light microscope,it can be seen that the interlayer structure of normal retinal tissue in SD rats from the inside to the outside is the inner limiting membrane,the nerve fiber layer,the ganglion cell layer,the inner lamella,the inner nuclear layer,the outer lamella,the outer nuclear layer,and photoreceptor cell layer,pigment epithelial layer,each layer has clear tissue structure and orderly arrangement on the 2nd day after Nd.YAG laser treatment.on the 2nd day after Nd.YAG laser treatment,under the light microscope,it can be seen that the structure of each layer of the retinal injury area is broken,the boundaries are not clear,inflammatory cells,red blood cells and necrotic tissues are present,and no new blood vessels.On the 5th day after treatment,under the light microscope,a large number of proliferating cells can be seen filling the retinal injury area,the proliferative cells are arranged disorderly,no polarity,no obvious necrotic tissue.On the 7th day,under the light microscope,it can be seen that the various layers of the retinal injury area are gradually formed.On the10 th day,under the light microscope,it can be seen that the structure of each layer of the retina tends to be clear and looser than normal tissue.3.SOX2 protein immunohistochemical staining: In the Control group,SOX2 protein is a weak positive expression of the retina as a geocyte.In the experimental groups,on the 2nd day after Nd.YAG laser treatment,SOX2 protein is strongly expressed in the cytoplasm of the cell mass in the injured retina.On the 5th day after treatment,SOX2 protein can be seen in the retina injury area that the cells with strong positive expression in cytoplasm are in the migration state.On the 7th day,SOX2 protein was weakly expressed in the loose area on the surface and strongly expressed in the cytoplasm in the deep.There are more cells migrated to the photoreceptor layer.On the10 th day,the expression of SOX2 protein is moderately positive in the cytoplasm of the retina,and the morphology of the retina tended to be normal.PRPH2 protein immunohistochemical staining: In the Control group,PRPH2 protein is positive in the photosensitive cell OS layer.In the experimental groups,on the 2nd day after Nd.YAG laser treatment,PRPH2 is negatively expressed in the injury area.On the 5th day,the PRPH2 protein is scattered and a small amount of positive expression is visible in the proliferative mass,and a small cluster of positive expression could be seen in the base portion.On the 7th day,PRPH2 protein is highly expressed.On the 10 th day after laser treatment,PRPH2 protein is irregularly expressed in the photoreceptor cell layer of the retina.4.WB results showed that the relative expression of SOX2 protein in the experimental group is higher than that in the Control group(p<0.05),especially on the second day after operation(p <0.01).5.PCR results showed that the relative expression of sox2 m RNA in the experimental group was higher than that in the Control group on the 1st,2nd,3rd and 5th day after surgery(p< 0.05),especially on the 2nd day after operation(p< 0.01).There was no significant difference between the experimental group on the 7th day after surgery and the Control group(p> 0.05).Conclusion1.The overexpression of SOX2 protein in the retina of SD rats after retinal injury proved that M(?)ller cells dedifferentiation into retinal progenitor cells may play a role in the structural remodeling of the injured retina.2.In the late stage of wound healing,the high expression of PRPH2 protein in OS layer proved that photoreceptor cells were formed. |
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