Study On The Role Of Antigen Presenting Function Of Microglia In Light-induced Photoreceptor Degeneration

Part One:Establishment of the rat photic-injury modelNormal retinal sections of SD rats show discernible layers, including3nuclear layers-GCL, INL, and ONL. Damages caused by photopic injury are mainly located in the ONL and in the inner and outer segment of photoreceptor (IS/OS layer). One day after light exposure, the retinal section presented normal ONL but a swelling and disorganized IS/OS layer compared with normal retina; Three days after light exposure, thickness of the ONL was markedly reduced, indicating significant photoreceptor cell loss. Seven days after light exposure, loss of photoreceptor cell was more severe; On14days, only one to two rows of photoreceptors remained with overall disappearance of the IS/OS layer.The TUNEL assay was used to explore cell apoptosis within the retina. Positive staining was rarely seen in the normal rat; however, TUNEL-positive cells were seen in the ONL as early as the termination of light exposure and peaked at1day. TUNEL-positive cells were less abundant in the ONL at3days and dramatically decreased at7days.Part Two:Activation, migration and antigen presenting function alteration of retinal microglia in the rat photic-injury modelRetinal microglia were specifically stained by the OX42antibody. In normal retinas, a few OX42-positive microglia with thin cellular processes were distributed in the GCL and in the IPL. ONL or subretinal space showed no positive fluorescence. Rightly after photic injury, some OX42-positive microglia began to appear in the outer retina. These cells increased rapidly and reached the highest number at3days. Then they gradually became less in number and disappear at14days.In physical conditions, MHC-11(+) cell was seldom seen in retinal sections of SD rat. Three days after photic injury, there were some MHC-Ⅱ (+) cells in the IS/OS layer and in the subretinal area. Then they gradually became less in number and almost disappeared14days after photic injury. As one of the upstream molecules of the MHC-II, expression of CIITA was similar to that of MHC-II.Part Three:Vaccination with IRBP R16peptides protects the photoreceptors retina from photic injuryDamage of the ONL in the PBS-treated group was similar to that in the blank group: The retinal section presented normal ONL but a swelling and disorganized IS/OS layer one day after light exposure. Thickness of the ONL was markedly reduced3days after light exposure, indicating significant photoreceptor cell loss. Loss of photoreceptor cell was more severe7days after light exposure, and only one to two rows of photoreceptors remained at14days, with overall disappearance of the IS/OS. In contrast, vaccination with30μg IRBP R16peptide provided significant protection against photic injury. In the IRBP R16-treated group, morphological changes of ONL on14d were similar to that on3d, and the thickness of the ONL in the IRBP R16group significantly higher than that in the other two groups at Id,3d, and14d.On the other hand, apoptosis in the PBS-treated group was also similar to that in the blank group:TUNEL-positive cells were seen in the ONL as early as the termination of light exposure and peaked at1day. They became less abundant in the ONL at3days and dramatically decreased at7days. In contrast, vaccination with IRBP R16peptide showed significantly fewer TUNEL positive cells at3and7days but not at1day after light exposure, suggesting that photoreceptor cell apoptosis caused by light-induced damage was inhibited by vaccination with this peptide.Part Four: Vaccination with IRBP R16peptides upregulated the antigen presenting function of the retinal microgliaActivation of the microglia in the PBS-treated group was similar to that in the blank group:A few OX42-positive microglia with thin cellular processes were distributed in the GCL and in the IPL in normal conditions. Some OX42-positive microglia began to appear in the outer retina rightly after photic injury. They increased rapidly and reached the highest number at3days. Then number of these OX42(+) cells gradually declined and disappeared at14days. Activation of the microglia in the IRBP R16treated group was similar to that in the other two groups, indicating that vaccination with this peptide did not influence the activity of the microglia.Expressions of the MHC-II and its upstream signal—the CIITA were not different in between the PBS-treated group and the blank group:There were some MHC-II (+) cells appeared in the IS/OS layer and in the subretinal area3days after photic injury. Then they gradually became less in number and almost disappeared14days after photic injury. Expression of CIITA was similar to that of MHC-II in both groups. However, in the IRBP R16treated group, MHC-II (+) CIITA (+) cells appeared and peaked in number as early as1day after photic injury. Three days later, the expression was not reduced. Results of the Western Blot also supported that, compared with the other two groups, the IRBP R16treated group expressed the MHC-II and the CIITA earlier and stronger, indicating the upregulation of microglial antigen presenting function.