The Study On The Role Of Activated Retinal Microglia In Light-induced Photoreceptor Degeneration

The outer retinal degenerative diseases such as age-related macular degeneration, and some forms of retinitis pigmentosa are most-common irreversible blinding ocular diseases. Their common pathological change is the irreversible apoptosis of photoreceptors. Much effort have been devoted to understanding the pathological cellular changes and the theropy method and understanding the process of photoreceptor apoptosis might provide evidence for how to interfere with photoreceptor loss and therefore loss of vision in these diseases. Recently, an increasing number of studies have focused on the role of retinal microglia. Some results indicate that damaged photoreceptors resulted in microglial activation and subsequent further degeneration of remaining photoreceptors.Although the involvement of microglia is certain, the relationship between photoreceptor damage and microglial activation remains poorly understood. The cross talk between photoreceptor and microglia appear to be a potential novel therapeutic target to regulate inflammation and photoreceptor survival in light-induced retinal degeneration. Recent evidence indicates that the ligand-receptor pair Fractalkine/CX3CR1might play an important role in mediating neural/microglial interaction.In this work, we try to investigate the cross-talk between injured photoreceptors and activated retinal microglia, focusing on the role of Fractalkine and its receptor CX3CR1in light-induced photoreceptor degenerative disease using both in vivo and in vitro models. We evaluate the spatial and temporal relationship among photoreceptor apoptosis, Fractalkine/CX3CR1impairment, and microglial activation/migration. Furthermore, we explore the downstream intracellular signal transduction pathways. This study aims to find a novel therapeutic target to regulate inflammation and photoreceptor survival in light-induced retinal degeneration. Part I:The expression of Fractalkine/CX3CR1and the relationship with microglia and neurons in light-induced photoreceptor degenerationPurpose:To investigate the expression pattern of Fractalkine/CX3CR1in light-induced photoreceptor degenerative using in vivo rat model and evaluate the spatial and temporal relationship among photoreceptor apoptosis, Fractalkine/CX3CR1impairment, and microglial activation/migration.Methods:In vivo, Sprague-Dawley rats were exposed to intense blue light for24hours.2h,6h,ld,3d,5d,7d were chosen as time points. HE staining was used to evaluate the photoreceptor number and outer nuclear layer (ONL) thickness and antibodies were used to label Fractalkine/CX3CR1and retinal microglia at different time points after exposure to light. TUNEL method was used to detect retinal neuron apoptosis. Expression of Fractalkine/CX3CR1and inflammatory cytokines (TNF-α, IL-1(3) was detected by real-time PCR, Western immunoblot analysis. Furthermore, the MAPK intracellular signal transduction pathways was detected. Taken together the examination results, the spatial and temporal relationship among photoreceptor apoptosis, Fractalkine/CX3CR1impairment, and microglial activation/migration was analyzed.Results:SD rats showed progressive photoreceptor cell loss and a decrease in the ONL thickness, with disorganization of the inner and outer segments of the photoreceptors. Compared with the weak level of expression observed in the normal retina, Fractalkine expression, located mainly at the photoreceptor segments and the ONL where was also the direction of microglial migration, was significantly up-regulated after light damage. The double-labeled immunofluorescence showed that CX3CR1-positive cells were exclusively microglia. The protein and mRNA expressions of Fractalkine and CX3CR1began to increase at two hours after exposure to light and peaked at one day and three days respectively, then decreased. TUNEL assay showed that positive cells were seen in the ONL as early as two hours after light exposure and peaked at one day, and then the number of TUNEL-positive cells gradually decreased due to photoreceptor loss. Compared with the low levels in the control retinas, the expression of IL-1β, TNF-α, p-p38, p-p44/22and p-SAPK/JNK was up-regulated in a time-dependent manner.Conclusions:These findings demonstrate that Fractalkine/CX3CR1interaction may play an important role in the photoreceptor-microglia cross-talk in light-induced photoreceptor degeneration. The MAPK family may be involved in the intracellular signal transduction pathways.Part II:Role of Fractalkine/CX3CR1Interaction in Light-Induced Photoreceptor Degeneration through Regulating Retinal Microglial Activation and MigrationPurpose:The present study was performed to investigate the cross-talk between injured photoreceptors and activated retinal microglia, focusing on the role of Fractalkine and its receptor CX3CR1in light-induced photoreceptor degeneration.Methods:In vitro, the co-culture of primary retinal microglia and a photoreceptor cell line (661W cells) was exposed to blue light for five hours.6h,12h,24h,36h,48h were chosen as time points. Some cultures were pretreated by the addition of anti-CX3CR1neutralizing antibody or recombinant full-length Fractalkine or soluble Fractalkine before light exposure. Elisa assay was used to detect the levels of soluble Fractalkine, TNF-α, IL-1β and IL-10in the supernatants. TUNEL method was used to detect photoreceptor apoptosis. Chemotaxis assay was also performed to evaluate the impact of soluble Fractalkine on microglial migration.Results:The appearance of661W cells changed from flat with small intercellular spaces to spindled with large intercellular spaces after exposure to blue light for5hours, and70%to80%of them succumbed to apoptosis. After exposure to light, the microglia in the co-cultures became rounder and took on a characteristic amoeboid shape as they were activated. ELISA assay detected the upregulated secretion of soluble Fractalkine, IL-1β and TNF-α in the661W/microglia co-culture supernatant. Compared with untreated co-cultures, the group pretreated with neutralizing CX3CR1antibody showed significantly more photoreceptors preserved and the group pretreated with recombinant full-length Fractalkine showed less photoreceptor apoptosis. Also the addition of soluble Fractalkine prompted the migration of microglia.Conclusions:In conclusion, changes in the expression of Fractalkine/CX3CR1in response to intense blue light injury that we have documented strongly suggest that this ligand-receptor signaling pathway may play a role in the process of retinal microglial activation and migration in light-induced photoreceptor degeneration. Our results indicate that blockage of soluble Fractalkine represents a decreased inflammatory response, whereas overexpression of full-length Fractalkine suggests a role in photoreceptor survival. In this regard, we will further explore potential downstream intracellular signal transduction pathways in the future work.