Study On The Role Of SAP97 In Light-induced Outer Retinal Edema

The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family are thought to play key roles in synapse assembly and synaptic plasticity. This study is to determine whether scaffolding protein family member——SAP97 is involved in the Miiller cell response to blue light injury.Part One:Establishment of rat photic-injury model and identification of outer retinal edemaAdult Sprague-Dawley (SD) rats weighing 190-210g were housed in a 12/12hour light/dark cycle. After 24 hours of dark adaptation,48 rats were randomly divided into four groups and three of them were placed separately in cages, allowing both eyes to be exposed to evenly-distributed bright blue light. The floor of each cage was illuminated by approximately 2500 lux. The temperature inside each cage was maintained at 24℃. All rats were placed under these conditions at the same time of day and returned to their normal light/dark cycle after a 24 hour intense blue light exposure. At Id,2d and 3d after exposure to light, transmission electron microscopy was used to observe the changes of retinal histology and apoptosis of photoreceptors. The results showed that nuclei of photoreceptors were normal in control retina, and their inner and outer segments were aligned uniformly with some space between one another. Tight junctions were found between normal retinal pigment epithelium (RPE) cells as well. Apoptosis of photoreceptors, mitochondrial swelling in inner segments of photoreceptors were found after blue light exposure. The outer segments of photoreceptors were markedly swollen, and their discs were no longer aligned, but rather, were disorganized at day 3. Tight junctions were not found between RPE cells at day 3 and their basement membrane was not intact as well. Hence the conclusions are blue light-injured rat retina exhibite intracellular edema in the outer retina as well as photoreceptor degeneration. And RPE cells are damaged in light-injured rat retina which would cause leakage in the outer retina.Part Two:Miiller cell response in blue light-injured rat retinaAfter successfully established the rat photic-injury model, we further explore the Muller cell response under such pathological condition. Cryosections and single isolated Muller cells were immunostained with SAP97, aquaporin-4 (AQP4) and inwardly rectifying potassium channel Kir4.1 antibodies to detect the immunolocalization changes by confocal microscopy. Western blot and quantitative real-time PCR (qRT-PCR) were applied to evaluate the retinal SAP97, AQP4 and Kir4.1 protein and mRNA levels respectively.The results showed that SAP97 was upregulated and concentrated in the dendrites of Muller cells after photic injury. The immunostaining of the AQP4 and Kir4.1 proteins was increased in the outer retina after light treatment which was similar with those changes of SAP97. Compared to control rat retina, retinal SAP97 mRNA and AQP4 mRNA were both upregulated at the first day after light exposure. Whereas the mRNA levels of Kir4.1 was increased at the third day following light exposure. SAP97 protein level was increased at the second day following light exposure compared to control group (P<0.05). AQP4 protein level was increased at the third day following light exposure compared to control group (P<0.05), whereas the alteration of Kir4.1 protein level had no statistical significance. Upregulation of SAP97 coincides with the redistribution of AQP4 and Kir4.1, suggesting that SAP97 plays a major role in the recruitment of such channels in light-induced outer retinal edema.