Study On The Role Of Activation And Inhibition Of Microglia In Light-induced Photoreceptor Degeneration

Part One:The role of retinal microglia in rat photic-injury modelSprague-Dawley rats accepted dark-adaptation for 24 h prior to blue light exposure of 24 h at 2.5 Klux.At 3d,7d and 14d after exposure to light,HE staining, TUNEL staining and transmission electron microscopy were used to observe the changes of retinal histology and apoptosis of photoreceptors.Dark-adapted flash ERG was used to evaluate the retinal funciton.The results showed that the inner and outer segment of photoreceptors were destroied,the outer nuclear layer(ONL) was disarranged and thinned with 60.09%reduction at 14d after exposure to light.There were a large amount of TUNEL(+) apoptotic cells in the ONL at 1d which was confirmed by transmission electron microscopy.Both a- and b-wave amplitude were significantly reduced 14d after light exposure.After successfully established the rat photic-injury model,we further explore the migration,activation of retinal microglia and its relationship with photoreceptor apoptosis in our model.At 2h,6h,1d,3d,7d,14d after light exposure,OX42 immunostaining was used to label the migration and activation of microglia,ED1 antibody was used to label possible blood-borne macrophages.The number of TUNEL(+) and OX42(+) cells in the outer retina were counted and a Time-Num curve were established.Electron micrographic image was used to observe the phagocytization of microglia.Real-time PCR and Western-blot were used to evaluate the retinal IL-1βmRNA and protein level.The results showed that TUNEL(+) cells were noted in the ONL as early as 2h, and their presence were noticeably increased to reach the peak at 1d but declined at 3d. In contrast,OX42(+) cells assumed a more ameboid configuration were seen in the ONL at 6h and their presence increased significantly at 1d and 3d,so as the retinal IL-1βmRNA and protein expression.Electron micrography showed that microglia migrating into the ONL phagocytized the ROS disc of photoreceptors.The possibility of invasion of circulating macrophages cannot be excluded because a few ED1(+) cells were observed in the superior region at 7d.Hence the conclusions are that activation and migration of retinal microglia,as well as expression of microglia-derived toxic factor(IL-1β),coincides with photoreceptor apopotosis and migrated microglia phagocytized the ROS disc of photoreceptors,suggesting activated microglia play a major role in the further degeneration ofphotoreceptors after exposure to intense light.Part Two:The effect of naloxone stereoisomers on LPS-activated retinal microgliaPrimary cultured and purified retinal microglia were activated by LPS after pretreatment with diverse concentrations of(-)-naloxone and its isomer(+)-naloxone. OX42 immunostaining was used to observe the effect of naloxone on LPS-induced morphological changes of microglia;ELISA assay was used to evaluate the effect of naloxone on LPS-stimulated release of IL-1β;MTT assay was used to observe the proliferation of microglia.The results showed that following treatment with LPS,the microglia became activated with a greatly enlarged cell body and the characteristic shapes of activation; naloxone significantly inhibited the LPS-induced morphological change as demonstrated by the return of the OX-42(+) cells to the morphology of untreated cells. In addition to preventing the activation of microglia,naloxone significantly inhibited the production and release of IL-1βand this inhibition was dose-and time-related as illustrated by the Dose-Effect curve with the 1μM,12h showed the best effect. Naloxone and its isomer were equally effective in inhibiting the LPS-induced activation of microglia.MTT results showed that 0.25μM～1.25μM naloxone had no influence on the proliferation of microglia treated by≤100ng/ml LPS.Hence the conclusions are naloxone can functionally inhibit LPS-induced activation of retinal microglia and the release of IL-1β.Both naloxone stereoisomers were equally effective indicating that the possible mechanisim of action may be unrelated to the opioid system.Part Three:Naloxone has neuroprotective effects against light-induced photoreceptor degeneration through inhibiting retinal microglial activationSD rats were exposed to intense blue light for 24h.Daily intraperitoneal injection of naloxone or PBS as control was given 2 days before exposure to light and continued for 2 weeks.Apoptotic cells were detected by the TUNEL assay,and anti-OX42 antibody was used to label retinal microglia.Western-blot was applied to evaluate the retinal IL-1βprotein level.Retinal histology and ERG were also performed to evaluate the effects of naloxone on the light-induced photoreceptor degeneration.Similar to the morphological findings in the untreated control animals,the thickness of the ONL was markedly reduced in the PBS-treated group at 14d after photic injury.In contrast,treatment with 1 mg/day of naloxone provided significant protection of photoreceptors against photic injury in both the superior and inferior quadrants of the retina,especially in the posterior area.The extent of restoration of ONL thickness by naloxone was dose-related,with the maximum effect seen in the 1 mg/day group.Naloxone-treated group showed significantly less loss of a- and b-wave amplitudes when compared with the PBS-treated group.Compared to the control,the number of microglia in the outer retina was significantly decreased in the naloxone-treated group at 3d,so as the retinal IL-1βprotein level.TUNEL assay showed that treatment with naloxone(1mg/day) showed significantly fewer TUNEL-positive cells at 3 and 7 days,but not at 1d post light exposure.Hence the conclusions are that systemic infusion of naloxone significantly reduced the further photoreceptor cell apoptosis after initial direct light-induced damage possibly through inhibiting the activation of microglia.