The Role Of The Microglia/NLRP3/IL-1? Pathway In Chronic Photodamage Of The Retina

Background and Aims:Age-related macular degeneration?AMD?is one of the main courses of vision loss or even blindness in elderly population in western developed countries.During the past decades,with the development of the national economy and improvement of people's life quality,which exacerbates the population aging,AMD has become the main course of serious vision impairment in China.Based on the differential retinal pathological features,AMD can be divided into dry-AMD which characterizes by fundus atrophy and wet-AMD which characterizes by choroidal neovascularization.Of note,death of retinal pigment epithelia?RPE?and photoreceptors in late stage of dry-AMD,and exudation and hemorrhage arising from choroidal neovascularization both cause serous vision impairment.Herein,it is the hot topic of great significance to deepen the investigation of mechanism behind the AMD pathology and to explore novel targets for future AMD intervention.However,the exact etiology of AMD still remains unclear.Typically,a series of genetic and environmental factors collectively activate the occurrence of this retinopathy,aging and oxidative stress exacerbate the progression,and chronic inflammation leads to an inevitable pathological change.Actually,those factors play their roles in a cross-talk instead of independent manner.Recently,chronic photo exposure has been reported to play an important role in AMD.Epidemiological studies have suggested an association between short-wavelength visible light exposure and AMD risk.In population with intraocular lens?IOL?or no lens after cataract surgery,the incidence of AMD increases because of the lack of filter of blue light.On the other hand,populations with dense opacity or IOL with filter of blue light,the incidence of AMD decreases.Growing evidence has also suggested inflammation and immune response may play important and interacting roles in AMD.As the main endogenous immune cells,microglia locates in inner retinal,monitoring and clearing the retinal metabolite products.Though there is a link between the microglia activation and chronic retinopathy,the exact role of microglia plays still uncertain.In the long-term blue light-induced retinopathy,whether microglia protects or exacerbates the retinopathy needs to be clarified.On the other hand,as another part of human immune system,inflammasome has drawn more and more attention in AMD pathology.NLR family pyrin domain containing 3?NLRP3?inflammasome is a multiprotein complex that recruits caspase-1 and mediates the production of cellular IL-1?.More recently,in AMD pathogenesis,NLRP3inflammasome was reported mainly activated in RPE cells.However,important questions remain,such as whether inflammasome activation is involved in microglia in retina under chronic light exposure,whether this activation leads to light-induced retinopathy,and can the retinopathy be alleviated through impairment of microglia recruitment.Therefore,this work,based on the previous theory and experimental findings,firstly explored the role of microglial activation in chronic blue light-induced retinopathy and whether the retinopathy is mediated by the NLRP3/caspase-1/IL-1?pathway.The findings of this work will explicate the role of microglial NLRP3 activation in chronic AMD pathogenesis and provide novel targets for future treatment of AMD.Methods:In the first party,we used a photo exposure device which had been designed with self-property right.The C57BL/6 mice were exposed to cool blue light-emitting diodes?LEDs,transmission peak wavelength:480 nm?,which were positioned aside the cages on a 12-hour light/dark cycle every day for up to 3 months.We applied the illuminance intensity of 500 lux as it is measured as the normal room light intensity.After blue light exposure?BLE?,we compared the electroretinogram?ERG?response after dark adaption between mice receiving BLE or not.H&E staining was used to quantify the changes of retinal anatomy.IBA-1immunofluorescence was performed to quantify the microglial activation.In the second part,we firstly compared the IBA-1 immunofluorescence among the groups of wild type?WT?mice,type?WT?mice+BLE,and CCR2^-/-+BLE.ERG,and H&E staining and transferase-mediated deoxyuridine triphosphate-biotin nick endlabeling?TUNEL?analysis were used to analyze the functional and anatomic changes in mice after blue light exposure.In the third part,we compared the location and semi-quantification of retinal IBA-1,NLRP3,and IL-1?in frozen retinal sections among WT Ctrl,WT+BLE,and CCR2^-/-+BLE groups.Further western blot was used to quantify the protein expression of NLRP3 and its downstream cytokines in retinal and RPE-choroidal complex.Results:In the first part,after low blue light exposure,the a-and b-wave in ERG of WT C57BL/6 mice significantly decreased.The average thickness of outer nuclear layer?ONL?became thinner after low blue light exposure and much more activated microglia were found in both inner and outer retina.In the second part,in CCR2^-/-mice after low blue light exposure,a damaged microglia recruitment was shown in retina and this could protect the visual function in electroretinogram?ERG?and alleviate the photoreceptor apoptosis,which thus helped attenuate the blue-light induced retinopathy.In the third part,we further found an increased co-location of NLRP3,Iba-1,and IL-1?in fluorescence and a concomitant increased protein expression of NLRP3,caspase-1 and IL-1?in western blot in chronic blue light-induced retinopathy.Moreover,the activation of microglia and their cellular NLRP3 inflammasomes occurred as an earlier step before the structural and functional damage of the mice retinas,which collectively supported that microglial NLRP3 inflammasome might be the key to the chronic blue light-induced retinopathy.Conclusions:The three-month low-energy blue light can induce retinopathy in mice.The chronic blue light-induced retinopathy were associated with microglia activation.With CCR2^-/-mice,we demonstrated the impairment of microglia recruitment can alleviate the chronic blue light induced retinopathy.Further investigation indicated the microglia may cause the chronic blue light induced retinopathy by NLRP3/Caspase-1/IL-1?pathway.Our data suggested that future therapeutic interventions aiming at preventing the photoreceptor cells loss can be through the modulation of microglia and the cellular NLRP3 inflammasome activation.