The Role Of The P2X7-NLRP3/Caspase-1 Pathway In Choroidal Neovascularization

[Purpose]To investigate the possible mechanism of microglia participating in CNV through P2X7-NLRP3/Caspase-1 pathway in the mouse model of choroidal neovascularization(CNV)induced by laser.[Method]90 C57BL/6J mice were randomly divided into normal control group and 4 groups of CNV experimental group induced by laser.The experimental group was taken and tested on the 1st,4th,7th and 14th days after the successful laser treatment.In each group,3 mice were randomly selected for FFA and OCT examination of fundus changes;3 mice(6 eyes)were selected for HE staining of the retina;4 mice(8 eyes)were selected for immunofluorescence staining of frozen sections to detect the expression of CD11b,P2X7,NF-κB,NLRP3,caspase-1,IL-1β,IL-18 in retina and choroids;4 mice(8 retinas)were selected for RT-qPCR and Western blot detecting the level of mRNA and protein expression.[Outcome]Compared with the normal control group,FFA showed high fluorescence and fluorescein leakage of choroidal neovascularization at the laser spot on the 1st,4th,7th and 14th day after the successful establishment of CNV model;OCT showed RPE/Bruch’s membrane rupture,spindle and(or)clump hyperreflexive signals in sub-retina,with or without subretinal fluid.HE staining of pathological sections showed that RPE/Bruch’s membrane was discontinuous,the structure of outer retina was disordered,RPE cells/blood cells were deposited in the subretinal cavity.The results of immunofluorescence staining,in the normal retina,Iba-1 and CDllb showed that microglia located in the ganglion cell layer,inner plexus layer and outer plexus layer exhibited branching shape with large nucleus.On the 1st,4th,7th and 14th day after the successful establishment of CNV mice model,a large number of CD11b positive cells in the subretina space could be seen in the laser spots,and they invaded into choroid and inner retina.In the subretina space,P2X7,NLRP3,caspase-1 antibody positive cells were co-labeled with some CDllb positive cells.Outcomes of Western Blot and qPCR showed that the difference among five groups was statistically significant:CD11b(p=0.000;p=0.000),P2X7(p=0.000;p=0.000),NF-κB(p=0.019;p=0.000),NLRP3(p=0.000;p=0.000),Caspase-1(p=0.004;p=0.002),IL-1β(p=0.000;p=0.010),IL-18(p=0.000;p=0.000)were significantly up-regulated.[Conclusion]In the model of CNV induced by laser,microglia were activated and migrated or gathered in the subretinal cavity;the activated microglia may induce and promote the formation and development of CNV through P2X7-NLRP3/Caspase-1/IL-1β/IL-18 inflammatory pathway.