| Objective:To study the changes of P2X7 and NLRP3/Caspase-1 inflammasome and IL-1β during the formation of choroidal neovascularization(CNV)induced by laser in C57BL/6J mice.To clarify the role of microglia cells mediated by P2X7-NLRP3/Caspase-1 inflammasome pathway in the occurrence of CNV,and provide scientific basis for selecting wet AMD prevention and treatment mode.Methods:40C57BL/6J mices were randomly divided into the normal control group and the CNV laser model group.We induced CNV model using the krypton red laser irradiation retina(laser parameters:wavelength 647nm,laser energy 200mW,spot diameter 80um,irradiation time 100ms),which each eye has 5 points around the optic disc at the distance between the 1 optic disc diameters.The mices in each group were collected in fourth days after laser irradiation,and the formation of CNV was observed by fundus fluorescein angiography(FFA),optical coherence tomography(OCT)and hematoxylin eosin staining(HE).The expression of P2X7 and NLRP3/Caspase-1 inflammasome in the frozen section of retina was detected by immunofluorescence.Western Blot detects the expression of CD11b、P2X7、NLRP3/Caspase-1 inflammasome and IL-1β.And quantitative real-time RCR was used to detect the expression of mRNA about CD11b、P2X7、NLRP3/Caspase-1 inflammasome and IL-1β.Results:The FFA examination showed that the laser spots in the model group showed strong fluorescence at early stage,and the late fluorescent leakage increased,indicating the formation of CNV,and there was no significant fluorescein leakage in the control group.HE staining observed that the local choroid membrane was damaged,and the cells in each layer of the outer layer of the retinal edema were arranged in disorder and the Bruch membrane and the RPE layer are destroyed;The pigment macrophages and migratory RPE cells are seen in the photocoagulation area,and the neovascularization is formed,and the retinal structure in the normal control group is arranged neatly.Immunofluorescence of retinal tissue showed that the enlargement of the cell body of microglia at the laser spot is amoeba;The positive cells marked by P2X7、NLRP3、Caspase-1、and IL-1β antibody were seen in the laser spot in the fourth days after laser damage.Compared with the normal control group,the mRNA content of CD11b、P2X7、NLRP3/Caspase-1 inflammasome and IL-1β in the CNV group was obviously increased by quantitative real-time PCR(P<0.05),and the protein content of CD11b、P2X7、NLRP3/Caspase-1 inflammasome and IL-1β in the CNV group was significantly increased by Western Blot(P<0.05).Conclusion:The mechanism of retinal microglia through the pathway of P2X7-NLRP3/Caspase-1 inflammasome in the process of CNV development is clarified. |
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