VALIDATION OF LASER-INDUCED CHOROIDAL NEOVASCULARIZATION (CNV) MOUSE MODEL: PARTICIPATION OF α₂M/LRP1 SYSTEM

Abstract

Purpose : Age-related macular degeneration (AMD) is the primary cause of legal blindness in the developed world. In neovascular (NV) AMD (also called wet AMD), abnormal blood vessels grow underneath the retina. In the present study, we validate an established procedure for induction of choroidal neovascularization (CNV) in mice. This procedure consists in the perforation of Bruch’s membrane by laser-induced photocoagulation, mimicking wet form of AMD. In this model, we characterized the neovascular process, its impact on the retinal functionality and the inflammatory profile. Due to, we previously demonstrated that α₂M and its receptor, LRP1, participate during retinal NV, we also focalized in the participation of α₂M/LRP1 system during CNV.

Methods : C57BL/6 mice (3–6 months) were previously anesthetized and treated with four spots of argon green laser photocoagulation per eye. After 7 days of laser burn, we analyzed the NV on choroid–RPE flatmounts by isolectin IB4 staining using confocal microscopy. At the same time, using specific cell markers we characterize cells in the lesion area: CD105 (ECs), NG2 (pericytes), F4/80 (microglia), as wells as LRP1 cell distribution. In addition, protein levels of α₂M and LRP1 were analyzed by WB and the inflammatory profile (IL1β, IL6, and TNFα) using qPCR assay. Finally, the retinal functionality was assessed by scotopic ERG.

Results : First, we had able to standardize the size of the lesion from isolectin stained choroidal flatmounts. In addition, a significant number of ECs and pericytes was observed around the lesion whereas microglia was localized in central and peripheral area. α₂M and LRP1 proteins showed a high expression pattern in cells close to the lesion site, accompanied by high level of pro-inflammatory and pro-angiogenic factors. The scotopic ERG a- and b-wave were decreased (p<0.05) in the eyes of this animals respect control and the latencies were not modified.

Conclusions : This study demonstrate the validation the CNV mouse model in our laboratory with the potential to analyze pathogenic mechanisms not yet described, as well as possible therapeutic strategies in the future.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.