A Two-Stage Laser-Induced Mouse Model of Subretinal Fibrosis Secondary to Choroidal Neovascularization

Purpose: To develop a model that can recapitulate key features of macular fibrosis in neovascular age-related macular degeneration (nAMD). Methods: Adult C57BL/6J mice received three laser burns/eye to induce choroidal neovascularization (CNV). Seven days later, a second laser burn was directed to each of the neovascular lesions. Traditional laser-induced CNV was used as a control. Lesions were monitored at 10, 20, 30, and 40 days post-laser (p.l) treatment by fundus imaging, fundus fluorescein angiography, optical coherence tomography (OCT), and immunohistochemistry. The expression of collagen-1 (COL-1), fibronectin, alpha-smooth muscle actin, F4/80, complement factor B (CFB), Complement component 3 (C3), transforming growth factor-beta (TGF-beta), and fibroblast growth factor 2 (FGF2) in retina and retinal pigment epithelium/choroid was examined by immunofluorescence and reverse transcription polymerase chain reaction. Results: The two-stage laser protocol induced significantly larger lesions than the traditional laser-CNV by OCT and immunohistochemistry at all time points. Confocal microscopy detected COL-1(+) fibers and IBA1(+)/CD31(+) blood vessels in lesions from the two-stage laser protocol 30 to 40 days p.l. Lesions from traditional laser-CNV contain only COL-1(+) fibers but not blood vessels at this time point. Higher levels of proinflammatory (inducible nitric oxide synthase (iNOS), C3, CFB) and profibrotic (TGF-beta, FGF2, and vascular endothelial growth factor) genes were detected in the retinas from the two-stage laser-induced lesions compared with the traditional laser-CNV lesion. Higher number of infiltrating F4/80 macrophages was also observed in and around the two-stage laser-induced fibrotic lesion. Conclusions: The two-stage laser treatment induced subretinal fibrovascular membranes that persist over 40 days. Translational Relevance: The model is a useful tool to study the mechanism of macular fibrosis in nAMD and test antifibrotic drugs.