Translational vision science & technology · 2020
A Two-Stage Laser-Induced Mouse Model of Subretinal Fibrosis Secondary to Choroidal Neovascularization.
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Little Karis, Llorián-Salvador María, Tang Miao, Du Xuan, O'Shaughnessy Órlaith, McIlwaine Gemma, Chen Mei, Xu Heping
Аннотация
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, α-smooth muscle actin, F4/80, complement factor B (CFB), Complement component 3 (C3), transforming growth factor-β (TGF-β), 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-β, 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.
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, α-smooth muscle actin, F4/80, complement factor B (CFB), Complement component 3 (C3), transforming growth factor-β (TGF-β), 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-β, 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.