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

ABSTRACT: 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, ?-smooth muscle actin, F4/80, complement factor B (CFB), Complement component 3 (C3), transforming growth factor-? (TGF-?), 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-?, 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.

SUBMITTER: Little K

PROVIDER: S-EPMC7396176 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Little Karis K Llorián-Salvador María M Tang Miao M Du Xuan X O'Shaughnessy Órlaith Ó McIlwaine Gemma G Chen Mei M Xu Heping H

Translational vision science & technology 20200309 4

<h4>Purpose</h4>To develop a model that can recapitulate key features of macular fibrosis in neovascular age-related macular degeneration (nAMD).<h4>Methods</h4>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 ...[more]

PMID: 32818091

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Project description:BackgroundVision loss in patients with wet/exudative age-related macular degeneration (AMD) is associated with choroidal neovascularization (CNV), and AMD is the leading cause of irreversible vision impairment in older adults. Interleukin-17A (IL-17A) is a component of the microenvironment associated with some autoimmune diseases. Previous studies have indicated that wet AMD patients have elevated serum IL-17A levels. However, the effect of IL-17A on AMD progression needs to be better understood. We aimed to investigate the role of IL-17A in a laser-induced CNV mouse model.MethodsWe established a laser-induced CNV mouse model in wild-type (WT) and IL-17A-deficient mice and then evaluated the disease severity of these mice by using fluorescence angiography. We performed enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS) to analyze the levels of IL-17A and to investigate the immune cell populations in the eyes of WT and IL-17A-deficient mice. We used ARPE-19 cells to clarify the effect of IL-17A under oxidative stress.ResultsIn the laser-induced CNV model, the CNV lesions were larger in IL-17A-deficient mice than in WT mice. The numbers of γδ T cells, CD3+CD4+RORγt+ T cells, Treg cells, and neutrophils were decreased and the number of macrophages was increased in the eyes of IL-17A-deficient mice compared with WT mice. In WT mice, IL-17A-producing γδ T-cell numbers increased in a time-dependent manner from day 7 to 28 after laser injury. IL-6 levels increased and IL-10, IL-24, IL-17F, and GM-CSF levels decreased in the eyes of IL-17A-deficient mice after laser injury. In vitro, IL-17A inhibited apoptosis and induced the expression of the antioxidant protein HO-1 in ARPE-19 cells under oxidative stress conditions. IL-17A facilitated the repair of oxidative stress-induced barrier dysfunction in ARPE-19 cells.ConclusionsOur findings provide new insight into the protective effect of IL-17A in a laser-induced CNV model and reveal a novel regulatory role of IL-17A-producing γδ T cells in the ocular microenvironment in wet AMD.

Project description:Choroidal neovascularization (CNV) is a major cause of severe visual loss in patients with age-related macular degeneration (AMD). Recently, itraconazole has shown potent and dose-dependent inhibition of tumor-associated angiogenesis. We evaluated the anti-angiogenic effect of itraconazole in a rat model of laser-induced CNV. After laser photocoagulation in each eye to cause CNV, right eyes were administered intravitreal injections of itraconazole; left eyes received balanced salt solution (BSS) as controls. On day 14 after laser induction, fluorescein angiography (FA) was used to assess abnormal vascular leakage. Flattened retinal pigment epithelium (RPE)-choroid tissue complex was stained with Alexa Fluor 594-conjugated isolectin B4 to measure the CNV area and volume. Vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression was determined 1, 4, 7, and 14 days after intravitreal injection by quantitative RT-PCR or Western blot. VEGF levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Intravitreal itraconazole significantly reduced leakage from CNV as assessed by FA and CNV area and volume on flat mounts compared with intravitreal BSS (p = 0.002 for CNV leakage, p<0.001 for CNV area and volume). Quantitative RT-PCR showed significantly lower expression of VEGFR2 mRNA in the RPE-choroid complexes of itraconazole-injected eyes than those of BSS-injected eyes on days 7 and 14 (p = 0.003 and p = 0.006). Western blots indicated that VEGFR2 was downregulated after itraconazole treatment. ELISA showed a significant difference in VEGF level between itraconazole-injected and BSS-injected eyes on days 7 and 14 (p = 0.04 and p = 0.001). Our study demonstrated that intravitreal itraconazole significantly inhibited the development of laser-induced CNV in rats. Itraconazole had anti-angiogenic activity along with the reduction of VEGFR2 and VEGF levels. Itraconazole may prove beneficial for treating CNV as an alternative or adjunct to other therapies.

Dataset Information

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

Publications

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

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