A modified laser-induced choroidal neovascularization animal model with intravitreal oxidized low-density lipoprotein

Abstract

AIM: To investigate whether intravitreal injection of oxidized low-density lipoprotein (OxLDL) can promote laser-induced choroidal neovascularization (CNV) formation in mice and the mechanism involved, thereby to develop a better animal model. METHODS: C57BL6/J mice were randomized into three groups. Immediately after CNV induction with 532 nm laser photocoagulation, 1.0 μL of OxLDL [100 μg/mL in phosphate-buffered saline (PBS)] was intravitreally injected, whereas PBS and the same volume low-density lipoprotein (LDL; 100 μg/mL in PBS) were injected into the vitreous as controls. Angiogenic and inflammatory cytokines were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) after 5d, and CNV severity was analyzed by choroid flat mount and immunofluorescence staining after 1wk. In vitro, retinal pigment epithelial (RPE) cell line (ARPE19) were treated with OxLDL (LDL as control) for 8h. Angiogenic and inflammatory cytokine levels were measured. A specific inhibitor of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) was used to evaluate the role of LOX1 in this process. RESULTS: At 7d after intravitreal injection of 1 μL (100 μg/mL) OxLDL, T15-labeled OxLDL was mainly deposited around the CNV area, and the F4/80-labeled macrophages, the CD31-labeled vascular endothelial cells number and CNV area were increased. Meanwhile, WB and qRT-PCR results showed that vascular endothelial growth factor (VEGF), CC chemokine receptor 2 (CCR2), interleukin-6 (IL-6), IL-1β, and matrix metalloproteinase 9 (MMP9) expressions were increased, which was supported by in vitro experiments in RPE cells. LOX1 inhibitors significantly reduced expressions of inflammatory factors IL-1β and VEGF. CONCLUSION: A modified laser-induced CNV animal model is established with intravitreal injection of 1 μL (100 μg/mL) of OxLDL at 7d, which at least partially through LOX1. This animal model can be used as a simple model for studying the role of OxLDL in age-related macular degeneration.

Keywords

• age-related macular degeneration
• choroidal neovascularization
• oxidized low-density lipoprotein
• animal model