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. 2023 Jun 12;17:1192464. doi: 10.3389/fncel.2023.1192464

FIGURE 1.

FIGURE 1

Dual angiopoietin-2 (Ang-2)/vascular endothelial growth factor-A (VEGF-A) inhibition reduces spontaneous choroidal neovascularization (CNV) and lesion leakage area in JR5558 mice. (A) General experimental protocol used to assess short-term treatments effects on limiting vascular leakage, neovascularization, and inflammation in the JR5558 mouse model. (B) Examples of lesion leakage area and total area of isolectin B4 signal quantification. (C) Representative isolectin B4 immunostaining images detected on retinal pigment epithelium (RPE)/choroid flatmounts from untreated JR5558 mice. Isolectin B4 is a marker for endothelial cells allowing visualization and quantification of spontaneous CNV (white asterisk). Scale bar = 500, 100 (1), and 25 (2) μm. (D) Representative analysis by immunofluorescence staining of isolectin B4 signal on RPE/choroid flatmounts in JR5558 mice 1 week after treatment with immunoglobulin G (IgG) control or anti–VEGF-A, anti–Ang-2, or bi-specific anti–Ang-2/VEGF-A antibodies. Scale bar = 500 μm. (E) Total area of isolectin B4 signal on RPE/choroid whole flatmounts in JR5558 mice 1 week after treatment with IgG control or anti–VEGF-A, anti–Ang-2, or bi-specific anti–Ang-2/VEGF-A antibodies (n = 20–24 flatmounts). (F) Representative infrared (IR) and fluorescein angiography (FA) images in JR5558 mice following treatment at day (D) 15 for CNV lesion leakage detection and quantification. (G) Lesion leakage area quantification in JR5558 mice 1 week after treatment following two weekly doses of IgG control or anti–VEGF-A, anti–Ang-2, or bi-specific anti–Ang-2/VEGF-A antibodies (n = 10 eyes). (H) Retinal vascular leakage detected in the vitreous of JR5558 mice, and corrected to plasma fluorescein concentration, using fluorescein fluorophotometry 1 week after treatment following two weekly doses of IgG control or anti–VEGF-A, anti–Ang-2, or bi-specific anti–Ang-2/VEGF-A antibodies (n = 7–8 eyes). Values are mean ± SD. One-way analysis of variance followed by Tukey’s multiple comparison test. **P < 0.01, ***P < 0.001, ****P < 0.0001. IHC, immunohistochemistry.