Figure 2. Intravitreal injection of CL1-R2 reduces retinal neovascularization in a murine model of oxygen-induced retinopathy. Retinal neovascularization was induced by exposing 7-d-old mice to 75 ± 2% oxygen for 5 d, and then to normoxia. Animals were injected intravitreally with control IgG1 or CL1-R2 or received no injection. (A) Flat-mounted retinas harvested from 17-d-old pups were perfused with FITC-dextran and observed by fluorescence microscopy to assess retinal vasculature. Representative flat-mounted retinas are from normal mice (Normal retina), mice exposed to hyperoxic conditions without injection (Mock), or mice injected intravitreally with 5 µg IgG1 (Ctrl) or 5 µg CL1-R2. Central avascular zone, arrowheads; neovascular tufts, asterisks. Data are representative of three independent experiments (n = 6–11 mice/group). Bars: (top) 770 µm; (bottom) 335 µm. (B) Histological analyses (periodic acid–Schiff staining) of whole eye tissue sections. Representative photomicrographs of eye sections from normal mice (Normal retina; n = 3) and mice exposed to hyperoxic conditions without injection (Mock; n = 6) or injected intravitreally with IgG1 (Ctrl; n = 9) or CL1-R2 (n = 11). Longitudinal and transverse aberrant microvessels, arrows; isolated nuclei of endothelial cells not involved in tube formation, asterisks. GCL, ganglion cell layer; INL, inner nuclear layer. Bar, 50 µm. (C) Von Willebrand Factor staining of whole eye tissue sections. Representative photomicrographs of eye sections from normal mice (Normal retina) and mice exposed to hyperoxic conditions without injection (Mock) or injected intravitreally with IgG1 (Ctrl) or CL1-R2 mAb. After paraffin removal, sections were rehydrated, treated with 0.3% Triton X-100, and incubated with polyclonal antibody against Von Willebrand factor, followed by Alexa Fluor 488 secondary antibody (green) and counterstaining with DAPI (blue). GCL, ganglion cell layer; INL, inner nuclear layer. Bar, 25 µm. (D and E). Quantitative assessment of the retinal vascularization illustrated in B for 6 (Mock), 9 (Ctrl), or 11 (CL1-R2) mice/group. Endothelial cell nuclei and vessel lumens were counted on four to eight sections per eye stained with periodic acid–Schiff reagent. The mean number of endothelial cell (EC) nuclei (D) and vessel lumen (E) presented in the histograms were determined using a Poisson regression model for clustered data. 95% confidence intervals of the mean number estimates are figured as error bars. P-values were corrected for post-hoc group comparisons by the Bonferroni method. **, P < 0.001.
Figure 2.

Intravitreal injection of CL1-R2 reduces retinal neovascularization in a murine model of oxygen-induced retinopathy. Retinal neovascularization was induced by exposing 7-d-old mice to 75 ± 2% oxygen for 5 d, and then to normoxia. Animals were injected intravitreally with control IgG1 or CL1-R2 or received no injection. (A) Flat-mounted retinas harvested from 17-d-old pups were perfused with FITC-dextran and observed by fluorescence microscopy to assess retinal vasculature. Representative flat-mounted retinas are from normal mice (Normal retina), mice exposed to hyperoxic conditions without injection (Mock), or mice injected intravitreally with 5 µg IgG1 (Ctrl) or 5 µg CL1-R2. Central avascular zone, arrowheads; neovascular tufts, asterisks. Data are representative of three independent experiments (n = 6–11 mice/group). Bars: (top) 770 µm; (bottom) 335 µm. (B) Histological analyses (periodic acid–Schiff staining) of whole eye tissue sections. Representative photomicrographs of eye sections from normal mice (Normal retina; n = 3) and mice exposed to hyperoxic conditions without injection (Mock; n = 6) or injected intravitreally with IgG1 (Ctrl; n = 9) or CL1-R2 (n = 11). Longitudinal and transverse aberrant microvessels, arrows; isolated nuclei of endothelial cells not involved in tube formation, asterisks. GCL, ganglion cell layer; INL, inner nuclear layer. Bar, 50 µm. (C) Von Willebrand Factor staining of whole eye tissue sections. Representative photomicrographs of eye sections from normal mice (Normal retina) and mice exposed to hyperoxic conditions without injection (Mock) or injected intravitreally with IgG1 (Ctrl) or CL1-R2 mAb. After paraffin removal, sections were rehydrated, treated with 0.3% Triton X-100, and incubated with polyclonal antibody against Von Willebrand factor, followed by Alexa Fluor 488 secondary antibody (green) and counterstaining with DAPI (blue). GCL, ganglion cell layer; INL, inner nuclear layer. Bar, 25 µm. (D and E). Quantitative assessment of the retinal vascularization illustrated in B for 6 (Mock), 9 (Ctrl), or 11 (CL1-R2) mice/group. Endothelial cell nuclei and vessel lumens were counted on four to eight sections per eye stained with periodic acid–Schiff reagent. The mean number of endothelial cell (EC) nuclei (D) and vessel lumen (E) presented in the histograms were determined using a Poisson regression model for clustered data. 95% confidence intervals of the mean number estimates are figured as error bars. P-values were corrected for post-hoc group comparisons by the Bonferroni method. **, P < 0.001.

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