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. 2013 Sep;33(18):3689–3699. doi: 10.1128/MCB.00343-13

Fig 6.

Fig 6

miR-329 inhibits retinal neovascularization in an OIR mouse model. (A) Schematic representation for OIR induction and miR-329 injection. Modified miR-329, control miRNA (miR ctrl) (5 μg, respectively), or control solution (0.9% NaCl) was injected intravitreously in the OIR mice at postnatal day 12 (P12). Retinal samples were collected at P17 for RNA isolation, flat mount staining, and immunohistochemical analysis. (B) Real-time PCR analysis of the endogenous CD146 and miR-329 expression in the retinas of normal or OIR mice at P12 and P17. GAPDH or U6 was used as an internal control. CD146/GAPDH or miR-329/U6 was normalized to that of normal retinas at P12 (n = 8 to 14). (C) The expression of endothelial CD146 in the normal retinas and in OIR retinas treated with control solution, control miRNA, or miR-329 (5 μg) was measured at P17. The relative fluorescence density of CD146/CD31 was analyzed in the confocal images of panel E using Image Pro Plus software (n = 5). (D) Flat-mounted retinas under indicated treatments were perfused with FITC-dextran and observed by fluorescence microscopy to assess retinal vasculature (green) at P17. Images were processed, and the areas of vaso-obliteration (VO) (yellow) and neovascularization (NV) (red) were added by using Adobe PhotoShop 7.0 software. The ratios of VO or NV to total retinal area were quantified and are presented in the graphs below (n = 8). The bar represents 500 μm. (E and F) The vascularization of the retinas in normal or OIR mice treated as indicated was analyzed by immunofluorescence. Staining of blood vessels was performed with anti-CD146 antibody (red) and anti-CD31 antibody (green). Nuclei were stained with DAPI (blue). The numbers of vessels in the inner retinas (ganglion cell layer [GCL] and inner nuclear layer [INL]) (E), and the migrated vessels to the vitreous cavity (F) were quantified (CD31 positive) and are presented in the histograms (9 to 14 mice per group). Arrows show longitudinal and transverse aberrant microvessels. VC, vitreous cavity. The bar represents 50 μm. NS., not significant, *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent SD. Data are representative of at least three experiments.