Flt3 Regulation in the Mononuclear Phagocyte System Promotes Ocular Neovascularization

Figure 7

Immunofluorescence staining for Flt3 and F4/80 in normal, ischemic retina, rho/VEGF transgenic, CNV, and rho/rtTA-TRE/VEGF transgenic retinas. Retinal preparation was conducted for staining F4/80 macrophages and Flt3+ cells using the perfusion procedure described earlier. Eyes of OIR mouse model at P18, subretinal NV mouse model at P21, CNV and RD mouse models were enucleated and submitted to fixation and preparation for frozen sections. Immunofluorescence staining of Flt3 (red) showed high expression of Flt3 in OIR, subretinal NV, CNV, and RD model retinas (arrowheads), which colocalized with F4/80 staining. A nonischemic retinal sample from a P18 mouse displayed Flt3 expression areas, as revealed by Cy3-conjugated secondary antibodies (red), throughout the inner retina. F4/80 expression in the same section as in (A) with FITC-conjugated secondary antibodies displayed reduced areas. Ocular section from a P18 mouse after oxygen-induced ischemic retinopathy displayed areas (arrowheads) of increased expression in the whole inner retina colocalizing with F4/80-stained macrophages or closely localized around macrophages in and on the retina (B). Ocular specimens from P21 transgenic mice overexpressing VEGF in photoreceptors (rho/VEGF mice) showing Flt3 expression that colocalizes with F4/80 (C). An ocular section from a D14 CNV mouse model showing Flt3 staining, while F4/80 staining of the same section showed macrophage localization (D). Ocular sections from 5- to 8-week-old transgenic mice (rho/rtTA-TRE/VEGF) showing an area of Flt3 staining in the inner layer (red) and green F4/80 staining (E). Merging of Flt3 and F4/80 staining of all NV model retinas showed colocalization (yellow), indicating that macrophages may express Flt3.