Simultaneous in vivo imaging of blood and lymphatic vessel growth in Prox1–GFP/Flk1::myr–mCherry mice

The ability to visually observe angiogenesis and lymphangiogenesis simultaneously and repeatedly in living animals could greatly enhance our understanding of the interdependence of these processes. To generate a mouse model that affords such visualization via in vivo fluorescence imaging, we bred Prox-1-GFP mice with Flk1::myrmCherry mice to generate Prox1-GFP/Flk1::myr-mCherry mice, in which lymphatic vessels emit green fluorescence and blood vessels emit red fluorescence. Corneal neovascularization was induced in these mice using three injury models: vascular endothelial growth factor (VEGF) pellet implantation, basic fibroblast growth factor (bFGF) pellet implantation, and alkali burn injury. Vessel growth was observed in vivo by stereomicroscopy on days 0, 3, 7, and 10 after pellet implantation or alkali injury as well as in flat-mounted corneas via confocal microscopy after the final in vivo imaging time point. We observed blood and lymphatic vessel growth in all three models, with the most significant growth occurring from days 0–7. Upon VEGF stimulation, the growth kinetics of blood and lymphatic vessels were similar. Blood vessels exhibited similar growth patterns in VEGF- and bFGF-stimulated corneas. Alkali burn injury induced robust angiogenesis and lymphangiogenesis. In this study, the intrinsic fluorescence of blood and lymphatic endothelial cells in Prox1-GFP/Flk1::myr-mCherry mice permitted simultaneous in vivo imaging of angiogenesis and lymphangiogenesis. This imaging ability allowed us to differentiate the processes as well as observe their interdependence and will be valuable in the development of therapies targeting angiogenesis and/or lymphangiogenesis.