The microglia began to migrate towards the aortic ring on approximately day 4 of culturing. Figure 6A illustrates the position of microglia at day 5 and 12 for cultures containing 3,125, 25,000 and 100,000 microglial cells. The distances between the front of the migrating microglia and the aortic ring decreased by approximately 1mm from day 5 to day 12, yielding a migration rate corresponding to about 140 mm per day. Parallel experiments in which MEFs replaced the microglia showed a strikingly different pattern of cell migration. In contrast to the oriented migration exhibited by microglia, the MEFs spread radially in all directions from the site of injection, as did microglia in the absence of an aortic ring. When approaching the aortic ring, the MEFs changed direction and turned away from the vessels. This supports the notion that the induced migration of microglial cells towards the endothelium aortic ring explant is cell type-specific. These results indicated that microglial cells secrete a soluble factor into the aortic ring culture medium that stimulated vessel branching in the explants. The results also suggest that the aortic rings affect microglial cell migration in the collagen gel. To address if aortic rings also influenced the release of angiogenesis stimulatory factor from microglial cells, the effects of cell-free microglia Nutlin-3 Mdm2 inhibitor Conditioned and Wortmannin abmole control medium were compared with embedded microglia in the aortic ring model. Conditioned medium was obtained from microglial cell cultures incubated in parallel with the aortic ring cultures in the same standard medium and with a similar number of cells. When comparing branch numbers on day 5, large differences in vessel sprouting were observed between cultures with embedded microglial cells and cultures supplemented with microglial cell conditioned medium. Furthermore, a smaller but significant difference in vessel sprouting was observed when comparing microglial cell conditioned medium with control medium. These results suggest that microglial cells secrete a soluble factor with a positive angiogenic effect on the aortic ring explants and that the secretory activity of the microglial cells is stimulated by the presence of aortic ring explants in the cultures. In this study, we used the developing mouse retina and the aortic ring model to address the role of microglial cells in angiogenesis. The retina is an organ where too many or to few vessels are associated with pathology. The retina is also subject to pharmacological application of anti-VEGF therapy, which is used to counteract the edema that compromises vision in agedependent macula degeneration.