Processing of fluorescence angiograms for the quantification of vascular effects induced by anti-angiogenic agents in...

The chicken embryo chorioallantoic membrane (CAM) is widely used as an in vivo model to study the vascular effects of angiogenesis modulating agents. The main goal of the present study was to develop and validate a quantitative method to characterize time-dependent changes mainly in the capillary network of the CAM. To that end, the CAM capillaries were monitored in ovo, between days 7 and 13 of embryo development, using an epi-fluorescence microscope equipped with a sensitive camera following the intravenous injection of a fluorescent agent. We present a method by which the fluorescence angiograms of the CAM vasculature were recorded and analyzed by a multistep mathematical procedure to obtain a skeleton representation of the vessels and capillaries. From this skeleton descriptors were extracted, including the number of branching points/mm(2), the mean area of the vessel network meshes, and the mean of the 3rd quartile of the mesh area histogram. A qualitative visual assessment of the vasculature based on the number and the size of the avascular zones was obtained for comparison. To illustrate this approach, the activity of a neutralizing anti-vascular endothelial growth factor antibody, Avastin (Bevacizumab), was then quantified. Blood vessel growth inhibition and changes in the architecture of the capillary plexus after topical application of this drug on the CAM surface were monitored using the three descriptors mentioned above.