Addressing the Three R principle in microvascular research – Measurements of Vascular development and proangiogenic factors with the HET cam assay

Considering animal experimentation, 3R (Replace, Reduce, Refine) strategies in microvascular research are urgently needed. The Henn"s Egg Chorioallantoic Membrane (CAM) assay offers immune deficiency and a highly perfused membrane where growth and vascularization of tumors can be observed. Yet, standards on vascular differentiation and levels of proangiogenic factors, especially concerning the influence of therapies like radiation, have yet to be established. After opening of the eggshell, the vascular network was daily documented using in vivo microscopy and quantified using the deep learning based IKOSA®​ CAM Assay Application. From day 6 till 14 of development CAM samples were analyzed via qPCR. To determine therapeutic effects on microvasculature, measurements from native CAM tissue were compared to tissue 6 h after irradiation with 8 Gy on day 10, 12, or 14, respectively by measuring the expression of 84 angiogenic factors with the RT² Profiler qPCR array from Qiagen®​. VEGF showed high expression from day 7-11 of embryonic development followed by a decrease towards day 14. The response to irradiation was characterized by a dynamic range of expression of proangiogenic factors. CDH5, KDR, TBX1, TEK, and TIE1 were significantly decreased by more than 1.5 in their fold change in comparison to the non-irradiated control group. The vascular analyses revealed a noticeable decrease in the total vessel area 6 h after irradiation. The CAM assay can be considered a legitimate alternative for rodent experimentation in microvascular research. Besides obvious ethical, pecuniary, and bureaucratic advantages, samples can be studied non-invasively in high numbers and in a reproducible setting.