Отработка подходов для изучения ангиогенеза in ovo и ex ovo на хориоаллантоисной оболочке яиц японского перепела ( Coturnix coturnix japonica

В результате исследований были отработаны методические подходы для изучения ангиогенеза в моделях in ovo и ex ovo с использованием хориоаллантоисной оболочки яйца перепела.

The development of in ovo and ex ovo models for studing angiogenesis on chorioallantoic membrane of Japanese quail''s eggs ( Coturnix coturnix japonica)

Angiogenesis - is a fundamental biological process providing the development and growth of blood vessels due to the proliferation and migration of endothelial cells to form a new or reconstract existing capillary network. The study of the phenomena and molecular and cellular mechanisms of angiogenesis in the experiment is of current interest. The assessment of angiogenic activity using chicken egg chorioallantois membrane of (HET-CAM test) is the most routine, because of its simplicity, accessibility and high reproducibility. However, when using this model, there are difficulties that can be avoided by utilizing of the Japanese quail eggs. The aim of this work was to develop an in ovo and ex ovo techniques for studying angiogenic activity using CAM of Japanese quail eggs. Eggs on the six day of incubation were used for in ovo techniques. The testing samples were inoculated into the eggs under sterile conditions through the holes made with dental drill at the blunt end of the egg. After the procedure, holes in eggs’ shell were sealed. The eggs were returned to the incubator for 24 hours, then dissected from a blunt end. The CAM was separated by tweezer and mounted on a glass slide for further morphometric analysis. Eggs on 3-rd day of incubation were used for ex ovo tests. Eggs’ content was transferred to the Petri dishes 60mm under sterile conditions in a laminar flow hood. Then, the petri dish was placed in a CO2 incubator (20 % O2, 5 % CO2, 37 ± 0,5 °C, humidity - 100 % ) for 72 hours. 30 mkl of the test sample was applied on the CAM of 6-th day old embryo and then CAM was returned to the CO2 incubator for 24 hours. After the procedure CAM was separated by tweezer and mounted on object plate for further morphometric analysis. AngioQuant program was used for analysis. The program measured the amount of branches and the number of vessels, which depart from the branches. Total number of vessels of all orders was calculated by summing the above means. The comparative evaluation of the effect of cell culture medium a-MEM before and after adding 10 % fetal calf serum was carried in both experimental models. Degree of branching vessels, the number of branches and the number of vessels from which branch is extended and also the total number of vessels in the in ovo and ex ovo micropreparations were higher for a-MEM +10 % FCS CAM in comparison with control (FBS) and a-MEM CAM. The embryo and CAM condition after a-MEM adding did not differ from control samples. Therefore, it can be concluded that the revealed effects on the development of blood vessels of the CAM may be due to the components of the FCS. This research allowed to improve methodological approaches for studying angiogenesis in ovo and ex ovo using CAM of quail eggs and to develop, experimental protocol for testing biological samles, as well as methods of morphometric analysis of the results. This work was supported in part by RFBR Grant 13-04-00791 и 14-04-00933.