Demonstration of Bartonella henselae-induced angiogenesis using chorioallantoic membrane assay on shell-less culture of the chick embryo as In Vivo model

Abstract

Objective: Angiogenesis induced by Bartonella henselae has been demonstrated in in vitro model systems such as HUVEC (Human umbilical vein endothelial cells). However, one of the in vivo model systems allowing angiogenesis studies in cancer and drug discovery studies is the chorioallantoic membrane (CAM) model. In this study, shell-less culture of the CAM has been utilized as the in vivo model for the demonstration of Bartonella henselae induced angiogenesis.

Material and Method: Eight shell-less CAM cultures were established 72 hours after first incubation of fertilized eggs at 37 degrees C and 5% CO2. One half of each CAM was used as the study group and in this half 3 B.henselae (ATCC 49882) soaked beads were planted in bifurcations of previously established vessels. The other half was used as a control, and sterile distilled water soaked beads were planted in bifurcations of previously established vessels. Beads were removed after 40 and 62 hours of incubation, and newly formed vessels were counted in a 4-mm(2) area under each bead.

Results: Neovascularization in the study group was significantly higher than that in control group (22.8 +/- 1.4 versus 8.3 +/- 0.7, respectively; p<0.001) at 40 hours of incubation. This significance was also observed at 62 hours of incubation (25.6 +/- 1.8 versus 9.4 +/- 0.9, respectively; p<0.001).

Conclusion: Bartonella henselae induced angiogenesis has been demonstrated in shell-less culture of CAM. This model has advantages for angiogenesis studies. These include: (a) cost efficiency (eggs, time, labor, etc.); (b) no mature immune system is available in these CAMs, thus the observed effect is attributed to the bacteria tested; (c) allowing multiple tests on individual CAMs; (d) easy reproduciblity; (e) tissue sampling and photography is easily performed. Thus, shell-less culture of the CAM may be utilized in studies testing angiogenic activity of many agents including other bacteria.