Contrast imaging in mouse embryos using high-frequency ultrasound.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or αvß3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

VEGFR2-targeted molecular imaging in the mouse embryo: an alternative to the tumor model.

As a tumor surrogate, the mouse embryo presents as an excellent alternative for examining the binding of angiogenesis-targeting microbubbles and assessing the quantitative nature of molecular ultrasound. We establish the validity of this model by developing a robust method to study microbubble kinetic behavior and investigate the reproducibility of targeted binding in the murine embryo. Vascular endothelial growth factor receptor 2 (VEGFR2)-targeted (MBV), rat immunoglobulin G2 (IgG2) control antibody-targeted (MBC) and untargeted (MBU) microbubbles were introduced into vasculature of living mouse embryos. Non-linear contrast-specific and B-mode ultrasound imaging, performed at 21 MHz with a Vevo-2100 scanner, was used to collect basic perfusion parameters and contrast mean power ratios for all bubble types. We observed a twofold increase (p < 0.001) in contrast mean power ratios for MBV (4.14 ± 1.78) compared with those for MBC (1.95 ± 0.78) and MBU (1.79 ± 0.45). Targeted imaging of endogenous endothelial cell surface markers in mouse embryos is possible with labeled microbubbles. The mouse embryo thus presents as a versatile model for testing the performance of ultrasound molecular targeting, where further development of quantitative imaging techniques may enable rapid evaluations of biomarker expression in studies of vascular development, disease and angiogenesis.