Denbeigh Janet M, Nixon Brian A, Puri Mira C, Foster F Stuart
Department of Medical Biophysics, University of Toronto; Sunnybrook Research Institute;
Department of Medical Biophysics, University of Toronto; Sunnybrook Research Institute.
J Vis Exp. 2015 Mar 4(97):52520. doi: 10.3791/52520.
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 αvβ3) 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.
超声造影成像可以传达有关组织血管生成和灌注的重要定量信息，并且在靶向应用中，有助于在分子水平上检测和测量血管生物标志物。在小鼠胚胎中，这种非侵入性技术可用于揭示小鼠早期循环系统和心血管疾病遗传模型中血管发育的基本机制。小鼠胚胎也是研究微泡与血管生成靶点（包括血管内皮生长因子受体2（VEGFR2）或αvβ3）的粘附以及评估分子超声定量特性的理想模型。因此，我们开发了一种将超声造影剂引入活的离体胚胎脉管系统的方法。这在注射控制和定位方面具有自由度，成像平面具有无阻碍和无运动的可重复性，并且简化了图像分析和定量。从子宫中分离出妊娠晚期（胚胎日（E）16.6和E17.5）的小鼠胚胎，将其从卵黄囊中轻轻取出，然后将微泡造影剂注入胎盘盘绒毛膜表面可触及的静脉中。然后采用非线性超声造影成像收集一些基本灌注参数（峰值增强、灌注速率和达峰时间），并在一个内皮糖蛋白小鼠模型中对靶向微泡结合进行定量。我们展示了微泡在活胚胎内的成功循环以及该方法在表征胚胎脉管系统和微泡行为方面的实用性。
