Weyers Jill J, Carlson Dara D, Murry Charles E, Schwartz Stephen M, Mahoney William M
Department of Pathology, Center for Cardiovascular Biology, and Institute for Stem Cell and Regenerative Medicine, University of Washington, USA.
J Vis Exp. 2012 Feb 10(60):e3740. doi: 10.3791/3740.
Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample. One method which circumvents these issues is micro-Computed Tomography (μCT). μCT imaging can scan at resolutions <10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry). However, imaging vessels by ex vivo μCT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by μCT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for μCT imaging. Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch), or ex vivo via a Langendorff perfusion system. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network--arteries, capillaries, and veins alike.
血管可视化对于理解多种不同疾病状态正变得越来越重要。虽然存在多种用于血管成像的技术，但很少有技术能够在扩展到包括较小血管的分辨率的同时，将血管网络作为一个整体进行可视化。此外，许多血管铸型技术会破坏周围组织，从而无法对样本进行进一步分析。一种能够规避这些问题的方法是微型计算机断层扫描（μCT）。μCT成像可以在小于10微米的分辨率下进行扫描，能够生成血管网络的三维重建图像，并且使组织保持完整以便后续分析（例如组织学和形态测量学）。然而，通过离体μCT方法对血管进行成像需要血管中充满不透射线的化合物。因此，μCT成像所产生的血管系统的准确呈现取决于血管的可靠且完全填充。在本方案中，我们描述了一种用于填充小鼠冠状动脉以准备进行μCT成像的技术。存在两种主要的填充冠状动脉血管系统的技术：通过插管在体内对主动脉（或主动脉弓分支）进行逆行灌注，或者通过Langendorff灌注系统在离体状态下进行灌注。在此，我们描述一种专门设计用于确保所有血管都被填充的体内主动脉插管方法。我们使用一种名为Microfil的低粘度不透射线化合物，它可以灌注通过最小的血管以填充所有毛细血管以及血管网络的动脉和静脉侧。使用加压灌注系统用缓冲液灌注血管，然后用Microfil填充。为确保Microfil填充较小的高阻力血管，我们结扎从主动脉发出的大分支，这会将Microfil引流到冠状动脉中。一旦填充完成，为防止心脏组织的弹性将Microfil挤出一些血管，我们在填充后立即结扎可触及的主要血管出口点。因此，我们的技术针对填充剂的完全填充和最大保留进行了优化，能够实现完整冠状动脉血管网络（动脉、毛细血管和静脉）的可视化。
