TECHNIQUES Quantitative Volumetric Analysis of Cardiac Morphogenesis Assessed Through Micro-Computed Tomography

We present a method to generate quantitative embryonic cardiovascular volumes at extremely high resolution without tissue shrinkage using micro-computed tomography (Micro-CT). A CT dense polymer (Microfil, Flow Tech, Inc.) was used to perfuse avian embryonic hearts from Hamburger and Hamilton stage (HH) 15 through HH36, which solidified to create a cast within the luminal space. Hearts were then scanned at 10.5 m3 voxel resolution using a VivaCT scanner, digital slices were contoured for regions of interest, and computational analysis was conducted to quantify morphogenetic parameters. The three-dimensional morphology was compared with that of scanning electron microscopy (SEM) images and serial section reconstruction of similarly staged hearts. We report that Microfil-perfused hearts swelled to maximum end-diastolic volume with negligible shrinking after polymerization. Comparison to SEM revealed good agreement of cardiac chamber proportions and intracardiac tissue structures (i.e., valves and septa) at the stages of development assessed. Quantification of changes in chamber volume over development revealed several notable results that confirm earlier hypotheses. Heart chamber volumes grow over two orders of magnitude during the 1-week developmental period analyzed. The atrioventricular canal comprised a significant proportion of the early heart volume. While left atrium/left ventricular volume ratios approached 1 in later development, right atrium/right ventricle ratios increase to over 2.5. Quantification of trabeculation patterns confirmed that the right and left ventricles are similarly trabeculated before HH27, after which the right ventricle became quantitatively coarser than that of the left ventricle. These results demonstrate that Micro-CT can be used to image and quantify cardiovascular structures durin