Quantitative volumetric analysis of cardiac morphogenesis assessed through micro‐computed tomography

Abstract

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 m 3 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 during development. Developmental Dynamics 236:802-809, 2007.

Quantitative Three‐Dimensional Analysis of Embryonic Chick Morphogenesis Via Microcomputed Tomography

“…At Day 5, the right ventricular myocardium was significantly larger than the left (0.83 AE 0.10 vs. 0.65 AE 0.05 mm 3 , P < 0.05), but Day 9 and onwards the left side has accumulated significantly more myocardium (9.6 AE 0.34 vs. 7.1 AE 0.03 mm 3 by Day 12, P < 0.01). These results paralleled our previously published results on cardiac chamber volume growth in the embryo, indicating that myocardial volume increases directly with lumen volume regardless of chamber (Butcher et al, 2007b). The relative deceleration of atrial myocardial accumulation compared to ventricular may be partially explained by the maturation of ventricular conduction patterns and increased proportion of diastolic inflow through ventricular suction.…”

“…This growth pattern may therefore be a conserved requirement for normal developmental progression. Our previous data quantifying cardiac lumen volumes with Micro-CT also showed exponential growth between Day 4 and Day 10 (Butcher et al, 2007b). Goedbloed reported slower exponential growth of the rodent eye in comparison to our chick data, which is expected since the rodent eye is proportionally smaller in the rodent than chick during this developmental window.…”

“…Atrial lumen volumes, in contrast, grew much larger than their respective myocardium. Our data suggests that the right atrial myocardium thins while the left thickens, which makes sense since it will eventually pump greater hemodynamic loads (Butcher et al, 2007b). It is important to note that growth occurs via cell proliferation, hypertrophy, and matrix synthesis and remodeling.…”

“…With scan times as low as a few minutes (for 25 l resolution) and the ability to image multiple embryos simultaneously, MicroCT is a versatile imaging platform well suited for small animal imaging. We previously established its utility for quantifying avian cardiovascular development, showing comparable resolution with scanning electron microscopy and better rendering of tortuous geometry like outflow tract and myocardial trabeculation (Butcher et al, 2007b). Higher resolution scans of as low as 1 lm voxel size are possible, which could help resolve finer anatomical details, but at a cost of field of view and the inability to image live specimens.…”

“…We and others used an injectable CT dense casting agents to visualize cardiovascular lumen development in chick embryos (Butcher et al, 2007b;Hu et al, 2009;Wang et al, 2009). More recently, we demonstrated using osmium tetroxide (OT) as a diffusive CT contrast agent that high resolution detailed visualization and segmentation of rodent embryos to newborns was possible with Micro-CT; these previous studies included early stage mouse and rabbit embryos (Johnson et al, 2006;Carney et al, 2007;Martinez et al, 2009).…”