Non-Destructive, High-Resolution 3-Dimensional Visualization of a Cardiac Defect in the Chick Embryo Resembling Complex Heart Defect in Humans Using Micro-Computed Tomography
Double Outlet Right Ventricle With Left Juxtaposition of Atrial Appendages
Nondestructive, high-resolution 3-dimensional (3D) imaging of the embryonic heart remains a challenge in cardiovascular development research. In the past, several imaging techniques (eg, magnetic resonance microscopy, optical coherence tomography) were tested for their suitability to visualize the 3D morphology of embryonic hearts. Most of these imaging tools have their drawbacks with respect to resolution and depth penetration. Here we present, to the best of our knowledge, the first high-resolution 3D images of normal and malformed embryonic chick hearts at the 10 μm level generated by microcomputed tomography (micro-CT) examination of critical point-dried heart specimens. Cardiac anatomy is demonstrated in great details with respect to myocardial fiber arrangement and trabeculations as well as atrioventricular (AV) and semilunar valves. Positions of great vessels with associated ventricular septal defects are visualized in high quality in malformed hearts.
Figure 1A shows the normal 4-chambered heart of a day 9 chick embryo with correct positioning of the heart chambers and great vessels. Figure 1B depicts an image of a malformed embryonic chick heart in which both great arteries are significantly shifted toward the right side with extreme dextroposition of the aorta, and both great arteries arise from the right ventricle (double outlet right ventricle [DORV]). Note also that both atrial appendages are located to the left of the great arteries (left juxtaposition of atrial appendages [LJAA]). The cardiac defect that we present here in the chick embryo resembles a rare complex heart defect known in humans, as depicted in a classic drawing by Frank Netter, MD, in the Netter Collection of Medical Illustrations – Heart (Figure 1C, reproduced with permission from netterimages.com).
DORV generally occurs as an isolated entity, but it is also seen in association with other heart defects, such as total anomalous pulmonary venous return, AV septal defect, mitral stenosis or atresia, juxtaposition of the right atrial appendage, or a persistent left superior caval vein. Juxtaposition of the atrial appendages itself is almost always associated with complex congenital heart disease.1,2 The condition in which both atrial appendages lie to the left side of the great vessels (LJAA) is more common than that in which the appendages lie to the right of the arteries (RJAA).1 Further, more complex and morbid anomalies are seen in those with LJAA than in those with RJAA.3 The developmental basis of LJAA remained unclear for a long time until an animal model for LJAA in the chick was demonstrated for the first time in 2003.4
Figure 2 demonstrates the dimensional relations of a malformed chick heart with DORV and LJAA (A) and a transverse image plane of a high-resolution 3D micro-CT reconstruction of such a heart showing details of cardiac anatomy of AV and semilunar valves at micrometer level (B). Figures 3 and 4 show separate sections of 3D micro-CT reconstructions of a normal chick heart versus the DORV heart with LJAA to demonstrate the relationship of the great vessels in both hearts by comparison as well as the significant differences at atrial, AV and chamber level.
Note the quality of resolution for both AV and semilunar valves in the normal (Figure 3C, E, G and Figure 4A, C) as well as in the DORV heart with LJAA (Figure 3D, F, H and Figure 4B, D) demonstrating fine details of the anatomic architecture. The high resolution of the micro-CT pictures allows the recognition of small structures such as the lumen of the coronary artery (Figure 2B) or the dysplastic aortic valve with a small fourth leaflet in the experimental heart (Figure 4B). The Data Supplement demonstrates further details of these hearts as fly-through movies (Movies I and II showing a fly-through of the right and left side of the normal heart, respectively, and Movie III demonstrating the relevant findings in detail as a fly-through in the DORV heart with LJAA).
In summary, we have shown that micro-CT facilitates nondestructive visualization of the anatomy of normal and malformed embryonic hearts at high resolution when the heart specimens have been dried by the critical point method (details of embryo preparation and the micro-CT unit together with images of the critical point dried specimens are presented in the Data Supplement (Figure I and Figure II, respectively). Micro-CT might be a valuable tool not only for nondestructive visualization of the 3D morphology of congenital heart defects at early stages of development but, additionally, might be used for exact volumetric measurements or for the determination of mechanical properties of the embryonic and fetal myocardium.
Sources of Funding
Supported by research grants from German Research Foundation (DFG; YE 42/3-1) and German Foundation for Cardiac Research (DSHF; F/19/07) to Dr. T.M. Yelbuz and internal funding from the Department of Pediatric Cardiology and Intensive Care Medicine at Hannover Medical School.
We thank K. Reccius at Hannover Medical School for her technical assistance in embryo preparations in a shell-less culture.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/122/22/e561/DC1.
- Received July 14, 2010.
- Accepted September 15, 2010.
- © 2010 American Heart Association, Inc.