Elfects of the size of lesions of the cardiac neural crest at various embryonic ages on incidence and type of cardiac defects

Removal of premigratory neural crest over somites 1 through 3 in chick embryos has been shown previously to result in a significant incidence of persistent truncus arteriosus. In the present study, single somite-length pieces of premigratory neural crest were removed unilaterally at slightly different embryonic ages. These lesions resulted in a number of different heart defects. Two defects, ventricular septal defect and double outlet right ventricle, were significantly correlated with the location of neural crest removed. The stage at which the ablation was performed was important in determining the incidence but not the type of defects. Circulation 73, No. 2, 360-364, 1986. NEURAL CREST plays a crucial role in the normal septation of the conotruncal region of the chick heart. It has been shown that neural crest cells from somite levels 1 through 3 migrate to pharyngeal arches 4 and 6, the aortic sac, and conotruncus.' Ablation of premigratory neural crest over somite levels 1 through 3 results in a high incidence of conotruncal abnormalities similar to ventricular septal defect (VSD) and persistent truncus arteriosus (PTA) seen in man. Recently, the area of "cardiac" neural crest was shown to extend from slightly cranial to somite 1 to somewhat caudal of somite 3.4 Ablation of bilateral pieces of single-somite lengths of neural crest or unilateral multiple somite-length pieces of neural crest often resulted in PTA and VSD. These were the only types of conotruncal malformations correlated with neural crest ablation in that study. Because PTA indicates a severe deficiency in conotruncal septation, removal of smaller lengths of premigratory neural crest might yield a larger variety of less severe forms of conotruncal malformations. The present study shows that removal of unilateral single-somite lengths of premigratory neural crest reFrom the Departments of Anatomy and Pathology, Medical College of Georgia, Augusta, and the Department of Pediatrics, University of Florida, Gainesville. Supported by USPHS grant HL32546. Dr. Kirby was an Established Investigator of the American Heart Association during the course of this study. Address for correspondence: Margaret L. Kirby, Ph.D., Department of Anatomy, Medical College of Georgia, Augusta, GA 30912. Received August 29, 1985; revision accepted Nov. 7. 1985. 360 sults in conotruncal malformations that are less severe than PTA. Materials and methods Arbor Acre chicken eggs (Seaboard Hatchery, Athens, GA) were incubated in a forced-draft incubator at 380 C and 97% relative humidity until stages 8 to 11.1 Embryos were prepared for microsurgery according to the method of Narayanan.' All operated eggs were returned to the same incubator until the circulatory system was well established (about 72 hr total incubation). The viable eggs were transferred to another forced-draft incubator maintained at 370 C and 70% relative humidity. Microsurgery was performed on experimental embryos (stages 8 to 11) through a window in the shell. A microcautery unit developed in collaboration with the Biomedical Engineering Department of the Medical College of Georgia was used to ablate neural crest cells. Various levels of the neural fold were ablated, with the somites used as reference. The ventral neural tube and underlying mesoderm were left intact. The neural fold consists of the presumptive dorsal part of the neural tube, the neural crest, and some adjacent surface ectoderm.7 Only neural crest cells migrate from this region. For control embryos, a window was made in the shell and was sealed with a glass coverslip. Control eggs were transferred to incubators in parallel with experimental eggs. All eggs were reincubated for an additional 6 to 7.5 days, bringing the total incubation time to 8 or 9 days. A number was assigned to each egg before it was returned to the incubator, and subsequent processing of specimens was done without knowledge of the group to which they belonged. After morphologic analysis of the hearts, numbers were decoded and the embryos grouped according to treatment. The embryos were removed from the shell and examined for defects of the thoracic wall. Thoracic wall defect was defined as a deficiency in development of the anterior portion of the thoracic wall so that the conus was visible. A midsagittal thoracic incision was made and the heart removed by cutting the great arteries above the origins of the brachiocephalic arteries. The CIRCULATION by gest on A ril 0, 2017 http://ciajournals.org/ D ow nladed from LABORATORY INVESTIGATION-CONGENITAL HEART DISEASE heart and accompanying vessels were transferred into heparinized saline at 370 C for 5 min and fixed in Perfix (Fisher). The pericardial sac was removed. By means of a calibrated ocular micrometer, the width of each great artery was measured at the level of origin of the right brachiocephalic artery (figure 1). Although this is a reliable method for measuring the outer diameters of the great arteries, it is flawed in that the pulmonary trunk has divided into right and left pulmonary arteries; thus only the outer diameter of the left pulmonary artery was measured by this method. A triangular flap was excised from the anterior right ventricular wall (figure 1). The thickness of the right ventricular wall was measured (figure 1). VSDs were classified as basilar or apical. Basilar VSDs were associated with the atrial (outflow, membranous) portion of the ventricular septum; apical VSDs occurred in the muscular portion of the septum. The crista is not clearly defined in 8 to 9 day chick hearts; therefore no attempt was made to classify basilar VSDs as supracristal or infracristal. The area and circumference of each VSD were measured with an Apple graphics tablet and Apple II Plus computer with appropriate software. The center of the VSD was described as being the intersection of its longest dimension and widest perpendicular (figure 1). It was related to the center of the pulmonary valve, the inferior limit of the right atrioventricular ostium, and, in cases of double outlet right ventricle (DORV), the midpoint of the aortic valve (figure 1). In hearts with PTA, the center of the ostium of the artery was related to the VSD intersect. The angle of VSD long-axis orientation was noted as being 45 degrees dextrorotated, 45 degrees levorotated, or horizontally intersecting a line passing from the apex of the heart to the arterial ostia. The origins of the great arteries were classified based on their position relative to the ventricles. The term PTA indicated origin of a common arterial trunk from the heart. In DORV both the aorta and pulmonary trunk arose from the right ventricle. The cusps of the semilunar valve(s) were counted and the positions of the atrioventricular valves were noted. The data obtained were entered into a computer (University of Georgia Systems), and means and standard deviations were determined for individual variables from the control group. Experimental hearts with variables outside the normal range were identified. Significant correlations were determined by chi-square analysis.