Diminished growth of atrioventricular cushion tissue in stage 24 retinoic acid-treated chicken embryos.

Stage 34 chicken hearts have shown a spectrum of looping disturbances, changed hemodynamics, and changed growth of both right ventricular myocardium and atrioventricular cushion tissue after retinoic acid treatment. To obtain more information about the onset of the malformations we studied stage 24, the stage between the previously studied stage 34 and the moment of treatment. Sixteen stage 24 chicken embryos were examined after treatment with 1 microg all-trans retinoic acid at stage 15 and compared with 6 sham operated embryos. Morphological examination was supported by graphic reconstructions. Absolute volumes of atrial, atrioventricular, and ventricular myocardia were measured by a point counting method. The absolute volumes of the endocardial cushions were measured as well. Fifteen (15/16) retinoic acid-treated hearts did not show marked malformations as far as could be detected with our current macroscopic and microscopic techniques. One (1/16) retinoic acid-treated heart showed an abnormal tubular C-shape with a less bended inner curvature and with an abnormal horizontally oriented atrioventricular canal. The dorsal cushion tissue of this atrioventricular canal was discontinuous with the dorsal mesocardium and covered the malpositioned myocardial border between the atrium and the atrioventricular canal. The volume measurements did show a difference between retinoic acid treatment and sham operations. The retinoic acid-treated hearts showed a significant volume decrease of the atrioventricular cushions. No significant differences were found in the volumes of the ventricular myocardium compared to the sham operated embryos. We hypothesize that, between stages 15 and 24, retinoic acid directly affects the myocardial wall and the cushion tissue formation. In the present material this has resulted in decreased atrioventricular cushion growth, in changed hemodynamics, and in a severe looping disturbance of one embryo. We further hypothesize that, between stages 24 and 34, the malformations with minor looping disturbances will become apparent. Thus, development beyond stage 24 would result in the spectrum of looping disturbances as has been found at stage 34. These latter morphological malformations would lead to increasing hemodynamic changes, resulting in changes in growth as a secondary effect.

Stereological study of stage 34 chicken hearts with looping disturbances after retinoic acid treatment: disturbed growth of myocardium and atrioventricular cushion tissue.

BACKGROUND: In a previous study retinoic acid treatment of chicken hearts has resulted in a spectrum of looping disturbances. Because of a decrease in contraction force of these hearts, the myocardial volume was hypothesized to be altered. Because retinoic acid has been suggested to influence endocardial cushion volumes, these were estimated as well. METHODS: The previously studied hearts were used for estimating the absolute volumes of the atrial and ventricular myocardium and of the endocardial cushions by means of Cavalieri's principle. To measure the surface density of the trabeculations according to the isector method, we used retinoic acid treated hearts, which were perfusion fixed and in which the sections were isotropic uniform random. The volumes and surface densities found in the three morphologically distinguished groups, i.e., intact septum, isolated ventricular septal defect and double outlet right ventricle, were compared with those in shams. RESULTS: A significant volume decrease was found in the right ventricular free wall myocardium of the double outlet right ventricle. No significant differences were found in the surface densities of the trabeculae. The volume of the atrioventricular cushion tissue in the double outlet right ventricle hearts was significantly increased. The morphological spectrum observed previously was also expressed in the right ventricular myocardial volume, which appeared to decrease from the least to the most malformed hearts, and in the volume of the atrioventricular cushion tissue, which appeared to increase. CONCLUSIONS: Several studies have shown pathology in myocardial and cushion tissue after retinoic acid treatment. In this study we have found a decreased growth of the right ventricular myocardium and an increased growth of the atrioventricular cushion tissue. We suggest that the previously found looping disturbance causes changed hemodynamics, as reported elsewhere, and that these result in changes in growth. We cannot exclude a direct effect of retinoic acid on the myocardium, which has to explain the looping disturbance.

Spectrum of looping disturbances in stage 34 chicken hearts after retinoic acid treatment.

BACKGROUND: In a recently developed chick model the teratogen retinoic acid has appeared to induce a spectrum of double outlet right ventricle, which needs further detailed evaluation. It is known that retinoic acid is able to induce cardiac malformations. Although the exact mechanism is not known, an interaction with neural crest cell function is thought to exist. METHODS: After treatment with 1 microgram all-trans retinoic acid at Hamburger and Hamilton stage 15 and reincubation until stage 34 of development 41 chicken embryos were evaluated macroscopically and microscopically, supported by graphic reconstructions. These retinoic acid treated embryos were compared with a control group (n = 8). RESULTS: The retinoic acid treated embryos could be divided in three groups. Group 1 (23/41) had an intact septum, group 2 (11/41) had an isolated ventricular septal defect (VSD), and group 3 (7/41) had a double outlet right ventricle (DORV). Besides, in the group with an intact septum 11 hearts showed an abnormal course of the subaortic outflow tract. In the group with DORV a straddling tricuspid orifice (7/8) and a double inlet left ventricle (1/8) could be distinguished. Considering the external contour, the hearts in the DORV group all showed a dextroposed arterial pole. Malformed pharyngeal arch arteries were found in all three groups (11/41) and with a great diversity. CONCLUSIONS: The present cardiac malformations in the chicken as a result of retinoic acid treatment are part of a continuous spectrum, varying from hearts with an intact ventricular septum and a normal course of the subaortic outflow tract to a double outlet right ventricle with a straddling tricuspid orifice or even a double inlet left ventricle. A remarkable observation in this spectrum concerns the correlation of malformations of the inflow and outflow tracts, which is explained as a cardiac looping disturbance. The disturbance of the looping process seems to lead to malalignment of septal components, although, in the chick, retinoic acid does not in general interfere with the formation of these septal components themselves.

Intracardiac blood flow patterns related to the yolk sac circulation of the chick embryo.

Intracardiac flow patterns during heart development were studied by injection of india ink into the yolk sac circulation of chick embryos at Hamburger-Hamilton stages 10 to 17. We injected india ink into a small venule or capillary, carefully preventing application of overpressure to the vascular system, and recorded the intracardiac route by video. From stage 12 onward, blood flow was laminar, and separate intracardiac currents were visualized. The yolk sac was divided into a left and a right half. Blood coursed through each half in concentric loops, ranging from the marginal sinus to the sinus venosus. This parallel array persisted within the heart. Bilateral to the embryo, two lateral regions arose that extended wedge-like within each half, resulting in six equally sized yolk sac regions at stage 16. The process of heart looping was not accompanied by a change in flow pattern. However, developmental changes of the yolk sac circulation were reflected in alteration of the intracardiac flow pattern. From stage 16 onward, the intracardiac flow pattern was no longer determined by the left- or right-hand side of the yolk sac but by bilateral anterior, lateral, and posterior regions of the yolk sac. Blood from the lateral regions of the yolk sac was preferentially distributed to the head. The results show that in preseptation stages a relatively stable flow pattern is present. We suggest that alterations in blood flow could influence the process of normal heart development.