The effect of isoproterenol on cardiovascular function in the stage 24 chick embryo

The developing cardiovascular system of the chick embryo is susceptible to teratogenic effects of catecholamines. Yet the mechanism for the teratogenetic action is unclear. Since catecholamines affect cardiovascular physiology, we studied the acute effect of the beta-agonist isoproterenol on mean atrial pressure, heart rate, mean dorsal aortic blood flow, mean arterial pressure and vascular resistance in stage 24 chick embryos. Dorsal aortic blood velocity was measured with a 20-MHz pulsed-Doppler velocity meter and intravascular pressure was measured with a servo-null pressure system. Isoproterenol in doses of 2 X 10(-4) micrograms (2.5 micrograms/kg), 8 X 10(-4) micrograms (10 micrograms/kg), and 1.2 X 10(-3) micrograms (15 micrograms/kg) was injected intravenously in 5-microliters aliquots of chick Ringer's solution. Additional groups of embryos were treated with the beta-antagonist propranolol, and isoproterenol plus propranolol. Control embryos received 5 microliters chick Ringer's solution to assess the hemodynamic effects of a volume injection. We found that isoproterenol caused no change in mean atrial pressure, heart rate, or mean arterial pressure. However, isoproterenol caused a dose-related decrease in dorsal aortic blood flow and a 2.5-fold increase in vascular resistance. The effects of isoproterenol were blocked by propranolol, which suggested that the increase in vascular resistance was mediated by beta-receptor stimulation.     

Cardiac mechanics in the stage-16 chick embryo.

A theoretical model is presented for the tubular heart of the stage-16 chick embryo (2.3 days of a 21-day incubation period). The model is a thick-walled, pseudoelastic cylindrical shell composed of three isotropic layers: the endocardium, the cardiac jelly, and the myocardium. The analysis is based on a shell theory that accounts for large deformation, material nonlinearity, residual strain, and muscle activation, with material properties inferred from available experimental data. We also measured epicardial strains from recorded motions of microspheres on the primitive right ventricles of stage-16 white Leghorn chick embryos. Relative to end diastole, peak axial and circumferential Lagrange strains occurred near end systole and had similar values. The magnitudes of these strains varied along the longitudinal axis of the heart (-0.16 +/- 0.08), being larger near the ends of the primitive right ventricle and smaller near midventricle. The in-plane shear strain was less than 0.05. Comparison of theoretical and experimental strains during the cardiac cycle shows generally good agreement. In addition, the model gives strong stress concentrations in the myocardial layer at end systole.     

Gamma ray induced genetic changes in different organs of chick embryo using peripheral blood micronucleus test and comet assay

Ionizing radiation is known to produce a variety of cellular and sub cellular damage in both prokaryotic and eukaryotic cells. Present studies were undertaken to assess gamma ray induced DNA damage in different organs of the chick embryo using alkaline comet assay and peripheral blood micronucleus test. Further the suitability of chick embryo, as an alternative model for genotoxicity evaluation of environmental agents was assessed. Fertilized eggs of Rhode island red strain were exposed to 0.5, 1 and 2 Gy of gamma rays delivered at a dose rate of 0.316 Gy/min using a ⁶⁰Co teletherapy machine. Peripheral blood smears were prepared from 8- to 11-day-old chick embryos for micronucleus test. Alkaline comet assay was performed on 11-day-old chick embryos in different organs such as the heart, liver, lung, blood, bone marrow, brain and kidney.Analysis of the data revealed a significant increase in the frequency of micronucleated polychromatic erythrocytes, micronucleated normochromatic erythrocytes and total micronucleated erythrocytes in the peripheral blood of gamma irradiated chick embryos at all the doses tested as compared to the respective controls. The polychromatic to normochromatic erythrocytes ratio which is an indicator of proliferation rate of hematopoetic tissue, decreased in the irradiated groups as compared to the controls. Data obtained from comet assay, clearly demonstrated a significant increase in DNA strand breaks in all the organs of irradiated chick embryos as compared to the respective controls. However, maximum damage was observed in the heart tissue on all the doses tested, followed by kidney, brain, lung, blood and liver. The lowest damage was observed in the bone marrow tissue. Both micronucleus test and comet assay were found to be suitable biomarkers for the evaluation of genotoxicity of gamma radiation in the chick embryo.     

Methyl mercury toxicity in the chick embryo

The toxicity of methyl mercury (mHg) in the developing chick embryo was investigated. The relationship of dose, time of administration (i.e., days 4-9 of development), and body levels of mercury was examined. The LD50 for mHg injected into the yolk sac on day 5 of incubation was 40-50 micrograms. Embryos dying within 24 hours showed increased total body mHg levels when compared to survivors (219 +/- 67 vs. 105 +/- 41 micrograms/gm, mean +/- SD). Absorption was dose-related, with a good correlation between mortality and body, blood, and brain levels. Daily analysis of body mHg levels after injection on day 5 showed continued mHg accumulation (0.88 +/- 0.35 micrograms/embryo/day). However, the rate of embryo growth exceeded the rate of mHg absorption, resulting in a progressive decrease in mHg in concentration in tissues (from 94.5 +/- 34.2 micrograms/gm on day 6 to 45.3 +/- 13.4 on day 9). Administration after day 5 resulted in a significant reduction in levels of mHg in the brain on day 18 (from 11.4 +/- 2.1 micrograms/gm when given on day 5 to 8.4 +/- 2.3 when given on day 9) and in mortality (from 64% to 33%). Because blood mHg levels remained unchanged, the increased brain levels and higher mortality early in embryogenesis may reflect facilitated transfer of mHg across a poorly developed blood-brain barrier. Later in development, the reduced mortality and lower brain mHg levels correspond to the formation of specialized interendothelial junctions and a more effective blood-brain barrier.     

Embryonic stroke volume and cardiac output in the chick

Cine recordings of the hearts of chick embryos of 3 days and 2 hr to 4 days and 21 hr incubation were projected and measured. The measurements were converted to volumes. Stroke volume was determined from the difference in end diastolic and end systolic volume and multiplied by heart rate to yield cardiac output. Mean stroke volume was 0.0058 (±0.00036 SEM) mm³ per mg body wt; mean cardiac output was 0.956 (± 0.061 SEM) mm³/min per mg body wt. Stroke volume and cardiac output rose above their control values after intravascular injection of Ringer's solution, and even more so after the injection of dextran solution. The increases in stroke volume were due to increases in end diastolic volume, in the case of dextran injected embryos they occurred in spite of a simultaneous increase in end systolic volume. It is concluded that the rise in cardiac output with growth of the embryo is in large part due to an increase in stroke volume, and that the increase in stroke volume depends in part on the known increase in embryonic blood volume. The experiments further suggest that a rapid hydrostatic and osmotic equilibrium exists between embryonic blood plasma and an extra vascular compartment.     

Uptake and blood pressure studies in the chick embryo following treatment with a teratogenic dose of dopamine

We report on the ventricular pressor response and regional (head, heart, and body) uptake of dopamine hydrochloride (DA) following topical application of a teratogenic dose to the stage 24 chick embryo. Embryos treated with DA exhibited a significant increase in mean ventricular blood pressure (MVBP) at 0.5 and 1 hour after treatment when compared to saline-treated control embryos. The time of the elevated MVBP correlated with the time of the peak uptake of DA in the embryonic heart. Even though the level of DA in the heart remained high for 9 hours, there was no measurable increase in blood pressure beyond 1.5 hours, suggesting unresponsiveness to DA or perhaps conversion of DA to its metabolites. These experiments have correlated a physiologic response of a known cardiovascular teratogen to its uptake in the embryo.     

Lipid and carbohydrate metabolism in euthyroid and hypothyroid chick embryo (Gallus domesticus).

1. The effect of propylthiouracil (PTU)-induced hypothyroidism on carbohydrate and lipid metabolism was studied in the chick embryo. 2. A single dose of PTU (250 micrograms/embryo) was administered on day 11 and embryos sacrificed on day 20 of incubation. 3. Thyroid glands were significantly enlarged (6 fold) by PTU administration. 4. Increased thyroid weight was associated with growth retardation and decreased plasma thyroxine levels. 5. Plasma glucose level was lower and phospholipids were significantly higher in the hypothyroid embryo. 6. Liver lipid concentrations in the control and hypothyroid embryos were not different but were significantly higher in both groups when compared to previously reported values in the young chick. 7. In contrast to PTU treatment after hatching, liver glycogen levels were not increased in the hypothyroid chick embryo. This was attributed to the high lipid nutrient condition of the chick embryo since a high lipid diet in the young chick decreased hepatic glycogen accumulation significantly.     

Sympathetic control of the cardiovascular response to acute hypoxemia in the chick embryo

In response to an acute hypoxemic insult, the mammalian fetus shows a redistribution of the cardiac output in favor of the heart and brain. Peripheral vasoconstriction contributes to this response and is partly mediated by the release of catecholamines. Two mechanisms of catecholamine release in the fetus are reported: 1) neurogenic sympathetic stimulation and 2) a nonneurogenic mechanism via a direct effect of hypoxemia on chromaffin tissues. In the present study, the effects of sympathetic blockade on plasma catecholamine release and cardiac output distribution in response to acute hypoxemia were studied in the chick embryo at different stages of incubation. Only at the end of the incubation period, sympathetic blockade markedly attenuated the increase in plasma catecholamine concentrations and resulted in a greater fraction of the cardiac output distributed to the carcass. However, these effects did not prevent a significant increase in cardiac output to the brain and heart during acute hypoxemia. These data imply that in the chick embryo the contribution of neurogenic mechanisms to the catecholaminergic response to acute hypoxemia becomes greater by the end of the incubation period.     

Lack of physiological plasticity in the early chicken embryo exposed to acute hypoxia

By exposing chicken embryos to hypoxia (10%) acutely (2, 4, and 6 hr) during early development (2, 3, and 4 days) we tested the hypothesis that hypoxia has an impact on embryonic growth and impairs cardiac development at the time cardiac morphogenesis is taking place. After the hypoxic perturbation, the embryos were allowed to develop until day 9, when embryo mass, heart mass, and rate of oxygen consumption were recorded. Four-day-old embryos exposed to 6 hr of hypoxia showed an increased mortality (38.9% versus 18% for controls), indicating the immediate effect of hypoxia on survivability. While only 8% of the controls displayed morphological abnormalities, 3- and 4-day-old embryos exposed for 6 hr showed more frequent developmental abnormalities (25% and 30% respectively). No significant differences in embryo or heart mass were found except in 4-day-old embryos exposed for 2 hr. Mass-specific oxygen consumption was not different between controls and embryos exposed to hypoxia at 2 or 3 days of development, but it was increased in 4-day-old embryos exposed for 4 hr (P < 0.05). These results suggest that an acute hypoxic episode does not have an impact when occurring very early in development (days 2 or 3). However, when the hypoxic episode occurs on day 4, survivability is largely decreased. Considering the lack of permanent effects on the surviving embryos, we suggest that the early embryo resorts to a simple strategy of death or survival, and the individual capacity for survival must be based on interindividual differences rather than the existence of compensatory mechanisms. J. Exp. Zool. 286:450-456, 2000.     

The antidysrhythmic effect of metoprolol in the epinephrine treated chick embryo

It was confirmed through electrocardiography that within two hours after epinephrine treatment, four day chick embryos either maintained normal rhythm or developed a severe cardiac dysrhythmia (22/93, 24% dysrhythmic). The ECG dysrhythmia in epinephrine treated embryos were characterized by periods of bradycardia, asystole, and various supraventricular or ventricular dysrhythmias. Within four hours after treatment, dysrhythmic embryos either reestablished normal rhythmicity or died. Electrocardiographic data also demonstrated that metoprolol pretreatment will block epinephrine induced dysrhythmias (0/46, 0% dysrhythmic). We conclude that metoprolol possesses antidysrhythmic properties in the epinephrine treated chick embryo.     

Preliminary investigation of the use of high frequency ultrasound imaging in the chick embryo

BACKGROUND: The purpose of this study was to investigate the feasibility of using high frequency ultrasound to study the chick embryo in a noninvasive and longitudinal fashion. METHODS: A total of 10 SPF White Leghorn chick embryos (GDs 11-17; Hamburger and Hamilton stage 37-43) were consecutively examined with a GE Logiq 400 ProSeries ultrasound unit using an 11-MHz small parts ultrasound probe. Access for ultrasound visualization of the embryos was accomplished by opening a 2-3-cm window either in the air cell over the blunt end of the egg or laterally over the embryo-dependent side of the egg. Warmed ultrasound coupling gel was used for imaging, and thermal regulation was maintained with infant heel warmers. The ultrasound images were recorded directly on digital video using a Sony TRV 900 DV camcorder. The images were directly converted to jpeg and mjeg2 files for further analysis. RESULTS: Effective visualization of each embryo was possible on each day of the study period. The embryos were best visualized through the opening made in the air cell at the blunt end of the egg. The extent of the anatomic survey of the chick embryo was dependent upon the position of the embryo in the egg relative to the opening in the air cell. Doppler color flow mapping studies were obtained of the embryonic and extraembryonic circulation. CONCLUSIONS: This preliminary investigation clearly shows the feasibility of high frequency ultrasound imaging to study chick embryo development in a longitudinal and noninvasive fashion. Further studies are presently ongoing regarding earlier embryo development, as well as to determine the stability and dynamics of the methodology.     

Thyrotropic and peripheral activities of thyrotrophin and thyrotrophin-releasing hormone in the chick embryo and adult chicken

The influence of an intravenous injection of thyrotrophin-releasing hormone (TRH) and bovine thyrotrophin (TSH) on circulating levels of thyroid hormones and the liver 5'-monodeiodination (5'-D) activity is studied in the chick embryo and the adult chicken. In the 18-day-old chick embryo, an injection of 1 microgram TRH and 0.01 I.U. TSH increase plasma concentrations of triiodothyronine (T3) and of thyroxine (T4). TRH, however, preferentially raises plasma levels of T3, resulting in an increased T3 to T4 ratio, whereas TSH preferentially increases T4, resulting in a decreased T3 to T4 ratio. The 5'-D-activity is also stimulated following TRH but not following TSH administration. The increase of reverse T3 (rT3) is much more pronounced following the administration of TSH. In adult chicken an injection of up to 20 micrograms of TRH never increased plasma concentrations of T4, but increases T3 at every dose used together with 5'-D at the 20 micrograms dose. TSH on the other hand never increased T3 or 5'-D, but elevates T4 consistently. It is concluded that TSH is mainly thyrotropic in the chick embryo or adult chicken whereas TRH is responsible for the peripheral conversion of T4 into T3 by stimulating the 5'-D-activity. The involvement of a TRH induced GH release in this peripheral activity is discussed.     

Gastrulating chick embryo as a model for evaluating teratogenicity: a comparison of three approaches

BACKGROUND: Teratology studies must be carefully designed to minimize potential secondary effects of vehicle and delivery routes. A systematic method to evaluate chick models of early embryogenesis is lacking. METHODS: We investigated 3 experimental approaches that are popular for studies of early avian development, in terms of their utility for teratogen assessment starting at gastrulation. These included in vitro embryo culture, egg windowing followed by direct application of a carrier vehicle to the embryo, and injection of a carrier vehicle into the egg yolk. We also developed a morphologically based scoring system to assess development of the early embryo. RESULTS: The in vitro culture and egg windowing approaches both caused an unacceptably high incidence of central nervous system and cardiac abnormalities in vehicle-treated embryos, which made it difficult to identify teratogen-specific defects. In contrast, exposing chick embryos to vehicle via direct egg yolk injection did not induce developmental anomalies. CONCLUSIONS: Optimization of the exposure route of potential toxicants to the embryo is critical because control treatments can cause developmental anomalies. In ovo yolk injection minimizes perturbation of young embryos and may be appropriate for teratogen delivery.     

Isotretinoin teratogenicity in mouse whole embryo culture

Recent clinical observations strongly suggest that isotretinoin [13-cis-retinoic acid (cis RA)] is a human teratogen causing primarily heart and craniofacial malformations including ear and palatal defects. The purpose of the present study was to determine if cis RA could induce similar craniofacial malformations in mouse embryo culture. Day 8 CD-1 mouse embryos were cultured for 48 hours in rat serum in the presence or absence of various concentrations of cis RA dissolved in DMSO. DMSO by itself had no effect on embryonic development; however, cis RA at 2 X 10(-5) M (6 micrograms/ml) was clearly toxic. At 2 X 10(-6) M cis RA, growth retardation was minimal, and approximately one-third of the embryos exhibited very specific defects including a dramatic reduction in the size of the first and second visceral arches, which eventually give rise to the maxilla, mandible, and ear. Similar observations were also made with 4-oxo-13-cis RA, which is a major metabolite of cis RA in the mouse and human. These malformations would be expected to result in defects similar to those observed in the human, and preliminary observations suggest these defects are due to cis RA-induced inhibition of cranial neural crest cell migration. Using day-10 mouse embryos cultured for 48 hours in Waymouth's medium containing 50% fetal calf serum, we observed that cis RA at 2 X 10(-5) M produced a high percentage of embryos with limb defects and median cleft lip. Our results demonstrate that labeled cis RA enters the tissues of the embryo both in vivo and in vitro. Cis RA inhibited proliferation of the frontonasal mesenchyme cells in primary culture with 31% inhibition occurring at 2 X 10(-5) M cis RA.     

Lack of effect of exogenous insulin-like growth factor-I (IGF-I) on chick embryo growth rate

Direct evidence that IGF-I has any significant effect on embryo growth is lacking. We therefore studied the effect of administration of IGF-I on the chick embryo in ovo. Five hundred ng pure IGF-I (purified from human plasma) were given to chick embryos on 2 occasions (7 and 14 d) by injection directly into the allantoic sac. Treated and control (saline injected) chicks hatched on the same day and were killed. IGF-I appeared to reach the tissues as the [35S]-sulphate uptake of treated sternal cartilage was significantly greater than that of control (P less than 0.02). However, there were no significant effects of treatment on total body weight, bone length measurements, organ (lung, liver, heart) weights, muscle DNA, RNA or protein levels. From these results we conclude that administration of exogenous IGF-I to the chick embryo at 7 and 14 d does not stimulate further growth of the chick embryo.     

Contribution of the superior atrioventricular cushion to the left ventricular infundibulum. Experimental study on the chick embryo

The role of the superior atrioventricular cushion in the normal development of the left ventricular infundibulum was experimentally studied in the chick embryo. 178 embryos at stages 19-24 of Hamburger and Hamilton were selectively labeled using gelatin-india ink; afterward embryos were reincubated until the mature heart stage, in which the final location of the labels was determined. In addition, anatomical microscopic studies were carried out on the chick embryo heart at different stages of the development. 91 embryos were obtained at the mature heart stage, 46 of which were normal. In 82,6% of these 46 embryos labels were found in the left ventricular infundibulum and were distributed in the following regions: (1) base of the free portion of the anteroseptal mitral leaflet (mitroaortic continuity); (2) the same region plus the left surface of the anterior basal portion of the ventricular septum, and (3) the left surface of the anterior basal portion of the ventricular septum. Anatomical microscopic studies showed that the superior atrioventricular cushion appears at stage 18, fusing with the inferior cushion at stage 28. Our results permit us to conclude that the superior atrioventricular cushion plays an important role in the normal development of the left ventricular infundibulum, and it contributes in the posterolateral and anteromedial wall formation.     

Effect of pituitary grafting on ovarian oestradiol secretion in the hypophysectomized chick embryo.

Chick embryos were hypophysectomized by partial decapitation at the stage of 42 h of incubation and grafted with a hypophysis from a 12-days-old donor embryo on the chorio-allantoic membrane at 9 1/2 days. Two days later, their ovary was removed for organ culture and its oestradiol secretion rate was compared to that of the ovary of hypophysectomized, non grafted control embryos. The oestradiol secretion rate in the grafted embryos was almost twice that in the hypophysioprivic embryos and in the range of that in normal embryos. This result suggests that the hypophysis controls oestradiol secretion of the ovary in the 11 1/2-days-old chick embryo.     

Chronic ethanol exposure in the embryonic chick heart: effect on myocardial function and structure

To investigate the effect of chronic ethanol exposure on the embryonic chick heart, chick embryos were exposed daily to one of seven graded doses of ethanol or to saline only (shams) from 0 to 96 hr of incubation. One hour before and after exposure at 72 hr, and 1 hr before and after exposure at 96 hr, embryos were analyzed for changes in heart function, embryo tissue ethanol content, occurrence of anomalies, and embryo weights. At both 71 and 73 hr of incubation (during cardiogenesis), when compared to shams, heart rate (HR) in embryos receiving ethanol doses greater than 0.0375 ml increased significantly (P less than .05) with commensurate increases in injected ethanol. Additionally, at 73 hr, depressed cardiac contractility, measured as shortening fraction, was noted at doses greater than or equal to .0375 when compared to shams. While slight increases in shortening fraction (SF) across dose were noted at 95 and 97 hr, only random doses were statistically significant from shams, with no specific trend in either HR or SF at this postcardiogenesis stage. Within each time group, gas chromatography analysis of embryo tissue ethanol content demonstrated a linear relationship between dose injected and tissue ethanol content retrieved. With increasing dose and stage, viability decreased. Weights of ethanol-injected embryos were not significantly different from shams within each time group. Our studies of the response of the embryonic chick heart to ethanol indicate both dose and stage susceptibility, with greater susceptibility to ethanol injury during active cardiogenesis.     

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein content

The role of the suspensor in the early development of the dicot embryo has not yet been defined. It has been described as merely an anchor and also as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor, early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos have been examined in tissue culture. It is known that Phaseolus embryos in culture at low osmotic potential will germinate precociously and that embryos in culture at high osmotic potential will either fail to grow or form callus. Optimum sucrose concentrations for continued, normal embryonic development of 0.2 mm and 0.5 mm P. vulgaris in tissue culture with Gamborg B5 medium were determined to be 12 and 6%, respectively. Protein content was examined in embryos and suspensors. Results showed that both 0.2- and 0.5-mm embryos required an attached suspensor for maximum protein content. Protein levels were substantially decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M restored the protein content to that of freshly excised embryos.