Ventilatory response to hyperoxia in the chick embryo

In the avian embryo at term we measured the ventilatory response to hyperoxia, which lowers the chemoreceptor activity, to test the hypothesis that the peripheral chemoreceptors are tonically functional. Measurements of pulmonary ventilation (VE) were conducted in chicken embryos during the external pipping phase, at 38 degrees C, during air and hyperoxia, and during hypercapnia in air or in hyperoxia. Hyperoxia (95% O2) maintained for 30 min lowered VE by 15-20%, largely because of a reduction in breathing frequency (f). The oxygen consumption and carbon dioxide production of the embryo were not altered. The hyperoxic drop of VE was more marked in those embryos, which had higher values of normoxic VE. Hypercapnia, whether 2 or 5% CO2, increased VE, almost exclusively because of the increase in tidal volume (VT). The increase in VT was less pronounced when hypercapnia was associated with hyperoxia, and f slightly decreased. Hence, in hyperoxia, the VE response to CO2 was less than in air. The results are in support of the hypothesis that in the avian embryo, after the onset of breathing, the peripheral chemoreceptors exert a tonic facilitatory input on . This differs from neonatal mammals, where the chemoreceptors have minimal or no activity at birth, presumably because the increased arterial oxygenation with the onset of air breathing is a much more sudden phenomenon in mammals than it is in birds.     

Level of ventilation influences the cardiovascular response to hypoxemia in lambs

Newborn animals of a number of species display a brisk increase in ventilation followed by a gradual drop toward or below baseline within minutes of exposure to acute hypoxemia. Heart rate and cardiac output (a determinant of systemic oxygen transport along with the arterial oxygen content) appear to follow a similar pattern, but whether or not the cardiovascular response is influenced by the respiratory response is unknown. We therefore carried out experiments in which the level of ventilation was controlled during normoxemia and hypoxemia to test the hypothesis that the level of ventilation influences the cardiovascular response to acute hypoxemia. Six lambs ranging in age from 17 to 22 days were anesthetized, tracheostomized, and instrumented for measurement of cardiovascular variables. A recovery period of at least 3 days was allowed before the study when each lamb was artificially ventilated with a mixture of 70% nitrous oxide and 30% oxygen in nitrogen. A control respiratory frequency (f) of 30 breaths per min was set and a control tidal volume (VT) was chosen to achieve normocapnia. Cardiovascular measurements were made during normoxemia and hypoxemia (FIO2 0.10) 5 min after f or VT was changed to simulate a decrease, no change, or an increase in ventilation. During normoxemia, the level of ventilation had little effect on the measured cardiovascular variables. At control levels of ventilation, hypoxemia caused an increase in cardiac output that was due solely to an increase in stroke volume as heart rate decreased; blood pressure was unchanged. Increasing ventilation during hypoxemia did not augment cardiac output or alter blood pressure as compared with that observed at control levels of ventilation. Decreasing ventilation during hypoxemia, however, decreased cardiac output due to a profound bradycardia; blood pressure increased significantly. Our data provide evidence that the level of ventilation significantly influences the cardiovascular response to hypoxemia in young lambs.     

Ethyl alcohol-induced cardiovascular malformations in the chick embryo

Chick embryos incubated for 72-80 hours were exposed to various volumes (0.20-0.40 m1/egg) of 50% ethyl alcohol. Examination of embryos at day 14 of incubation showed that higher doses of ethanol decreased the survival rate of embryos compared with control embryos. Three major categories of cardiovascular malformations were observed in this study: intracardiac anomalies characterized primarily by isolated ventricular septal defect, ventricular septal defect with overriding aorta, double outlet right ventricle or common aorticopulmonary trunk; aortic arch anomalies; and subclavian artery anomalies. Frequencies of embryos with intracardiac anomalies were equal to or greater than 64.8% in the six groups exposed to ethanol. Administration of ethanol also induced high frequencies of embryos with subclavian artery anomalies (11.2-89.1%). Absence or hypoplasia of the right and/or left secondary subclavian artery was commonly associated with persistence of the corresponding primary subclavian artery. Bilateral absence and/or hypoplasia of the secondary subclavian arteries was more common than unilateral anomalies, whereas absence of the left secondary subclavian artery was more commonly observed than an absent right secondary subclavian artery. No embryos in the two control groups combined (n = 94) demonstrated aortic arch or subclavian artery anomalies.     

Effects of acute acidemia on the fetal cardiovascular defense to acute hypoxemia

In complicated pregnancy, fetal hypoxemia rarely occurs in isolation but is often accompanied by fetal acidemia. There is growing clinical concern about the combined effects of fetal hypoxemia and fetal acidemia on neonatal outcome. However, the effects on the fetal defense responses to acute hypoxemia during fetal acidemia are not well understood. This study tested the hypothesis that fetal acidemia affects the fetal defense responses to acute hypoxemia. The hypothesis was tested by investigating, in the late-gestation sheep fetus surgically prepared for long-term recording, the in vivo effects of acute fetal acidemia on 1) the fetal cardiovascular responses to acute hypoxemia and 2) the neural and endocrine mechanisms mediating these responses. Under general anesthesia, five sheep fetuses at 0.8 gestation were instrumented with catheters and Transonic flow probes around the femoral and umbilical arteries. After 5 days, animals were subjected to an acute hypoxemia protocol during intravenous infusion of saline or treatment with acidified saline. Treatment with acidified saline reduced fetal basal pH from 7.35 +/- 0.01 to 7.29 +/- 0.01 but did not alter basal cardiovascular variables, blood glucose, or plasma concentrations of catecholamines, ACTH, and cortisol. During hypoxemia, treatment with acidified saline increased the magnitude of the fetal bradycardia and femoral vasoconstriction and concomitantly increased chemoreflex function and enhanced the increments in plasma concentrations of catecholamines, ACTH, and cortisol. Acidemia also reversed the increase in umbilical vascular conductance during hypoxemia to vasoconstriction. In conclusion, the data support our hypothesis and show that acute acidemia markedly alters fetal hemodynamic, metabolic, and endocrine responses to acute hypoxemia.     

Pharmacology of dextroamphetamine-induced cardiovascular malformations in the chick embryo

We have observed dextroamphetamine sulfate to cause cardiovascular malformations in the 4-day-old chick embryo. Essentially all malformations were of the heart and great vessels. About one-half of these were the abnormal persistence of the left fourth aortic arch. Ventricular septal defects comprised the vast majority of the other malformations. Since d-amphetamine has both a direct and, more importantly, an indirect mode of alpha and beta adrenergic stimulation, three drugs were used to try to inhibit malformation production: alpha-methyl-p-tyrosine (AMT), a catecholamine synthesis inhibitor; metoprolol, a beta 1 blocker; and phentolamine, an alpha blocker. When given with d-amphetamine, all three drugs significantly reduced the malformation rate resulting from d-amphetamine alone. We speculate that the embryonic chick is capable of responding to the alpha and/or beta properties of dextroamphetamine sulfate. These properties may be causally related to the malformations observed.     

The response of the early chick embryo heart to anoxia

Isolated 3- and 5-day chick embryo hearts contain sufficient endogenous substrates to maintain their pulsatile activity for several hours under aerobic conditions, and even after five hours in substrate-free medium the rates are 40 to 50% of the original rates. Carbohydrate appears to be an important component of the endogenous substrates since 1 mM 2-deoxyglucose causes rapid failure of rate, and glycolysis appears to be a major energy pathway since the rate is depressed only about 50% by 2-hour's exposure to 10 mM fluoroacetate. In nitrogen the hearts rapidly become asystolic in the absence of added substrate. Recovery of the rate occurs if oxygen is reintroduced within one hour, but longer periods of anoxia result in progressively less recovery, especially with the 3-day hearts which appear to be particularly susceptible to irreversible damage. With 5.55 mM glucose as substrate there is little decrease in the original aerobic heart rate during five hours, and the hearts can tolerate total anoxia for five hours with rates only slightly less than the aerobic rates. The hypothesis of a preferential pentose phosphate pathway of glucose catabolism in the very young chick embryo heart is discussed, but no direct evidence in support of its existence is revealed in this study.     

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.     

Cardiovascular response to acute hypoxemia induced by prolonged breath holding in air

Prolonged breath hold (BH) represents a valid model for studying the cardiac adaptation to acute hypoxemia in humans. Cardiac magnetic resonance (CMR) allows a three-dimensional, high-resolution, noninvasive, and nonionizing anatomical and functional evaluation of the heart. The aim of the study was to assess the adaptation of the cardiovascular system to prolonged BH in air. Ten male volunteer diving athletes (age 30 +/- 6 yr) were studied during maximal BH duration with CMR. Four epochs were studied: I, rest; II and III, intermediate BH; and IV, peak BH. Oxygen saturation (So(2)), heart rate (HR), blood pressure (BP), systemic vascular resistance (VR), end-diastolic (EDV) and end-systolic volumes (ESV), stroke volume (SV), cardiac output (CO), ejection fraction (EF), maximal elastance index (EL), systolic wall thickening (SWT), and end-systolic wall stress (ESWS) of the left ventricle (LV) were measured in all four BH epochs. Average BH duration was 3.7 +/- 0.3 min. So(2) was reduced (I: 97 +/- 0.2%, range 96-98%, vs. IV: 84 +/- 2.0%, range 76-92%; P < 0.00001). BP, EDV, ESV, SV, CO, and ESWS linearly increased from epochs I to IV, whereas EF, EL, and SWT showed an opposite behavior, decreasing from resting to epoch IV (all trends are P < 0.01). During prolonged BH in air, a marked enlargement of the LV chamber occurs in healthy diving athletes. This response to acute hypoxemia allows SV,CO, and arterial pressure to be maintained despite the severe reduction in LV contractile function.     

Differential response among cells in the chick embryo myogenic lineage to photosensitization by Merocyanine 540

Primary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10-day embryos (E10 cells), concentrations of MC540 greater than or equal to 60 micrograms/ml resulted in death of nearly all myogenic cells upon exposure to light, but non-myogenic cells survived and replicated. Below 60 micrograms/ml, there was a dose-dependent reduction in muscle differentiation. At concentrations less than 40 micrograms/ml, there was no effect on myogenesis. Cultures of cells from 18-day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub-populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540-resistant myogenic population is present in low numbers in 10-day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.     

Contribution of infralimbic cortex in the cardiovascular response to acute stress

The infralimbic region of the medial prefrontal cortex (IL) modulates autonomic and neuroendocrine function via projections to subcortical structures involved in the response to stress. We evaluated the contribution of the IL to the cardiovascular response evoked by acute stress. Under anesthesia (80 mg/kg ketamine-11.5 mg/kg xylazine), rats were implanted with telemetry probes or arterial lines for recording heart rate and blood pressure. Guide cannulas were implanted to target the IL for microinjection of muscimol (100 pmol/100 nl), N-methyl-d-aspartate (NMDA) (6 pmol/100 nl), or vehicle (100 nl). Microinjection of muscimol, an agonist of GABA(A) receptors, into the IL had no effect on stress-evoked cardiovascular and thermogenic changes in any of the paradigms evaluated (cage switch, restraint plus air-jet noise, or air-jet stress). However, microinjection of the excitatory amino acid NMDA into the IL attenuated the pressor and tachycardic response to air-jet stress. Pretreatment with the selective NMDA antagonist dl-2-amino-5-phosphonopentanoic acid (AP-5, 100 pmol/100 nl) blocked the effect of NMDA on the cardiovascular response to air-jet stress. We conclude that 1) the IL region is not tonically involved in cardiovascular or thermogenic control during stress or under baseline conditions, and 2) activation of NMDA receptors in the IL can suppress the cardiovascular response to acute stress exposure.     

The effects of L- and D-carnitine administration on cardiovascular development of the chick embryo

A single 1.0-ml volume of L- or D-carnitine solution, at several selected mmole concentrations, was applied to the extraembryonic membranes of 3- and 4-day chick embryos in ovo. Hamburger-Hamilton stages of chick development ranged from 17 to 23. During the 17-18th days of incubation, embryos were dissected, and both survival and intracardiac anomaly rates were determined. Only at extremely high doses, both stereoisomers of carnitine exhibited a statistically significant toxigenic effect (p less than 0.001) as measured by a sharp decrease in survival rate when compared to chick Ringer's saline controls. Furthermore, since the anomaly rates became significant only near the LD50's, this indicated that intracardiac anomalies were induced only at toxic doses. Therefore, it is suggested that cardiovascular teratogenicity may be the result of toxicity. Below the LD50, anomaly rates were not significantly different from those of control embryos. In comparison, L- and D-carnitine were significantly different from one another (p less than 0.001) both in survival rate and percent affected embryos at a dose of 0.5 mmole. In summary, exogenous carnitine administration to the chick embryo does not appear to be deleterious to the developing cardiovascular system.     

Ectodermal control of the avascular zone of the peripheral mesoderm in the chick embryo

Prospective skin ectoderm is underlaid by a relatively thick (100 +/- 20 micrometer) avascular zone of mesoderm in most regions of the early embryo. To determine whether or not the ectoderm exercises a role in the establishment and maintenance of the avascular zone, trypsin-isolated pieces of backskin ectoderm from chick or quail embryos were implanted as a sheet into a slit cut deep into the capillary bed of the wing bud of host chick embryos of stages 19-23. In sham operations, slits were cut at various anteroposterior levels, and the wing was allowed to heal. At intervals of 3-48 hr after these operations, embryos were injected with India ink, fixed, and cleared. Implants formed flattened vesicles, usually in continuity with host ectoderm, but sometimes completely internalized. Periderm cells from each side of the vesicle faced each other, and the cells of the cuboidal layer faced an avascular mesodermal layer at least 100 micrometer thick at all points. The implantation of prospective skin ectoderm resulted in the formation of an avascular zone in normally vascularized mesoderm of the wing bud. In contrast, the vascular bed of the limb bud abutted directly on implants of Millipore filters or of Silastic silicone (Dow Corning). Likewise, the capillary bed came in direct contact with implants of retinal pigment epithelium, an ectodermal derivative normally in close contact with the vascular choroid coat of the eye. These results, taken in conjunction with earlier experiments that show the necessity of the apical ectodermal ridge for the formation of the marginal veins of the limb bud, suggest that epithelial-mesenchymal interactions are involved in important aspects of vasculogenesis in early embryos.     

Erythropoiesis in the developing chick embryo

The types of erythroid cells of chick embryos developing in ovo have been correlated with the hemoglobins of the embryos. Prior to 5 days, when primitive cells constitute the only erythroid cells, two hemoglobins can be resolved by polyacrylamide gel electrophoresis. The two adult hemoglobins and a minor hemoglobin found only in embryos and young chicks first appear simultaneously with initiation of definitive erythropoiesis.