Background-Epidemiological findings suggest an association between low-for-age birth weight and the risk to develop coronary heart diseases in adulthood. During pregnancy, an imbalance between fetal demands and supply may result in permanent alterations of neuroendocrine development in the fetus. We evaluated whether chronic prenatal hypoxia increases arterial sympathetic innervation. Methods and Results-Chicken embryos were maintained from 0.3 to 0.9 of the 21-day incubation period under normoxic (21% O 2 ) or hypoxic conditions (15% O 2 ). At 0.9 incubation, the degree of sympathetic innervation of the embryonic femoral artery was determined by biochemical, histological, and functional (in vitro contractile reactivity) techniques. Chronic hypoxia increased embryonic mortality (32% versus 13%), reduced body weight (21.9Ϯ0.4 versus 25.4Ϯ0.6 g), increased femoral artery norepinephrine (NE) content (78.4Ϯ9.4 versus 57.5Ϯ5.0 pg/mm vessel length), and increased the density of periarterial sympathetic nerve fibers (14.4Ϯ0.7 versus 12.5Ϯ0.6 counts/10 4 m 2 ). Arteries from hypoxic embryos were less sensitive to NE (pD 2 , 5.99Ϯ0.04 versus 6.21Ϯ0.10). In the presence of cocaine, however, differences in sensitivity were no longer present. In the embryonic heart, NE content (156.9Ϯ11.0 versus 108.1Ϯ14.7 pg/mg wet wt) was also increased after chronic hypoxia. Conclusions-In the chicken embryo, chronic moderate hypoxia leads to sympathetic hyperinnervation of the arterial system. In humans, an analogous mechanism may increase the risk for cardiovascular disease in adult life. (Circulation.

The present study aimed to characterize the contractile reactivity of the chicken ductus arteriosus (DA) from the last stage of prenatal development and throughout the perinatal period. Isolated DA rings from 15-day, noninternally-pipped 19-day, and externally-pipped 21-day embryos were studied using myograph techniques. On embryonic day 15, the chicken DA did not respond to O 2 (0 to 21%), norepinephrine (NE), or phenylephrine (Phe) but contracted in response to high-K ϩ solution, the inhibitor of voltage-gated channels 4-aminopyridine, U-46619, and endothelin (ET)-1. These responses increased with advancing incubation age. Contractile responses to O 2, NE, and Phe were present in the 19-and 21-day embryo. Oxygen-induced contraction was restricted to the pulmonary side of the DA and was augmented by the nitric oxide synthase inhibitor N -nitro-L-arginine methyl ester and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and reduced by the peptidic ET A and ETB-receptor antagonist PD-142,893. Transmural electrical stimulation of nerves, the nonselective cyclooxygenase (COX) inhibitor indomethacin, the COX-1 inhibitor valeryl salicylate, the COX-2 inhibitor nimesulide, the inhibitor of ATP-sensitive K ϩ channels glibenclamide, and the inhibitor of Ca 2ϩ -activated K ϩ channels tetraethylammonium did not cause contraction of the DA rings at any age. We conclude that transition to ex ovo life is accompanied by dramatic changes in chicken DA reactivity. At 0.7 incubation, excitation-contraction and pharmacomechanical coupling for several contractile agonists are already present, whereas the constrictor effects of O 2 and cathecolamines appear later in development and are located in the pulmonary side of the DA. ductus arteriosus; chicken embryo; potassium channels; oxygen sensing; cathecolamines.THE DUCTUS ARTERIOSUS (DA) is a vessel that connects the pulmonary artery to the aorta and provides, during the fetal life, a pulmonary-to-systemic diversion that shunts more than half of the right ventricle output away from the nonventilated lungs into the systemic circulation (43, 47). The main factors maintaining patency of the DA in utero are low O 2 tension, high levels of circulating PGE 2 , and locally produced PGE 2 and PGI 2 (24, 43). Failure of DA closure after birth is a common complication of premature delivery that is still presenting challenges in terms of diagnosis, assessment, and treatment options (43).Although the isolated DA is sensitive to a wide range of contractile agonists, the major factor actively stimulating contraction at birth is increasing O 2 tension, which has a profound effect on the DA, both directly and by modulating its response to vasodilators and vasoconstrictors (43). To constrict properly after birth, the DA prepares itself for this specific task from a quite early onset during development (3). This preparation is reflected by changes in responsiveness with advancing gestational age. These have been extensively characterized in numerous mammalian specie...

Although chronic prenatal hypoxia is considered a major cause of persistent pulmonary hypertension of the newborn, experimental studies have failed to consistently find pulmonary hypertensive changes after chronic intrauterine hypoxia. We hypothesized that chronic prenatal hypoxia induces changes in the pulmonary vasculature of the chicken embryo. We analyzed pulmonary arterial reactivity and structure and heart morphology of chicken embryos maintained from days 6 to 19 of the 21-day incubation period under normoxic (21% O(2)) or hypoxic (15% O(2)) conditions. Hypoxia increased mortality (0.46 vs. 0.14; P < 0.01) and reduced the body mass of the surviving 19-day embryos (22.4 +/- 0.5 vs. 26.6 +/- 0.7 g; P < 0.01). A decrease in the response of the pulmonary artery to KCl was observed in the 19-day hypoxic embryos. The contractile responses to endothelin-1, the thromboxane A(2) mimetic U-46619, norepinephrine, and electrical-field stimulation were also reduced in a proportion similar to that observed for KCl-induced contractions. In contrast, no hypoxia-induced decrease of response to vasoconstrictors was observed in externally pipped 21-day embryos (incubated under normoxia for the last 2 days). Relaxations induced by ACh, sodium nitroprusside, or forskolin were unaffected by chronic hypoxia in the pulmonary artery, but femoral artery segments of 19-day hypoxic embryos were significantly less sensitive to ACh than arteries of control embryos [pD(2) (= -log EC(50)): 6.51 +/- 0.1 vs. 7.05 +/- 0.1, P < 0.01]. Pulmonary vessel density, percent wall area, and periarterial sympathetic nerve density were not different between control and hypoxic embryos. In contrast, hypoxic hearts showed an increase in right and left ventricular wall area and thickness. We conclude that, in the chicken embryo, chronic moderate hypoxia during incubation transiently reduced pulmonary arterial contractile reactivity, impaired endothelium-dependent relaxation of femoral but not pulmonary arteries, and induced biventricular cardiac hypertrophy.

We previously observed arterial sympathetic hyperinnervation and endothelial dysfunction in the chicken embryo after exposure to chronic hypoxia. We now investigate whether changes in arterial properties could also be observed at 14-15 weeks of life. Eggs of White Leghorn chicken were incubated under normoxic or moderately hypoxic (15% O2 from days 6-19 of a 21-day incubation) conditions. Experiments were performed at 14-15 weeks of life under standard conditions (Hm: males exposed to hypoxia; Hf: females exposed to hypoxia; Nm: males exposed to normoxia; Nf: females exposed to normoxia). Body weight at hatching and at 14-15 weeks was not affected by in ovo exposure to hypoxia. Mean arterial pressure and heart rate were not significantly altered by chronic in ovo hypoxia. However, isolated femoral arteries were more sensitive to electrical stimulation (frequency in Hz of half-maximal contraction, Hm: 1.62+/-0.33, Hf: 1.92+/-0.88, Nm: 2.49+/-0.49, Nf: 2.83+/-0.31) and pharmacological stimulation of peri-arterial sympathetic nerves (contraction in N/m in response to tyramine: Hm: 5.27+/-0.85, Hf: 4.10+/-0.9, Nm: 2.26+/-0.67, Nf: 3.65+/-0.51, p=0.07) after in ovo hypoxia. In side branches of the femoral artery, the effect of NO synthase blockade with L-NAME on contraction (in N/m) in response to high K+ (Hm: 0.35+/-0.91, Hf: 1.29+/-0.36, Nm: 2.88+/-0.19, Nf: 2.79+/-0.58) and on the sensitivity to acetylcholine (DeltapD2, H: 0.32+/-0.11, N: 0.62+/-0.05) was reduced after in ovo hypoxia. The present study shows that exposure to chronic moderate hypoxia during development affects the contractile and relaxing arterial responses of 14- to 15-week-old chickens. Although hypoxia did not lead to changes in blood pressure at this age, the observed effects on arterial sympathetic and endothelial function may represent early signs of future cardiovascular abnormalities.

. Direct effects of acute hypoxia on the reactivity of peripheral arteries of the chicken embryo. Am J Physiol Regulatory Integrative Comp Physiol 283: R331-R338, 2002. First published April 4, 2002 10.1152/ajpregu.00675.2001.-In the chicken embryo, acute hypoxemia results in cardiovascular responses, including an increased peripheral resistance. We investigated whether local direct effects of decreased oxygen tension might participate in the arterial response to hypoxemia in the chicken embryo. Femoral arteries of chicken embryos were isolated at 0.9 of incubation time, and the effects of acute hypoxia on contraction and relaxation were determined in vitro. While hypoxia reduced contraction induced by high K ϩ to a small extent (Ϫ21.8 Ϯ 5.7%), contractile responses to exogenous norepinephrine (NE) were markedly reduced (Ϫ51.1 Ϯ 3.2%) in 80% of the arterial segments. This effect of hypoxia was not altered by removal of the endothelium, inhibition of NO synthase or cyclooxygenase, or by depolarization plus Ca 2ϩ channel blockade. When arteries were simultaneously exposed to NE and ACh, hypoxia resulted in contraction (ϩ49.8 Ϯ 9.3%). Also, relaxing responses to ACh were abolished during acute hypoxia, while the vessels became more sensitive to the relaxing effect of the NO donor sodium nitroprusside (pD2: 5.81 Ϯ 0.21 vs. 5.31 Ϯ 0.27). Thus, in chicken embryo femoral arteries, acute hypoxia blunts agonist-induced contraction of the smooth muscle and inhibits stimulated endothelium-derived relaxation factor release. The consequences of this for in vivo fetal hemodynamics during acute hypoxemia depend on the balance between vasomotor influences of circulating catecholamines and those of the endothelium.catecholamine; endothelium-derived relaxation factor IN THE FETUS, AN ACUTE DECREASE in arterial oxygen tension leads to cardiovascular responses, involving an elevation in blood pressure and redistribution of the cardiac output in favor of vital organs. In fetal lambs (10), fetal llamas (9), and chicken embryos (24, 25), increased levels of circulating catecholamines take part in this response. Early in gestation, the chromaffin cells in the primitive adrenal medulla are directly sensitive to low oxygen tension. Later in gestation, activation of efferent sympathetic nerves also contributes to the response (38). Antagonists of ␣-adrenoreceptors blunt the hypoxia-induced increase in fetal total peripheral resistance (9, 10, 25).Neurohumoral mechanisms have been proposed to be important regulators of blood flow in the hypoxemic fetus, but it remains to be established whether local and direct effects of decreased oxygen tension participate in the fetal cardiovascular response to hypoxemia. In previous studies, we have shown that chronic exposure to hypoxia affects both sympathetic innervation (34) and endothelium-dependent relaxation (33) of femoral arteries of the chicken embryo, but acute effects of hypoxia in isolated systemic arteries were not studied. Moreover, acute effects of low oxygen tension have been studied i...