Carolina G. A. Kessels

Chronic Hypoxia Stimulates Periarterial Sympathetic Nerve Development in Chicken Embryo

BackgroundEpidemiological 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 ResultsChicken embryos were maintained from 0.3 to 0.9 of the 21-day incubation period under normoxic (21% O2) or hypoxic conditions (15% O2). 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 (21.9±0.4 versus 25.4±0.6 g), increased femoral artery norepinephrine (NE) content (78.4±9.4 versus 57.5±5.0 pg/mm vessel length), and increased the density of periarterial sympathetic nerve fibers (14.4±0.7 versus 12.5±0.6 counts/104 &mgr;m2). Arteries from hypoxic embryos were less sensitive to NE (pD2, 5.99±0.04 versus 6.21±0.10). In the presence of cocaine, however, differences in sensitivity were no longer present. In the embryonic heart, NE content (156.9±11.0 versus 108.1±14.7 pg/mg wet wt) was also increased after chronic hypoxia. ConclusionsIn 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.

Role of superoxide anion on basal and stimulated nitric oxide activity in neonatal piglet pulmonary vessels

The superoxide anion (O2·−) appears to be an important modulator of nitric oxide bioavailability. Enzymatic scavenging of O2·− is carried out by superoxide dismutase (SOD). The present study was designed to characterize the developmental changes on pulmonary vascular reactivity induced by 1) exogenous Cu/Zn SOD, 2) several putative SOD mimetics, and 3) endogenous SOD inhibition. We also analyzed age-related changes on pulmonary SOD activity and vascular O2·− levels. SOD (1–300 U/mL) produced endothelium-dependent relaxation of U46619-contracted intrapulmonary arteries (fourth branch) and veins from 12- to 24-h-old and 2-wk-old piglets. SOD-induced relaxation was greater in pulmonary arteries and was abolished by the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester. SOD induced a greater pulmonary artery relaxation in the 2-wk-old than in the 12- to 24-h-old piglet. SOD (100 U/mL) did not modify acetylcholine-induced relaxation in pulmonary arteries. In contrast, endogenous SOD inhibition by diethyldithiocarbamate (3 mM) impaired acetylcholine-induced relaxation in pulmonary arteries from newborn but not from 2-wk-old piglets. Total SOD activity in lung tissue did not change with postnatal age. With the use of dihydroethidium, an oxidant-sensitive fluorescent probe, we did not find significant age- or vessel-related differences in O2·− presence. From the putative SOD mimetics tested, only the metal salts MnCl2 and CuSO4 reproduced the vascular effects of SOD. In summary, SOD produces endothelium-dependent pulmonary vascular relaxation by protecting nitric oxide from destruction by O2·−. This effect was less marked in newborns than in 2-wk-old piglets. In contrast, pulmonary arteries from newborn piglets are more sensitive to the inhibition of endogenous SOD.

Cardiopulmonary Effects of Chronic Administration of the NO Synthase Inhibitor L-NAME in the Chick Embryo

Background: Experimental observations in mammalian models suggest that endothelial nitric oxide (NO) synthase (NOS) content and activity are decreased in persistent pulmonary hypertension of the newborn. Objectives: To test the hypothesis that disruption of NO signaling in the developing chick embryo lung may contribute to pulmonary hypertension. Methods: We analyzed pulmonary arterial reactivity and structure and heart morphology of 19-day chick embryos (incubation time 21 days) that received a daily injection of the NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 20 µg per gram egg) or vehicle from day 12 until day 18. Results: Exposure to L-NAME did not affect embryonic survival or body mass of the embryos. The contractile responses to KCl, endothelin-1, the thromboxane A2 mimetic U46619, noradrenaline, and electrical-field stimulation were not affected by exposure to L-NAME. In contrast, in ovo L-NAME exposure reduced the sensitivity of pulmonary arteries to acetylcholine (pD2: 6.53 ± 0.14 vs. 6.96 ± 0.13; p < 0.05) and this effect was reversed by the NOS substrate L-arginine. Relaxations induced by sodium nitroprusside or forskolin were not altered by chronic L-NAME. Pulmonary vessel density was not different, but the percentage medial wall area of small pulmonary arteries (external diameter 10–50 µm) was slightly but significantly increased in the embryos exposed to L-NAME. In addition, hearts of L-NAME-exposed embryos showed an increase in right and left ventricular wall area. Conclusions: Chronic inhibition of NOS produced, in the chick embryo, impairment of endothelium-dependent relaxation, structural remodeling of the pulmonary vascular bed and biventricular cardiac enlargement.

185 Mesenteric Artery Reactivity and Small Intestine Morphology in a Chicken Model of Hypoxia-Induced Fetal Growth Restriction

Background and aims Infants with intrauterine growth retardation are prone to intestinal dysfunction which is manifested by feeding intolerance and, in the most severe cases, necrotizing enterocolitis. The morphological and molecular mechanisms that lead to these complications are not completely understood and suitable experimental models are necessary. We aimed to characterize mesenteric artery (MA) reactivity, small intestine morphometry and intestinal expression of VEGF in a chicken model of hypoxia-induced fetal growth restriction. Methods Chicken embryos (15 and 19 days) and hatchlings (< 3-h-old and 1-d-old) were incubated under hypoxic (15% O2 from day 0 to day 19 of incubation) or normoxic conditions. Vascular reactivity was studied using wire miography. Intestinal morphometry was assessed in hematoxyline-eosine-stained sections. The expression of VEGF mRNA was determined by RT-PCR analysis. Results Hypoxia altered the responsiveness of chicken embryo MAs to acetylcholine, the NO donor sodium nitroprusside and the constrictor polypeptide ET-1. However, the majority of these alterations, with the exception of the hyperresponsiveness to ET-1, were not present in the hypoxic hatchlings. When intestinal histology was analyzed, subtle hypoxia-induced changes were noted in the muscularis propia and the villi from the hatchlings. Hypoxic incubation also diminished the expression of VEGF mRNA in the terminal ileum of the hatchlings. Conclusions Chronic moderate hypoxia during incubation results in subtle but significant alterations in chicken MA reactivity, small intestine morphology and VEGF expression. Whether these alterations may have a direct effect on the functional status of the intestine remains to be investigated.