1 Department of Anesthesiology and Cardio-thoracic Surgery, 2 Department of Perinatal Medicine, 3 EA1049, Department of Biophysics and 4 Departement Hospitalo-Universitaire de Recherche Experimentale, Centre Hospitalier et Universitaire, Lille, France
* Corresponding author. E-mail: lstorme{at}chru-lille.fr
Accepted for publication August 4, 2004.
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Abstract |
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Methods. Chronically instrumented fetal lambs were randomized into two groups: (i) a group with PH obtained by antenatal partial ligation of the ductus arteriosus (DA) (n=9) and (ii) a control group without DA ligation (n=6). Pulmonary vascular responses to norepinephrine (1.5 µg min1) were measured in utero 7 days after surgery. At day 8 post-surgery, after delivery, animals were ventilated for 3 h with oxygen 100%. The group with PH was randomly assigned to receive norepinephrine or saline.
Results. Mean pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR) were higher in the PH group (P<0.01). Norepinephrine-induced decrease in PVR was more pronounced in the PH group than in the control group (63 vs 35%, respectively; P<0.01). In the PH group, the decrease in PVR during mechanical ventilation was greater in the animals receiving norepinephrine than in the animal receiving saline (from 1.05 (0.12) to 0.1 (0.02) vs from 1.04 (0.1) to 0.2 (0.04) mm Hg ml1 min1, respectively; P<0.01). After 3 h of ventilation, mean PVR in the PH lambs treated by norepinephrine was similar to those measured in the control lambs. Aortic pressure was higher in the group treated with norepinephrine.
Conclusion. The data suggest that norepinephrine may improve post-natal pulmonary adaptation in the newborn with persistent PH both by increasing systemic vascular pressure and by increasing pulmonary blood flow.
Keywords: arteries, small pulmonary arteries ; complications, pulmonary hypertension ; pharmacology, nitric oxide ; sympathetic nervous system, norepinephrine
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Introduction |
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An experimental model of chronic intrauterine PH can be obtained by partial compression or ligation of the DA in the late gestation ovine fetus.4 5 In this model, chronic DA compression causes sustained intrauterine pulmonary hypertension (PH) without chronically increased pulmonary blood flow or hypoxaemia.4 5 Past studies have also demonstrated increased smooth muscle thickness in small pulmonary arteries,6 and blunted endothelium-dependent vasodilation after 912 days of DA compression.79
An increase in plasma norepinephrine concentration has been observed at the end of gestation. Birth induces a marked surge in catecholamine secretion. We have reported previously that norepinephrine infusion induces a potent nitric oxide (NO)-dependent pulmonary vasodilation in fetal lambs.10 11
Therefore, we hypothesize that norepinephrine may alter pulmonary vascular tone during fetal life and may improve circulatory adaptation at birth in a model of PPH. To test this hypothesis, we compared pulmonary vascular effects of norepinephrine in chronically prepared fetal lambs with and without PPH obtained by antenatal partial ligation of the DA for 8 days.
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Methods |
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At the end of the experiments, the animals were killed with i.v. pentobarbital (1 g) and a thoracotomy was rapidly performed to remove, in bloc, the heart and lungs.
Physiologic measurements
The flow transducer cable was connected to an internally calibrated flowmeter (T201, Transonic Systems, Ithaca, NY), for continuous measurements of LPA blood flow. Catheters were connected to an arterial pressure transducer (Merlin monitor, Hewlett-Packard, USA). Pressures were referenced to the amniotic cavity pressure. PVR in the left lung was calculated as the difference between mean pulmonary artery and left atrial pressures divided by mean left pulmonary blood flow. Blood samples from the main pulmonary artery catheter were used for blood gas analysis and oxygen saturation measurements.
Experimental design
Animals were assigned to: (i) a group with PH obtained by antenatal partial ligation of the DA (n=9) and (ii) a control group without DA ligation (n=6).
Four different experimental protocols were performed.
Protocol 1. Fetal haemodynamic effects of chronic partial ligation of the DA. To investigate the effects of DA ligation on the fetal pulmonary circulation, we studied haemodynamic parameters at days 2, 4, 7, and 8 after surgery in both control and PH groups. Haemodynamic measurements included mean pulmonary artery pressure (PAP), mean aortic pressure (AoP), LAP, amniotic pressure, LPA blood flow (Qp), and heart rate (HR). PVR in the left lung was calculated and daily blood gas analysis was performed. Data were compared between the two groups.
Protocol 2. Fetal pulmonary haemodynamic responses to norepinephrine after partial DA ligation. To determine whether chronic intrauterine PH alters the pulmonary vasodilator response to norepinephrine, we used an infusion protocol described previously.12 We studied the haemodynamic response to norepinephrine infusion 7 days after surgery in both control and PH groups. Duration of each experiment was at least 240 min and drugs were infused into the venous catheter. Saline (6 ml h1) was first infused for at least 30 min. After 30 min of stable baseline measurements, norepinephrine was infused at a rate of 1.5 µg min1 (=0.5 µg kg1 min1): (6 ml h1) for 120 min (from 30 to 150 min). The catheter was then flushed with saline (6 ml h1) for 30 min (from 150 to 180 min). Mean PAP, LAP, AoP, Qp, and HR were recorded at 5-min intervals and PVR in the left lung was calculated.
Protocol 3. Effects of norepinephrine on the postnatal circulatory adaptation at birth after partial ligation of the DA. To determine whether norepinephrine would increase pulmonary vasodilation at birth in lambs with chronic intrauterine PH, the group with antenatal partial DA ligation (n=9) was randomly assigned to receive norepinephrine (1.5 µg min1 started at the beginning of the ventilation) or saline. The norepinephrine infusion rate was chosen for consistency with earlier studies on norepinephrine effects in the late-gestation fetal lamb. In particular, we found that a rate of 0.5 µg kg1 min1 increases pulmonary blood flow in the ovine fetus.10 The control group received only saline. Norepinephrine was infused for 3 h until the end of the experiment. Mean PAP, LAP, AoP, Qp, and HR were recorded at 5-min intervals and PVR in the left lung was calculated.
Protocol 4. Effects of partial antenatal DA ligation on muscle of small pulmonary arteries, and on the development of right ventricular hypertrophy. To determine the degree of change in the muscle of small pulmonary arteries and right ventricular hypertrophy after 8 days of chronic antenatal PH, 5 µm contiguous sections of distal left lung were stained with haematoxylin and eosin. In each microscopic specimen, small pulmonary arteries land-marked by their association with small terminal bronchioles were measured. Two blinded observers performed morphometric analysis of at least 20 consecutive pulmonary arteries per animal. External diameter (ED) and wall thickness (WT) were measured and per cent wall thickness (2WTx100/ED) was calculated.
To assess development of right ventricular hypertrophy, the free wall of the right ventricle (RV) was removed from the fetal heart and weighed. Then, the left ventricle plus septum (LV+S) was weighed separately. The ratio (RV)/(LV+S) was calculated.
Data analysis
Results are presented as means (SEM) and the data were analysed using repeated-measures and factorial analysis of variance (ANOVA). Inter-group differences were analysed with the Fisher's, Scheffe's, and Bonferroni/Dunn's least significant test (Stat View for PC, Abacus Concepts, Berkeley, CA, USA). MannWhitney test (independent values) and paired Wilcoxon rank test (paired values) were used for the comparison of groups of lambs. A P<0.05 was considered as statistically significant.
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Results |
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Percentage change in mean PVR during norepinephrine infusion was higher in the DA ligation group than in the control group (65 vs 35%, respectively; P<0.05) (Fig. 3). In the two groups, mean left atrial pressure before infusion was 2 (1) mm Hg and did not change during the study period.
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Discussion |
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PPHN results from the failure of birth-related mechanisms that contribute to the normal decline in PVR at birth. PPHN may reflect a decreased production or responsiveness to vasodilator stimuli and/or increased production or responsiveness to vasoconstrictor stimuli.1 Structural change such as distal extension of smooth muscle and thickening of the media and adventitia in the pulmonary vessel walls have also been shown in PPHN. The relative role of abnormal pulmonary vascular reactivity and of pulmonary vascular remodelling in PPHN is unknown. Chronic partial compression of the DA in fetal lambs causes sustained intrauterine PH leading to structural changes in small pulmonary arteries and right ventricular hypertrophy, which show similarities to those described in human neonates.13 Vascular and cardiac remodelling has been observed as early as 5 days after DA ligation.4 5 Beside increased smooth muscle thickness in small pulmonary arteries,6 PH obtained by chronic DA compression,4 or ligation5 impairs pulmonary vasodilation to birth-related stimuli, including ventilation with oxygen 100%. Our results show that the norepinephrine-induced increase in pulmonary blood flow is preserved after 8 days of sustained PH despite attenuated responses to ventilation with oxygen 100% and despite vascular remodelling.
Previous experimental studies reported that right ventricular hypertension in the newborn may be associated with a significant decrease in AoP and cardiac output.2 3 Mechanisms may include leftward shift of the interventricular septum impairing the left ventricular filling, decreased left ventricular preload resulting from decreased pulmonary venous flow and/or decreased coronary artery perfusion resulting from increased right ventricular transmural pressure.14 Norepinephrine activates both (
1 and
2) and ß1-adrenoceptors. Alpha-adrenoceptors participate in sympathetically mediated vasoconstriction of human vessels.15 Activation of ß1-adrenoceptors may increase cardiac contractility and cardiac output.16 Both
1- and ß1-adrenoceptor activation may explain the increase in AoP observed in our study. In adult dogs with acute right ventricular hypertension, elevation of AoP reversed right ventricular failure by restoring right coronary flow.14 In newborn piglets with right ventricular hypertension, an elevation in systemic arterial pressure reduced right to left foramen ovale shunt and increased pulmonary blood flow and systemic oxygen delivery.17 Our results are consistent with these studies, suggesting that the norepinephrine-induced raise in AoP may be associated with increased pulmonary blood flow. A pulmonary vasodilator response to norepinephrine was observed after acute hypoxia in isolated perfused cat lung,18 and in isolated intra-pulmonary arteries of neonatal and adult pigs.19 20 Norepinephrine decreased pulmonary vascular tone in a canine model of pulmonary embolism with PH.21 Moreover, norepinephrine induces an NO-dependent pulmonary vasodilation in the ovine fetus.10 In in vitro studies, preconstriction of the pulmonary vessels is clearly a prerequisite for norepinephrine to induce pulmonary vessels relaxation.1821 We could speculate that, in vivo, the more elevated the basal pulmonary vascular tone, the greater would be the norepinephrine-induced pulmonary vasodilation. Our results support this hypothesis as the pulmonary vasodilator response to norepinephrine was enhanced in lambs with the highest basal PVR (DA ligation).
Further evidence demonstrates that norepinephrine induces pulmonary vasodilation through NO release in pulmonary arteries in experimental models from fetal to adult,10 19 20 and that NO synthase inhibition modulates the norepinephrine vascular response. Norepinephrine-induced NO release mechanisms are uncertain but may include activation of 2-adrenoceptors. As
1-adrenoceptor agonists raise pulmonary vascular tone,20 pulmonary vascular response to norepinephrine may result from the balance between activation of
1-adrenoceptor-induced vasoconstriction and
2-adrenoceptor-mediated NO release and vasodilation.11 22 Thus, the degree of vascular response to norepinephrine may depend on the ratio of
1- to
2-adrenoceptors at the surface of the endothelium or of the smooth muscle cells.23 Previous studies have demonstrated that norepinephrine-induced vasodilation is related, at least in part, to NO release.10 11 Moreover, NO release was decreased in the DA compression model as shown by alteration of endothelium dependent vasodilation.9 Collectively, these results suggest that mechanisms other than NO release might also contribute to the observed norepinephrine-induced vasodilation.
Surprisingly high were found in the newborn lambs despite sustained PH at 3 h of mechanical ventilation with oxygen 100%. Lower
was observed after 2 h of mechanical ventilation with oxygen 100% in fetal lambs whose DA was compressed for 912 days.4 Degree of vascular remodelling and altered reactivity depend on the duration the PH.4 Thus, it is likely that shorter duration of PH (8 vs 9 to 12 days) may explain this discrepancy. Whether or not norepinephrine-induced increase in pulmonary blood flow is impaired after more prolonged chronic antenatal PH is at present unclear.
In conclusion, norepinephrine-induced increase in pulmonary blood flow observed in the normal fetal lamb is preserved, before and at birth, in lambs with PH. It is possible that norepinephrine improves postnatal circulatory adaptation at birth in newborn infants with persistent PH both by increasing systemic pressure and by increasing pulmonary blood flow.
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Acknowledgments |
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References |
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