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Pulmonary vasodilator effects of norepinephrine during the development of chronic pulmonary hypertension in neonatal lambs
British Journal of Anaesthesia 93 (6): 818–24 (2004)
doi:10.1093/bja/aeh278
Advance Access publication October 1, 2004
Pulmonary vasodilator effects of norepinephrine during the development of chronic pulmonary hypertension in neonatal lambs S. Jaillard1
3 4
, F. Elbaz1, S. Bresson-Just2, Y. Riou2 3, V. Houfflin-Debarge2 3, T. Rakza2, B. Larrue1 and L. Storme2–4 *
1
*Corresponding author. E-mail: [email protected] Background. This experimental study was performed to determine the effects of norepinephrine on: (i) the pulmonary vascular tone during the development of pulmonary hypertension (PH) in the fetus and (ii) the circulatory adaptation at birth after chronic intrauterine PH. 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 mg 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. Br J Anaesth 2004; 93: 818–24 Keywords: arteries, small pulmonary arteries; complications, pulmonary hypertension; pharmacology, nitric oxide; sympathetic nervous system, norepinephrine Accepted for publication: August 4, 2004
Persistent pulmonary hypertension of the newborn (PPHN) results from the failure of the pulmonary circulation to dilate at birth. This syndrome is characterized by sustained elevation of pulmonary vascular resistance (PVR),1 causing extrapulmonary right-to-left shunting of blood across the ductus arteriosus (DA) and foramen ovale and severe hypoxaemia.1 PPHN is also usually associated with low systemic pressure and low cardiac output because of increased right ventricular afterload and myocardial dysfunction.2 3 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
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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 9–12 days of DA compression.7–9 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
The Board of Management and Trustees of the British Journal of Anaesthesia 2004
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Department of Anesthesiology and Cardio-thoracic Surgery, 2Department of Perinatal Medicine, 3 EA1049, Department of Biophysics and 4Departement Hospitalo-Universitaire de Recherche Experimentale, Centre Hospitalier et Universitaire, Lille, France
Vasodilator effects of norepinephrine in PH
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.
Methods Animal preparation
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
Fig 1 Fetal partial DA ligation. DA was partially ligated with a graduated umbilical tape (width: 0.5 cm): circumference of the DA was measured and reduced to one-third. MPA: main pulmonary artery, LPA: left pulmonary artery, Ao: aorta, Fm: flowmeter around the LPA (size 6, Transonic Systems, Ithaca, NY).
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
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All animal procedures and protocols used in this study were reviewed and approved by the French ‘Ministe`re de l’Agriculture, de la Peˆche et de l’Alimentation’ before the studies were conducted. Fifteen mixed-breed pregnant ewes between 130 and 133 days gestation (term=147 days) were fasted for 48 h before surgery. Ewes were sedated with i.v. pentobarbital sodium (total dose: 2–4 g) and anaesthetized with bupivacaine 1% hydrochloride (4 mg) by lumbar puncture. Under sterile conditions, the fetal lamb’s left forelimb was delivered through uterine incision. I.M. injection of 6 mg of SufentanilTM was performed. A skin incision was made after local infiltration with lidocaine. Polyvinyl catheters (20 gauge) were advanced into the aorta and the vena cava through the axillary vessels. A left thoracotomy exposed the heart and great vessels. Catheters were inserted into the left pulmonary artery (LPA) (22 gauge), main pulmonary artery (20 gauge), and left atrium (20 gauge). An ultrasonic flow transducer, size 6 (Transonic Systems, Ithaca, NY), was placed around the LPA to measure blood flow. In nine animals, the DA was partially ligated (Fig. 1). The fetus was gently replaced into the uterus.
Catheters were exteriorized through a s.c. tunnel to an external flank pouch. Estimated weight of the fetal lambs was 3000 g. Eight days after the initial surgery, ewes were sedated with i.v. pentobarbital sodium (total dose: 2–4 g) and anaesthetized with bupivacaine 1% hydrochloride (4 mg) by lumbar puncture. Then, the fetal lambs were carefully delivered through an abdominal incision and hysterotomy. After an injection followed by a continuous infusion of nalbuphine (0.6 mg then 0.15 mg h1) and an i.v. injection of pancuronium bromide (200 mg), the animals were dried and warmed (heating table) throughout the study. Temperature was continuously monitored (target temperature=39.5 C). Haemodynamic variables were measured continuously thereafter for the duration of the delivery study. The baseline measurements were recorded after haemodynamic parameters had stabilized for 30 min. Saline (6 ml h1) was infused via the venous catheter. A tracheostomy was then performed after local infiltration with lidocaine. The animals were intubated with a 3.5-mm tracheal tube and ventilated for 3 h with a time-cycled, pressure-limited neonatal ventilator (Babylog 8000, Draegger, Germany). Initial settings were: peak inspiratory pressure=32 cm H2O; positive endexpiratory pressure=5 cm H2O; ventilator rate=40 cycles min1; inspiratory time=0.6 s; and fraction of inspired oxygen concentration=1.00. After blood gas stabilization, the umbilical cord was ligated. Subsequent ventilator adjustments were made based on arterial blood gas values. Target blood gas parameters were pH=7.35–7.45 and Paco2 ¼ 35-45mmHg. Blood gas analyses were performed at 15 min intervals. 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.
Jaillard et al.
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.
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.
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 mg min1 (=0.5 mg 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.
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). Mann–Whitney 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.
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 mm 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 (2WT·100/ ED) was calculated.
Results Protocol 1. Fetal haemodynamic effects of chronic partial ligation of the DA. From day 2 to day 8 following DA ligation, mean PAP increased by 35% (from 52 (3) to 67 (4) mm Hg; P<0.05) and mean PVR increased by 25% (from 0.85 (0.06) to 1.1 (0.1) mm Hg ml1 min1; P<0.05). In this group, mean AoP increased from 41 (4) to 48 (9) mm Hg (P<0.05). Conversely, mean PAP and PVR did not change from day 2 to day 8 after the initial surgery in the control group. Mean PAP and PVR were higher and mean left pulmonary blood flow (Qp) was lower in the DA ligation group than in the control group (P<0.05) (Fig. 2). Mean left atrial pressure before infusion was 2 (1) mm Hg and did not change during the study period. HR did not change during the study. Protocol 2. Fetal pulmonary hemodynamic responses to norepinephrine after partial ligation of the DA. In the two groups, norepinephrine infusion induced a similar increase in AoP (by 12% for the DA ligated vs 13% in controls). In the DA ligation group, norepinephrine infusion increased PAP by 10% (P<0.05), and progressively increased Qp by 220% after 80 min of drug infusion (P<0.05). Mean PVR progressively decreased by 65% after 80 min of drug infusion (P<0.05). In the control group, norepinephrine infusion increased PAP by 15% after 20 min of drug infusion (P<0.05), and progressively increased Qp by 60% after 60 min of drug infusion (P<0.05). Mean PVR progressively decreased by 35% after 40 min of drug infusion (P<0.01). 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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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 mg 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 mg 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.
Data analysis
Vasodilator effects of norepinephrine in PH
80
80
75 AoP (mm Hg)
AoP (mm Hg)
70 DA ligation (n = 9)
65
*
Control (n = 6)
60 55 50 45
DA ligation (n = 9) Control (n = 6)
70 60 50 40
40 35
80 PAP (mm Hg)
80 75 70 65 60
*
55 50 45 40 35 30
Flow (ml min–1)
40 300
110
250 200 150 100 50
Norepinephrine 1.5 µg min–1
100 90
PVR (mm Hg ml–1 min–1)
Flow (ml min–1)
60 50
120
80 70 60 50 40 Day2
Day4
Day7
1.2 PVR (mm Hg ml–1 min–1)
70
Day8
0 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30
*
0.20 0
1
20
40
60
80 100 Time (min)
120
140
160
0.8 Fig 3 Pulmonary haemodynamic response to norepinephrine 1.5 mg min1 (=0.5 mg kg1 min1) 7 days after DA ligation. Mean PVR decreased by 30% in control lambs n=6 and by 63% in pulmonary hypertensive lambs n=8. Vasodilation was higher in hypertensive lambs, with decrease of the PVR to control levels. PAP: pulmonary artery pressure. Values are expressed as mean (SEM).
0.6 0.4 0.2 Day 2
Day 4
Day 7
Day 8
Fig 2 Fetal haemodynamic effects of chronic partial compression of the DA. *P<0.05 at 2 vs 8 days after DA ligation. AoP was similar in the two groups during the study period. Mean PAP and mean PVR were higher and mean left pulmonary flow was lower in the pulmonary hypertensive lambs. Values are expressed as mean (SEM).
Protocol 3. Effects of norepinephrine on the postnatal circulatory adaptation at birth after partial ligation of the DA. Baseline blood gas parameters (after birth and before mechanical ventilation) were similar in both groups
(Table 1). PAP and PVR rapidly decreased and AoP and Qp increased in both groups after starting mechanical ventilation (P<0.05) (Fig. 4). After an immediate and similar increase of AoP in the two groups, the increase in AoP was higher in the hypertensive group treated by norepinephrine than in the hypertensive group infused with saline (by 40% in the norepinephrine group vs 10% in the saline group 2 h after the beginning of the ventilation (P<0.001)). Within the DA ligation group, the decrease in PAP and PVR and increase in Qp during mechanical ventilation
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PAP (mm Hg)
30
Jaillard et al.
Table 1 Blood gas parameters, mean plasma lactate concentration, mean AoP, mean PAP, mean left pulmonary blood flow (Qp), mean PVR, at baseline (before starting mechanical ventilation with oxygen 100%), in control group (without DA ligation), and in group with DA ligation and after 3 h of ventilation * P<0.05 compared to control group Parameters
Baseline Control group n=6
7.3 (0.03) 19 (2) 41 (2) 23 (1) 2 (1) 1.6 (0.9) 47 (4) 49 (4) 108 (15) 0.44 (0.06)
Group with DA ligation n=9 Saline n=5
Norepinephrine n=4
7.39 (0.02) 21 (2) 46 (5) 25 (2) 0 (2) 3.2 (1.6) 51 (5) 74 (6)* 71 (6)* 1.12 (0.16)*
7.36 (0.01) 17 (4) 53 (12) 28 (9) 2 (9) 2.2 (0.5) 46 (3) 71 (3)* 69 (7)* 1.06 (0.06)*
were higher in the sub-group treated by norepinephrine than in the sub-group infused with saline, despite similar baseline blood gas and haemodynamic parameters (P<0.05) (Table 1 and Fig. 4). Mean PVR in both the control group and the sub-group with DA ligation treated with norepinephrine was lower than mean PVR in the subgroup with DA ligation infused with saline after 3 h of mechanical ventilation (P<0.05) (Fig. 5). HRs were similar in both groups during the study. Protocol 4. Effects of partial antenatal DA ligation on muscle of small pulmonary arteries, and on the development of right ventricular hypertrophy. Lamb body weights were comparable between the two groups (3700 (400) vs 3600 (640) g). Macroscopic examination of the DA at the time of death revealed a narrow (n=4) or no (n=5) residual vascular lumen. Histologic examination and morphometric analysis revealed increased muscularization of small pulmonary arteries associated with terminal bronchioles in the DA ligation group when compared with the control group. Wall thickness was 30% greater in the DA ligation group than in the control group (17 (1) vs 12 (2)%, respectively; P<0.05). The ratio of right ventricular weight to left ventricular +septum weight was higher in the DA ligation group than in the control group (0.75 (0.05) vs 0.50 (0.04), respectively; P<0.05).
Discussion This study provides information regarding the potential beneficial haemodynamic effects of norepinephrine in PPHN. This syndrome is usually associated with low systemic pressure and low cardiac output because of increased right ventricular afterload and myocardial dysfunction.2 3 Cardiac failure further impairs tissue oxygen delivery. With inhaled NO—a selective pulmonary vasodilator—catecholamines, especially dopamine or dobutamine, are largely used in PPHN. However, evidence exists that dopamine may elevate pulmonary arterial pressure or PVR in the perinatal period.12
Control group n=6
7.38 (0.03) 468 (86) 41 (4) 23 (1) 2 (2) 2.5 (0.6) 61 (6) 36 (4) 325 (64) 0.12 (0.02)
Group with DA ligation n=9 Saline n=5
Norepinephrine n=4
7.43 (0.08) 415 (45) 39 (3) 29 (9) 2 (5) 2.7 (0.4) 64 (6) 55 (7)* 250 (85) 0.24 (0.08)*
7.43 (0.04) 390 (120) 36 (2) 22 (3) 2 (3.5) 2.5 (0.3) 75 (4)* 41 (4) 370 (16) 0.12 (0.03)
Furthermore, dobutamine has no or limited effects on systemic pressure although it increases cardiac output.12 Although norepinephrine induces a potent pulmonary vasodilation in the normal fetus,10 the effects of norepinephrine on the pulmonary circulatory adjustments at birth especially in PPHN remained to be studied. Our data suggest that norepinephrine may have two potentially beneficial effects in PPHN: (i) it increases AoP and (ii) it increases pulmonary blood flow. 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
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PH PO2, mm Hg PCO2, mm Hg HCO3, mEq litre1 Excess base Lactate, mmol litre1 AoP, mm Hg PAP, mm Hg Qp, ml min1 PVR, mm Hg ml1 min1
3 h after starting ventilation
Vasodilator effects of norepinephrine in PH
*
80
*
70
50 40 30 20 80 70
* 0.6 * 0.4
50 0 Baseline
30 20
2h3clampage After h of mechanical ventilation
Fig 5 Effects of norepinephrine infusion and chronic intrauterine hypertension on the changes of PVR after Caesarean section. After birth, the decrease of PVR was similar in control lambs to PH lambs treated with norepinephrine. Values are expressed as mean (SEM). *P<0.05.
500 Flow (ml min–1)
Control n=6
0.8
0.2
60 40
400 300 200 100 0 1.40 0
PVR (mm Hg ml–1min–1)
1
20 40 60 80 100 120 140 160 180 200 220 240
1.20 1.00 DA ligation + saline (n=5)
0.80
DA ligation + NE (n=4)
0.60 0.40 0.20 0.00 0
20 40 60
80 100 120 140 160 180 200 200 240
Mechanical ventilation Time (min) Fig 4 Effect of norepinephrine infusion 0.5 mg kg1 min1 on postnatal adaptation after chronic ductal ligation. Increase of the AoP was higher in the norepinephrine-treated group. Decrease of PAP and PVR and increase of left pulmonary artery blood flow were higher in the norepinephrinetreated lambs. Values are expressed as mean (SEM).
venous flow and/or decreased coronary artery perfusion resulting from increased right ventricular transmural pressure.14 Norepinephrine activates both a (a1 and a2) and b1-adrenoceptors. Alpha-adrenoceptors participate in sympathetically mediated vasoconstriction of human vessels.15 Activation of b1-adrenoceptors may increase cardiac contractility and cardiac output.16 Both a1- and b1-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.18–21 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. Norepinephrineinduced NO release mechanisms are uncertain but may include activation of a2-adrenoceptors. As a1-adrenoceptor agonists raise pulmonary vascular tone,20 pulmonary vascular response to norepinephrine may result from the balance between activation of a1-adrenoceptor-induced
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PAP (mm Hg)
90
DA ligation + saline n=5 DA ligation + norepinephrine n=4
1.2
60 PVR (mm Hg ml–1min–1)
PAo (mm Hg)
90
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Acknowledgements
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We thank Professor Steven H. Abman for help in developing the experimental ovine model. This work was supported by the cardiovascular committee of the ‘Fondation de France’, and by the ‘Centre Hospitalier Regional Universitaire’-CHRU-de Lille.
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References 1 Levin DL, Heyman MA, Kitterman JA, Gregory GA, Phibbs RH, Rudolph AM. Persistent pulmonary hypertension of the newborn. J Pediatr 1976; 89: 626–30 2 Belik J, Light RB. Effect of increased afterload on right ventricular function in the newborn pig. J Appl Physiol 1989; 66: 868–9 3 Belik J, Baron K, Light RB. Central hemodynamic and regional blood flow changes in the newborn with right ventricular hypertension. Pediatr Res 1989; 26: 548–53 4 Abman SH, Shanley PF, Accurso FJ. Failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs. J Clin Invest 1989; 83: 1849–59 5 Morin FC. Ligating the DA before birth causes persistent pulmonary hypertension in the newborn lamb. Pediatr Res 1989; 25: 245–50 6 Levin DL, Hyman AI, Heymann MA, Rudolph AM. Fetal hypertension and the development of increased pulmonary vascular
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smooth muscle: a possible mechanism for persistent pulmonary hypertension of the newborn infant. J Pediatr 1978; 92: 265–9 Villamor E, Le Cras TD, Horan M, Halbower AC, Tuder RM, Abman SH. Chronic hypertension impairs endothelial NO synthase in the ovine fetus. Am J Physiol 1997; 16: L1013–20 Shaul PW, Yuhanna IS, German Z, Chen Z, Steinhorn RN, Morin FC. Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension. Am J Physiol 1997; 272: L1005–12 McQueston JA, Kinsella JP, Ivy DD, McMurtry IF, Abman SH. Chronic pulmonary hypertension in utero impairs endotheliumdependent vasodilation. Am J Physiol 1995; 268: H288–94 Jaillard S, Houfflin-Debarge V, Riou Y, et al. Effects of catecholamines on the pulmonary circulation in the ovine fetus. Am J Physiol 2001; 281: R607–14 Magnenant E, Jaillard S, Deruelle P, et al. Mechanisms of the norepinephrine-induced pulmonary vasodilation in the ovine fetus. Pediatr Res 2003; 54: 44–51 Cheung PY, Barrington K, Bigam D. The hemodynamic effects of dobutamine infusion in the chronically instrumented newborn piglet. Crit Care Med 1999; 27: 558–64 Murphy JD, Rabinovitch M, Goldstein JD, Reid LM. The structural basis of persistent pulmonary hypertension. J Pediatr 1981; 98: 962–7 Vlahakes GJ, Turley K, Hoffman JIE. The pathophysiology of failure in acute right ventricular hypertension: hemodynamic and biochemical correlation. Circulation 1981; 63: 87–95 Parkinson NA, Thom SM, Hughes AD, Sever PS, Mulvany MJ, Nielsen H. Neurally evoked responses of human isolated resistance arteries are mediated by both alpha 1- and alpha 2-adrenoceptors. Br J Pharmacol 1992; 106: 568–73 O’Laughlin MP, Fisher DJ, Dreyer WJ, Smith EO. Augmentation of cardiac output with intravenous catecholamines in anaesthetized hypoxemic newborn lambs. Pediatr Res 1987; 22: 667–74 Belik J, Baron K, Light RB. The effect of an increase in systemic arterial pressure in the newborn with right ventricular hypertension. Pediatr Res 1990; 28: 603–8 Cutaia M, Friedrich P. Hypoxia-induced alterations of norepinephrine vascular reactivity in isolated perfused cat lung. J Appl Physiol 1987; 63: 982–7 Tulloh RM, Dyamenahalli U, Stuart SK, Haworth SG. Adrenoceptor-stimulated endothelium-dependent relaxation in porcine intrapulmonary arteries. Pulm Pharmacol 1994; 7: 299–303 Wilson LE, Levy M, Stuart-Smith K, Haworth SG. Postnatal adrenoreceptor maturation in porcine intrapulmonary arteries. Pediatr Res 1993; 34: 591–5 Hirsch LJ, Rooney MW, Wat SS, Kleinmann B, Mathru M. Norepinephrine and phenylephrine effects on right ventricular function in experimental canine pulmonary embolism. Chest 1991; 100: 796–801 MacLean MR, McCulloch KM, McGrath JC. Influences of the endothelium and hypoxia on a1 and a2-adrenoceptor-mediated responses in the rabbit isolated pulmonary artery. Br J Pharmacol 1993; 108: 155–61 Wright IK, Blaylock NA, Kendall DA, Wilson VG. The relationship between density of a adrenoceptor binding sites and contractile responses in several porcine isolated blood vessels. Br J Pharmacol 1995; 109: 678–88
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vasoconstriction and a2-adrenoceptor-mediated NO release and vasodilation.11 22 Thus, the degree of vascular response to norepinephrine may depend on the ratio of a1- to a2-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 norepinephrineinduced vasodilation. Surprisingly high Pao2 were found in the newborn lambs despite sustained PH at 3 h of mechanical ventilation with oxygen 100%. Lower Pao2 was observed after 2 h of mechanical ventilation with oxygen 100% in fetal lambs whose DA was compressed for 9–12 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.
doi:10.1093/bja/aeh278
Advance Access publication October 1, 2004
Pulmonary vasodilator effects of norepinephrine during the development of chronic pulmonary hypertension in neonatal lambs S. Jaillard1
3 4
, F. Elbaz1, S. Bresson-Just2, Y. Riou2 3, V. Houfflin-Debarge2 3, T. Rakza2, B. Larrue1 and L. Storme2–4 *
1
*Corresponding author. E-mail: [email protected] Background. This experimental study was performed to determine the effects of norepinephrine on: (i) the pulmonary vascular tone during the development of pulmonary hypertension (PH) in the fetus and (ii) the circulatory adaptation at birth after chronic intrauterine PH. 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 mg 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. Br J Anaesth 2004; 93: 818–24 Keywords: arteries, small pulmonary arteries; complications, pulmonary hypertension; pharmacology, nitric oxide; sympathetic nervous system, norepinephrine Accepted for publication: August 4, 2004
Persistent pulmonary hypertension of the newborn (PPHN) results from the failure of the pulmonary circulation to dilate at birth. This syndrome is characterized by sustained elevation of pulmonary vascular resistance (PVR),1 causing extrapulmonary right-to-left shunting of blood across the ductus arteriosus (DA) and foramen ovale and severe hypoxaemia.1 PPHN is also usually associated with low systemic pressure and low cardiac output because of increased right ventricular afterload and myocardial dysfunction.2 3 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
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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 9–12 days of DA compression.7–9 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
The Board of Management and Trustees of the British Journal of Anaesthesia 2004
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Department of Anesthesiology and Cardio-thoracic Surgery, 2Department of Perinatal Medicine, 3 EA1049, Department of Biophysics and 4Departement Hospitalo-Universitaire de Recherche Experimentale, Centre Hospitalier et Universitaire, Lille, France
Vasodilator effects of norepinephrine in PH
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.
Methods Animal preparation
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
Fig 1 Fetal partial DA ligation. DA was partially ligated with a graduated umbilical tape (width: 0.5 cm): circumference of the DA was measured and reduced to one-third. MPA: main pulmonary artery, LPA: left pulmonary artery, Ao: aorta, Fm: flowmeter around the LPA (size 6, Transonic Systems, Ithaca, NY).
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
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All animal procedures and protocols used in this study were reviewed and approved by the French ‘Ministe`re de l’Agriculture, de la Peˆche et de l’Alimentation’ before the studies were conducted. Fifteen mixed-breed pregnant ewes between 130 and 133 days gestation (term=147 days) were fasted for 48 h before surgery. Ewes were sedated with i.v. pentobarbital sodium (total dose: 2–4 g) and anaesthetized with bupivacaine 1% hydrochloride (4 mg) by lumbar puncture. Under sterile conditions, the fetal lamb’s left forelimb was delivered through uterine incision. I.M. injection of 6 mg of SufentanilTM was performed. A skin incision was made after local infiltration with lidocaine. Polyvinyl catheters (20 gauge) were advanced into the aorta and the vena cava through the axillary vessels. A left thoracotomy exposed the heart and great vessels. Catheters were inserted into the left pulmonary artery (LPA) (22 gauge), main pulmonary artery (20 gauge), and left atrium (20 gauge). An ultrasonic flow transducer, size 6 (Transonic Systems, Ithaca, NY), was placed around the LPA to measure blood flow. In nine animals, the DA was partially ligated (Fig. 1). The fetus was gently replaced into the uterus.
Catheters were exteriorized through a s.c. tunnel to an external flank pouch. Estimated weight of the fetal lambs was 3000 g. Eight days after the initial surgery, ewes were sedated with i.v. pentobarbital sodium (total dose: 2–4 g) and anaesthetized with bupivacaine 1% hydrochloride (4 mg) by lumbar puncture. Then, the fetal lambs were carefully delivered through an abdominal incision and hysterotomy. After an injection followed by a continuous infusion of nalbuphine (0.6 mg then 0.15 mg h1) and an i.v. injection of pancuronium bromide (200 mg), the animals were dried and warmed (heating table) throughout the study. Temperature was continuously monitored (target temperature=39.5 C). Haemodynamic variables were measured continuously thereafter for the duration of the delivery study. The baseline measurements were recorded after haemodynamic parameters had stabilized for 30 min. Saline (6 ml h1) was infused via the venous catheter. A tracheostomy was then performed after local infiltration with lidocaine. The animals were intubated with a 3.5-mm tracheal tube and ventilated for 3 h with a time-cycled, pressure-limited neonatal ventilator (Babylog 8000, Draegger, Germany). Initial settings were: peak inspiratory pressure=32 cm H2O; positive endexpiratory pressure=5 cm H2O; ventilator rate=40 cycles min1; inspiratory time=0.6 s; and fraction of inspired oxygen concentration=1.00. After blood gas stabilization, the umbilical cord was ligated. Subsequent ventilator adjustments were made based on arterial blood gas values. Target blood gas parameters were pH=7.35–7.45 and Paco2 ¼ 35-45mmHg. Blood gas analyses were performed at 15 min intervals. 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.
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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.
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.
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 mg min1 (=0.5 mg 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.
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). Mann–Whitney 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.
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 mm 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 (2WT·100/ ED) was calculated.
Results Protocol 1. Fetal haemodynamic effects of chronic partial ligation of the DA. From day 2 to day 8 following DA ligation, mean PAP increased by 35% (from 52 (3) to 67 (4) mm Hg; P<0.05) and mean PVR increased by 25% (from 0.85 (0.06) to 1.1 (0.1) mm Hg ml1 min1; P<0.05). In this group, mean AoP increased from 41 (4) to 48 (9) mm Hg (P<0.05). Conversely, mean PAP and PVR did not change from day 2 to day 8 after the initial surgery in the control group. Mean PAP and PVR were higher and mean left pulmonary blood flow (Qp) was lower in the DA ligation group than in the control group (P<0.05) (Fig. 2). Mean left atrial pressure before infusion was 2 (1) mm Hg and did not change during the study period. HR did not change during the study. Protocol 2. Fetal pulmonary hemodynamic responses to norepinephrine after partial ligation of the DA. In the two groups, norepinephrine infusion induced a similar increase in AoP (by 12% for the DA ligated vs 13% in controls). In the DA ligation group, norepinephrine infusion increased PAP by 10% (P<0.05), and progressively increased Qp by 220% after 80 min of drug infusion (P<0.05). Mean PVR progressively decreased by 65% after 80 min of drug infusion (P<0.05). In the control group, norepinephrine infusion increased PAP by 15% after 20 min of drug infusion (P<0.05), and progressively increased Qp by 60% after 60 min of drug infusion (P<0.05). Mean PVR progressively decreased by 35% after 40 min of drug infusion (P<0.01). 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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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 mg 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 mg 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.
Data analysis
Vasodilator effects of norepinephrine in PH
80
80
75 AoP (mm Hg)
AoP (mm Hg)
70 DA ligation (n = 9)
65
*
Control (n = 6)
60 55 50 45
DA ligation (n = 9) Control (n = 6)
70 60 50 40
40 35
80 PAP (mm Hg)
80 75 70 65 60
*
55 50 45 40 35 30
Flow (ml min–1)
40 300
110
250 200 150 100 50
Norepinephrine 1.5 µg min–1
100 90
PVR (mm Hg ml–1 min–1)
Flow (ml min–1)
60 50
120
80 70 60 50 40 Day2
Day4
Day7
1.2 PVR (mm Hg ml–1 min–1)
70
Day8
0 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30
*
0.20 0
1
20
40
60
80 100 Time (min)
120
140
160
0.8 Fig 3 Pulmonary haemodynamic response to norepinephrine 1.5 mg min1 (=0.5 mg kg1 min1) 7 days after DA ligation. Mean PVR decreased by 30% in control lambs n=6 and by 63% in pulmonary hypertensive lambs n=8. Vasodilation was higher in hypertensive lambs, with decrease of the PVR to control levels. PAP: pulmonary artery pressure. Values are expressed as mean (SEM).
0.6 0.4 0.2 Day 2
Day 4
Day 7
Day 8
Fig 2 Fetal haemodynamic effects of chronic partial compression of the DA. *P<0.05 at 2 vs 8 days after DA ligation. AoP was similar in the two groups during the study period. Mean PAP and mean PVR were higher and mean left pulmonary flow was lower in the pulmonary hypertensive lambs. Values are expressed as mean (SEM).
Protocol 3. Effects of norepinephrine on the postnatal circulatory adaptation at birth after partial ligation of the DA. Baseline blood gas parameters (after birth and before mechanical ventilation) were similar in both groups
(Table 1). PAP and PVR rapidly decreased and AoP and Qp increased in both groups after starting mechanical ventilation (P<0.05) (Fig. 4). After an immediate and similar increase of AoP in the two groups, the increase in AoP was higher in the hypertensive group treated by norepinephrine than in the hypertensive group infused with saline (by 40% in the norepinephrine group vs 10% in the saline group 2 h after the beginning of the ventilation (P<0.001)). Within the DA ligation group, the decrease in PAP and PVR and increase in Qp during mechanical ventilation
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PAP (mm Hg)
30
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Table 1 Blood gas parameters, mean plasma lactate concentration, mean AoP, mean PAP, mean left pulmonary blood flow (Qp), mean PVR, at baseline (before starting mechanical ventilation with oxygen 100%), in control group (without DA ligation), and in group with DA ligation and after 3 h of ventilation * P<0.05 compared to control group Parameters
Baseline Control group n=6
7.3 (0.03) 19 (2) 41 (2) 23 (1) 2 (1) 1.6 (0.9) 47 (4) 49 (4) 108 (15) 0.44 (0.06)
Group with DA ligation n=9 Saline n=5
Norepinephrine n=4
7.39 (0.02) 21 (2) 46 (5) 25 (2) 0 (2) 3.2 (1.6) 51 (5) 74 (6)* 71 (6)* 1.12 (0.16)*
7.36 (0.01) 17 (4) 53 (12) 28 (9) 2 (9) 2.2 (0.5) 46 (3) 71 (3)* 69 (7)* 1.06 (0.06)*
were higher in the sub-group treated by norepinephrine than in the sub-group infused with saline, despite similar baseline blood gas and haemodynamic parameters (P<0.05) (Table 1 and Fig. 4). Mean PVR in both the control group and the sub-group with DA ligation treated with norepinephrine was lower than mean PVR in the subgroup with DA ligation infused with saline after 3 h of mechanical ventilation (P<0.05) (Fig. 5). HRs were similar in both groups during the study. Protocol 4. Effects of partial antenatal DA ligation on muscle of small pulmonary arteries, and on the development of right ventricular hypertrophy. Lamb body weights were comparable between the two groups (3700 (400) vs 3600 (640) g). Macroscopic examination of the DA at the time of death revealed a narrow (n=4) or no (n=5) residual vascular lumen. Histologic examination and morphometric analysis revealed increased muscularization of small pulmonary arteries associated with terminal bronchioles in the DA ligation group when compared with the control group. Wall thickness was 30% greater in the DA ligation group than in the control group (17 (1) vs 12 (2)%, respectively; P<0.05). The ratio of right ventricular weight to left ventricular +septum weight was higher in the DA ligation group than in the control group (0.75 (0.05) vs 0.50 (0.04), respectively; P<0.05).
Discussion This study provides information regarding the potential beneficial haemodynamic effects of norepinephrine in PPHN. This syndrome is usually associated with low systemic pressure and low cardiac output because of increased right ventricular afterload and myocardial dysfunction.2 3 Cardiac failure further impairs tissue oxygen delivery. With inhaled NO—a selective pulmonary vasodilator—catecholamines, especially dopamine or dobutamine, are largely used in PPHN. However, evidence exists that dopamine may elevate pulmonary arterial pressure or PVR in the perinatal period.12
Control group n=6
7.38 (0.03) 468 (86) 41 (4) 23 (1) 2 (2) 2.5 (0.6) 61 (6) 36 (4) 325 (64) 0.12 (0.02)
Group with DA ligation n=9 Saline n=5
Norepinephrine n=4
7.43 (0.08) 415 (45) 39 (3) 29 (9) 2 (5) 2.7 (0.4) 64 (6) 55 (7)* 250 (85) 0.24 (0.08)*
7.43 (0.04) 390 (120) 36 (2) 22 (3) 2 (3.5) 2.5 (0.3) 75 (4)* 41 (4) 370 (16) 0.12 (0.03)
Furthermore, dobutamine has no or limited effects on systemic pressure although it increases cardiac output.12 Although norepinephrine induces a potent pulmonary vasodilation in the normal fetus,10 the effects of norepinephrine on the pulmonary circulatory adjustments at birth especially in PPHN remained to be studied. Our data suggest that norepinephrine may have two potentially beneficial effects in PPHN: (i) it increases AoP and (ii) it increases pulmonary blood flow. 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
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PH PO2, mm Hg PCO2, mm Hg HCO3, mEq litre1 Excess base Lactate, mmol litre1 AoP, mm Hg PAP, mm Hg Qp, ml min1 PVR, mm Hg ml1 min1
3 h after starting ventilation
Vasodilator effects of norepinephrine in PH
*
80
*
70
50 40 30 20 80 70
* 0.6 * 0.4
50 0 Baseline
30 20
2h3clampage After h of mechanical ventilation
Fig 5 Effects of norepinephrine infusion and chronic intrauterine hypertension on the changes of PVR after Caesarean section. After birth, the decrease of PVR was similar in control lambs to PH lambs treated with norepinephrine. Values are expressed as mean (SEM). *P<0.05.
500 Flow (ml min–1)
Control n=6
0.8
0.2
60 40
400 300 200 100 0 1.40 0
PVR (mm Hg ml–1min–1)
1
20 40 60 80 100 120 140 160 180 200 220 240
1.20 1.00 DA ligation + saline (n=5)
0.80
DA ligation + NE (n=4)
0.60 0.40 0.20 0.00 0
20 40 60
80 100 120 140 160 180 200 200 240
Mechanical ventilation Time (min) Fig 4 Effect of norepinephrine infusion 0.5 mg kg1 min1 on postnatal adaptation after chronic ductal ligation. Increase of the AoP was higher in the norepinephrine-treated group. Decrease of PAP and PVR and increase of left pulmonary artery blood flow were higher in the norepinephrinetreated lambs. Values are expressed as mean (SEM).
venous flow and/or decreased coronary artery perfusion resulting from increased right ventricular transmural pressure.14 Norepinephrine activates both a (a1 and a2) and b1-adrenoceptors. Alpha-adrenoceptors participate in sympathetically mediated vasoconstriction of human vessels.15 Activation of b1-adrenoceptors may increase cardiac contractility and cardiac output.16 Both a1- and b1-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.18–21 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. Norepinephrineinduced NO release mechanisms are uncertain but may include activation of a2-adrenoceptors. As a1-adrenoceptor agonists raise pulmonary vascular tone,20 pulmonary vascular response to norepinephrine may result from the balance between activation of a1-adrenoceptor-induced
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PAP (mm Hg)
90
DA ligation + saline n=5 DA ligation + norepinephrine n=4
1.2
60 PVR (mm Hg ml–1min–1)
PAo (mm Hg)
90
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Acknowledgements
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We thank Professor Steven H. Abman for help in developing the experimental ovine model. This work was supported by the cardiovascular committee of the ‘Fondation de France’, and by the ‘Centre Hospitalier Regional Universitaire’-CHRU-de Lille.
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References 1 Levin DL, Heyman MA, Kitterman JA, Gregory GA, Phibbs RH, Rudolph AM. Persistent pulmonary hypertension of the newborn. J Pediatr 1976; 89: 626–30 2 Belik J, Light RB. Effect of increased afterload on right ventricular function in the newborn pig. J Appl Physiol 1989; 66: 868–9 3 Belik J, Baron K, Light RB. Central hemodynamic and regional blood flow changes in the newborn with right ventricular hypertension. Pediatr Res 1989; 26: 548–53 4 Abman SH, Shanley PF, Accurso FJ. Failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs. J Clin Invest 1989; 83: 1849–59 5 Morin FC. Ligating the DA before birth causes persistent pulmonary hypertension in the newborn lamb. Pediatr Res 1989; 25: 245–50 6 Levin DL, Hyman AI, Heymann MA, Rudolph AM. Fetal hypertension and the development of increased pulmonary vascular
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smooth muscle: a possible mechanism for persistent pulmonary hypertension of the newborn infant. J Pediatr 1978; 92: 265–9 Villamor E, Le Cras TD, Horan M, Halbower AC, Tuder RM, Abman SH. Chronic hypertension impairs endothelial NO synthase in the ovine fetus. Am J Physiol 1997; 16: L1013–20 Shaul PW, Yuhanna IS, German Z, Chen Z, Steinhorn RN, Morin FC. Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension. Am J Physiol 1997; 272: L1005–12 McQueston JA, Kinsella JP, Ivy DD, McMurtry IF, Abman SH. Chronic pulmonary hypertension in utero impairs endotheliumdependent vasodilation. Am J Physiol 1995; 268: H288–94 Jaillard S, Houfflin-Debarge V, Riou Y, et al. Effects of catecholamines on the pulmonary circulation in the ovine fetus. Am J Physiol 2001; 281: R607–14 Magnenant E, Jaillard S, Deruelle P, et al. Mechanisms of the norepinephrine-induced pulmonary vasodilation in the ovine fetus. Pediatr Res 2003; 54: 44–51 Cheung PY, Barrington K, Bigam D. The hemodynamic effects of dobutamine infusion in the chronically instrumented newborn piglet. Crit Care Med 1999; 27: 558–64 Murphy JD, Rabinovitch M, Goldstein JD, Reid LM. The structural basis of persistent pulmonary hypertension. J Pediatr 1981; 98: 962–7 Vlahakes GJ, Turley K, Hoffman JIE. The pathophysiology of failure in acute right ventricular hypertension: hemodynamic and biochemical correlation. Circulation 1981; 63: 87–95 Parkinson NA, Thom SM, Hughes AD, Sever PS, Mulvany MJ, Nielsen H. Neurally evoked responses of human isolated resistance arteries are mediated by both alpha 1- and alpha 2-adrenoceptors. Br J Pharmacol 1992; 106: 568–73 O’Laughlin MP, Fisher DJ, Dreyer WJ, Smith EO. Augmentation of cardiac output with intravenous catecholamines in anaesthetized hypoxemic newborn lambs. Pediatr Res 1987; 22: 667–74 Belik J, Baron K, Light RB. The effect of an increase in systemic arterial pressure in the newborn with right ventricular hypertension. Pediatr Res 1990; 28: 603–8 Cutaia M, Friedrich P. Hypoxia-induced alterations of norepinephrine vascular reactivity in isolated perfused cat lung. J Appl Physiol 1987; 63: 982–7 Tulloh RM, Dyamenahalli U, Stuart SK, Haworth SG. Adrenoceptor-stimulated endothelium-dependent relaxation in porcine intrapulmonary arteries. Pulm Pharmacol 1994; 7: 299–303 Wilson LE, Levy M, Stuart-Smith K, Haworth SG. Postnatal adrenoreceptor maturation in porcine intrapulmonary arteries. Pediatr Res 1993; 34: 591–5 Hirsch LJ, Rooney MW, Wat SS, Kleinmann B, Mathru M. Norepinephrine and phenylephrine effects on right ventricular function in experimental canine pulmonary embolism. Chest 1991; 100: 796–801 MacLean MR, McCulloch KM, McGrath JC. Influences of the endothelium and hypoxia on a1 and a2-adrenoceptor-mediated responses in the rabbit isolated pulmonary artery. Br J Pharmacol 1993; 108: 155–61 Wright IK, Blaylock NA, Kendall DA, Wilson VG. The relationship between density of a adrenoceptor binding sites and contractile responses in several porcine isolated blood vessels. Br J Pharmacol 1995; 109: 678–88
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vasoconstriction and a2-adrenoceptor-mediated NO release and vasodilation.11 22 Thus, the degree of vascular response to norepinephrine may depend on the ratio of a1- to a2-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 norepinephrineinduced vasodilation. Surprisingly high Pao2 were found in the newborn lambs despite sustained PH at 3 h of mechanical ventilation with oxygen 100%. Lower Pao2 was observed after 2 h of mechanical ventilation with oxygen 100% in fetal lambs whose DA was compressed for 9–12 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.