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Eur J Cardiothorac Surg 2000;18:334-341
© 2000 Elsevier Science NL

Effects of inhaled nitric oxide on gas exchange and acute lung injury in premature lambs with moderate hyaline membrane disease

Sophie Jaillarda, Yvon Rioub, Serge Klosowskic, Fabrice Cneudec, Patrice Fialdèsd, Xavier Codaccionie, Pierre Lequienc, Laurent Stormec

a Department of Thoracic Surgery, Centre Hospitalier Régional et Universitaire, 59037 Lille Cédex, France
b Department of Physiology, Centre Hospitalier Régional et Universitaire, 59037 Lille Cédex, France
c Department of Neonatology, Hôpital Jeanne de Flandre, Centre Hospitalier Régional et Universitaire, 59037 Lille Cédex, France
d Department of Biophysics, Centre Hospitalier Régional et Universitaire, 59037 Lille Cédex, France
e Department of Obstetrics, Centre Hospitalier Régional et Universitaire, 59037 Lille Cédex, France

Received 22 December 1999; received in revised form 11 April 2000; accepted 18 April 2000.

Corresponding author. Tel.: +33-3-2044-6467; fax: +33-3-2044-6236
e-mail: lstorme{at}chru-lille.fr


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The purpose of this study was to examine whether inhaled nitric oxide (iNO) may change lung injury in moderate hyaline membrane disease (HMD). Methods: Fifteen moderately premature lambs (128 days gestation, term=147 days) were randomly assigned to treatment with 20 ppm inhaled NO (n=7) from the onset of ventilation or control (n=8). Except for inhaled NO, treatments were intentionally similar to those applied in clinical situations. After porcine surfactant administration (Curosurf, 100 mg/kg), mechanical ventilator settings were modified during the course of the study to maintain PaCO2 between 40 and 50 mmHg and post-ductal SpO2 between 90 and 95%. The main studied parameters were gas exchanges parameters, respiratory mechanics (static compliance and functional residual capacity) and pulmonary vascular permeability and/or filtration rate indices. Results: We found that 20 ppm of inhaled NO for 5 h significantly reduce ventilatory and oxygen requirements, but only during the first hour of mechanical ventilation. No increase in extravascular lung water content (5.41±0.96 vs. 5.46±1.09 ml/g bloodless dry lung in the control group and in the NO group, respectively) and no impairment of the respiratory mechanics could be found in the NO-treated group. However, inhaled NO increased the albumin lung leak index in this model (6.09±1.51 in the NO-treated group vs. 4.08±1.93 in the control group; P<0.05). Conclusions: Our results do not therefore support a detrimental effect of short-term exposure to low doses of NO inhalation in moderate HMD. However, it may induce an increase in lung vascular protein leakage. The pathophysiological consequences of this finding remain to be elucidated.

Key Words: Pulmonary vascular permeability • Respiratory distress syndrome • Lung leak index • Extravascular lung water content


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Inhaled NO (iNO) improves arterial PO2 (PaO2), and decreases the need for extracorporeal membrane oxygenation in full-term newborn infants with persistent pulmonary hypertension of the newborn (PPHN) [1]. Gas exchange improvement by low dose iNO was also reported in experimental severe hyaline membrane disease [2], and in premature newborn infants with severe respiratory failure [3]. However, respiratory effects of iNO in moderate respiratory distress syndrome have not been fully investigated.

Therapeutic benefits of iNO could be expected. iNO could result in decreased oxygen and ventilatory requirements, and thus in reduced lung injury [4]. In addition, some reports showed that iNO may prevent the increase of pulmonary vascular permeability caused by oxidative stress, and may prevent pulmonary edema by inhibiting neutrophil adhesion, and by reducing pulmonary artery and capillary pressures [5]. Conversely, biochemical mechanisms exist by which iNO may induce oxidant-mediated tissue injury. NO is a free radical that can oxidized to nitrogen dioxide, and is also able to react with the free radical superoxide (O2-) to form peroxynitrite (ONOO-) [6]. These compounds have the potential to cause significant cellular damage. Furthermore, studies demonstrated that superoxide anion production is increased during mechanical ventilation with high FiO2 [7], and that pulmonary antioxidant defenses in premature infants are lower than in full-term newborn infants [8]. Thus, premature infants might be at greater risk for NO mediated oxidant lung injury.

The purpose of this investigation was to examine whether inhaled NO may change lung injury in moderate HMD. The premature lambs, extracted at 128 days of gestational age (term=147 days gestation), were chosen to test this hypothesis because they have been extensively studied as a model of HMD. The main studied parameters were gas exchanges and haemodynamic parameters. Respiratory mechanics and pulmonary vascular permeability and/or filtration rate indices were measured to quantify oxidant and mechanical ventilation-induced acute lung injury.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Surgical preparation
The following procedures were approved by the French Ministère de l'Agriculture, de la Pêche et de l'Alimentation before the studies were conducted. By the method previously described [2], ewes were sedated with i.v. thiopental sodium (initial dose 500 mg followed by 250 mg/30 min: 2 g total dose) and anaesthetized with intrathecal marcaine (0.5% solution, 4 ml). Foetal lambs were delivered by caesarean section under sterile conditions. A skin incision was made in the axilla of the left forelimb of the foetal lamb after local infiltration with lidocaine (0.5% solution, 4 ml). Polyvinyl catheters (05 Ch, 40 cm length; Vygon Medical Products, Ecouen, France) were placed in the ascending aorta through the axillary artery for systemic arterial pressure and heart rate monitoring and in the right atrium through the axillary vein for continuous administration of a 5% dextrose solution at a rate of 4 ml/kg per h. The foetal head was then exteriorized, and a tracheotomy was performed with placement of an endotracheal tube (ETT; 4.0 mm inner diameter). Special care was taken to tie the trachea on the ETT to avoid air leakage. Before mechanical ventilation, lambs were treated with exogenous surfactant (Curosurf, Serono, Italy) at an estimated dose of 1.5 ml/kg (120 mg phospholipid/kg). The tracheal tube was then connected to the ventilator circuit. The animals were ventilated with a time-cycled, pressure-limited ventilator (Babylog 1HF, Dragger, Germany). No thoracotomy for pulmonary vascular catheterizations was performed to avoid effects of acute surgery on lung fluid balance as previously reported [9].

First, we determined the optimal gestational age to obtain an experimental ovine model with moderate HMD defined by an oxygenation index between 5 and 20. Two lambs were extracted at each of the following gestational ages: 115, 120, 125 and 130 days (term=145 days gestation), and the oxygen and ventilatory requirements at 1 h of life were determined. An appropriate model of moderate HMD was established in the delivery of premature lambs at 128 days’ gestation. Then, 18 foetal lambs were extracted at 128 days’ gestation for the study. Except for inhaled NO, treatments were intentionally similar to those applied in clinical situations. The ventilator was set initially to provide the following parameters: peak inspiratory pressure (PIP), 30 cmH2O; positive end expiratory pressure (PEEP), 5 cmH2O; rate, 60 breaths/min; inspiratory time, 0.5 s; and FiO2, 1.00. Mechanical ventilator settings were modified during the course of the study to maintain PaCO2 and post-ductal SpO2 between predetermined values. The usual ‘heater humidifier’ was replaced by a ‘heat and moisture exchanger’ (Vygon Medical Products; dead-space 0.6 ml). No tracheal suction was performed during the study.

NO in nitrogen was purchased at a concentration of 450 ppm (Air Liquide Santé, Paris, France). This source was certified to contain less than 1 ppm of other nitrogen oxides (chemiluminescent analyzer). The NO cylinder was equipped with a stainless-steel diffusion-free regulator and low flow meter (Air Liquide Santé) for delivering gauged flow rate. This source gas was connected to the inspiratory limb of the breathing circuit –15 cm before the Y piece – via a polyvinyl line (unitube Bruneau, France). NO and NO2 concentrations were continuously measured by a chemiluminescence analyzer (Topaze, Cosma, France) at the Y piece of the breathing circuit. The rate of NO flow was set to obtain a NO concentration of 20 ppm. This concentration of iNO is the one usually applied in newborn infants with refractory hypoxaemia [10,11]. The fractional concentration of O2, measured at the inspiratory part of the breathing circuit using an O2-analyzer (Beckman OM11, USA), was >95% in the NO-treated group and in the control group.

After 5 min of mechanical ventilation, the umbilical cord was ligated. The foetus was weighed, dried, and placed in an incubator. The animal's core temperature was measured rectally and maintained at 38°C. Pancuronium bromide (80 µg/kg per h) was then administered continuously with a 5% dextrose solution.

2.2. Ventilator management strategy
Arterial blood gas (ABL 520, Radiometer, Copenhagen, Denmark) values were recorded before the onset of mechanical ventilation and then at 30 min and 1, 2, 3, 4 and 5 h of mechanical ventilation. The aim was to maintain a PaCO2 and a PaO2, respectively, between 35 and 45 mmHg, and between 50 and 80 mmHg. Mechanical ventilator settings were modified during the course of the study based on PaCO2 and PaO2 values according to the following protocol: if PaCO2 was <35 mmHg, then the PIP was reduced to 20 cmH2O; if subsequent measurements of PaCO2 were <35 mmHg, then the respiratory rate was reduced to 40 breaths/min; if subsequent measurements of PaCO2 were still <35 mmHg, then the PIP was reduced to 15 cmH2O; conversely, if PaCO2 was >45 mmHg, then the PIP was raised to 35 cmH2O; if subsequent measurements of PaCO2 were >50 mmHg, then the respiratory rate was increased to 80 breaths/min; PaO2 values were controlled by the FiO2, PEEP and inspiratory time. If PaO2 was >80, then the FiO2 was first reduced progressively to 60%; if subsequent PaO2 was >80, then the PEEP was reduced to 4 cmH2O, the inspiratory time to 0.4 s, and the FiO2 was lowered progressively to 30%; if subsequent PaO2 was still >80, then the PEEP was lowered to 3 cmH2O, the inspiratory time to 0.3 s, and the FiO2 was lowered progressively to 21%; conversely, if PaO2 was <50, then the PEEP was increased to 6 cmH2O and the inspiratory time to 0.6 s.

2.3. Pulmonary function testing
Static compliance of the respiratory system was calculated at 20 ml/kg. Briefly, one calibrated syringe was filled with 20 ml/kg of a gas mixture sampled at the expiratory part of the breathing circuit and tightened with a three-way tap. Airway pressure was measured at the airway opening with a pressure transducer (Validyne, ±50 cmH2O, USA) calibrated with an electronic calibrator (Premier, Dimelco, Vendeville, France). Airway pressure values were recorded on a four-channel polygraph (Linseis, Germany). The ETT was disconnected from the breathing circuit for 3 s to ensure complete passive gas exsufflation. Then the tap was connected to the ETT and opened. Special care was taken to avoid any gas leakage during the manoeuvre. Gas was injected rapidly and manually (average injection time=1.5 s). At the end of inflation, end inspiratory volume was maintained at least 15 s until obtaining a plateau pressure (elastic recoil pressure). Obtaining a plateau pressure was considered evidence that there were no gas leaks around the endotracheal tube. Three tests were recorded to ensure reproducibility. Static compliance of the respiratory system was calculated as the gas volume/plateau pressure ratio.

Functional residual capacity (FRC) and pulmonary diffusing capacity (DLCO) were measured by using the ‘Helium-dilution’ and the ‘single-breath’ methods, respectively. This method of end-expiratory lung volume measurement is part of the ‘single-breath’ manoeuvre and evaluates only the gas volume that readily communicates with the airways. Five calibrated syringes were filled with 20 ml/kg of a gas mixture containing 10% helium, 0.1% carbon monoxide, 21% oxygen, Qsp Azote (Air Liquide Santé) and tightened with a three-way tap. Special care was taken to avoid any gas contamination or leakage during tap manipulations. The ETT was disconnected from the breathing circuit for 3 s to ensure complete passive gas exsufflation. Then the tap was connected to the ETT and opened. Twenty ml/kg (Vinsp) of gas mixture was injected rapidly and manually (average injection time=1.5 s). After 10 s of breath-holding, the same volume of gas mixture was rapidly aspirated. The duration of each step of the manoeuvre (inflation, breath-holding, and deflation) was measured by recording the airway pressure. Disconnection from the ventilator for the test lasted 20 s. Three tests were recorded to ensure reproducibility. Inspiratory and expiratory helium (FEHe and FIHe) and carbon monoxide (FECO and FICO) concentrations were measured on the five syringes (helium and carbon monoxide analyzer, Morgan, UK). FRC (ml/kg) was normalized for the lambs’ weight. Specific FRC was calculated according to the following equations:

DLCO (ml/min per mmHg) were normalized for the FRC. Specific DLCO was calculated according to the following equations:

where FA=FICOx(FEHe/FIHe), VA=Vinspx(FIHe/FEHe), t is the time elapsed from the half of inflation to the half of deflation, PB (mmHg) is the barometric pressure, and PH2O, equal to 47 mmHg, represents water vapour pressure at 37°C.

Static compliance of the respiratory system, FRC, and DLCO were calculated at 30 min and 5 h of mechanical ventilation.

2.4. Measurement of edema and albumin leak lung index
Lung vascular permeability was measured using the method of Dreyfuss et al. [4]. Briefly, 2 mm of blood were sampled 3 h after the onset of mechanical ventilation to label the red blood cells, and reinjected at 4 h with labelled albumin. At 5 h, the animals were killed. This 1-h delay was necessary to ensure sufficient mixing and exchange times for the tracers. Pulmonary edema was assessed by measurement of the extravascular lung water content (Qwl). Qwl was obtained from the difference between wet and dry lung weights while taking into account blood content. Red blood cells, obtained after centrifugation, were mixed in vitro with sodium chromate (Cis Bio-international, Gif, France) for 20 min at room temperature. The labelled cells were washed twice with saline and 51Cr activity was determined in the supernatant of the last wash. Less than 1% free activity was considered satisfactory. Pulmonary vascular permeability was assessed by the 1-h 125iodine-albumin (125I-Alb: Cis Bio-international) uptake by lungs. Free iodine in the injected solution, measured by chromatography, was less than 1% of the 125I-Alb activity. Two hundred kBq of 51Cr-labelled red blood cells suspended in 50% normal saline and 30 kBq of 125I-Alb were thus injected intravenously at 4 h after the onset of mechanical ventilation. Extravascular lung water content (Qwl) and bloodless dry lung weight (Dlw) were estimated by using red blood cells labelled with 51Cr to calculate lung blood content. One hour later, 10 ml of blood were sampled from the arterial line and a lethal dose of potassium was administered. The thorax was rapidly opened and the lungs were carefully dissected from mediastinal tissue. The right and left lungs and the blood were weighed. Tracers activities were measured by gamma-counting for the lungs and the blood sample.

The fraction of blood in the lung was:

Then, the lungs and the blood sample were desiccated for 8 days at 70°C. The bloodless wet lung weight (Wlw), the bloodless dry lung weight (Dlw), and the extravascular lung water content (Qwl) were calculated according to the following equation:



Qwl was normalized for the body weight.

Vascular permeability was assessed by the albumin lung leak index (LLI) measurement defined by:

2.5. Experimental design
The premature lambs were mechanically ventilated for 5 h. They were randomly assigned treatment of 20 ppm inhaled NO from the onset of ventilation (H0) or control. In the NO treatment group, inhaled NO was administered throughout the 5-h study period. We did not attempt to correct metabolic acidaemia with infusion of a base or to correct arterial hypotension with macromolecule infusion to avoid acute changes in plasma osmotic or oncotic pressure. Arterial blood samples for pH, PaO2, PaCO2, SaO2, and percent methaemoglobin, and systemic arterial pressure measurement were performed before birth and mechanical ventilation (baseline measurements) and at 30 min and 1, 2, 3, 4 and 5 h of mechanical ventilation. The ventilator settings were adjusted in accordance with PaCO2 and SpO2. Sus-ductal oxygenation index values, i.e. OI=mean airway pressurexFiO2/PaO2, were calculated from the blood gas values.

2.6. Statistical analysis
Results are expressed as mean±1 SD. As the distribution of the studied parameters could not be established as normal, non-parametric tests were used. Differences between the NO treatment group and the control group were performed using two-way analysis of variance for repeated measures. The Student–Newman–Keul's test was used as post hoc analysis to compare the studied parameters. Comparisons in pulmonary vascular permeability and/or filtration rate indices were performed by using the Mann–Whitney test. A P-value of less than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Ten lambs were enrolled in the control group and eight in the NO-treated group. Both groups (control and NO-treated) were similar in gestational age (128±1 vs. 128±1 days) and in birth weights (3070±730 vs. 2950±450 g, respectively). Baseline physiological variables – haemodynamic and blood parameters – and mechanical ventilation before birth did not differ between control and NO-treated lambs (Table 1).


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  		Table 1. Baseline physiological variables before birtha

 
Arterial blood gas were similar in control and treated lambs (Fig. 1) . Furthermore, ventilatory parameters PIP, PEEP, ti, Paw and ventilator rate, were not significantly different between the two groups (Table 2). However, FiO2 and OI were significantly lower in the NO group than in the control group at 30 min and 1 h of mechanical ventilation (38±16% vs. 54±15% and 6.6±2.7 vs. 13.3±5.6, respectively; P<0.05) (Fig. 2) . DLCO decreased significantly in the control group during the course of the study (P<0.05). On the contrary, DLCO did not change during the experiment and was higher in the NO group than in the control group at 5 h of mechanical ventilation. iNO had no significant effect on specific static compliance (Fig. 3) of the respiratory system and on specific functional residual capacity (Fig. 4) .



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  		Fig. 1. Time course of PaO2 and PaCO2 in the iNO treatment and in the control groups (mean±SD). (•) PaO2/iNO; ({circ})=PaO2/control; ({blacksquare}) PaCO2/iNO; ({square}) PaCO2/control. *P<0.05.

 

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  		Table 2. Summary of the ventilatory parameters during the study perioda

 


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  		Fig. 2. Oxygenation index (OI=mean airway pressurexFiO2/PaO2) measured in the iNO treatment and in the control groups. Except during the first hour of mechanical ventilation, no difference could be found between the two groups during the following 4 h of the mechanical ventilation. Mean±SD; *P<0.05.

 


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  		Fig. 3. Static compliance measured in the iNO-treated lambs and in the control lambs. iNO did not change static compliance. Mean±SD; *P<0.05.

 


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  		Fig. 4. Specific functional residual capacity (FRC) measured in the iNO-treated lambs and in the control lambs. iNO did not the FRC. Mean±SD; *P<0.05.

 
NO inhalation did not change the extravascular lung water content (5.41±0.96 ml/g bloodless dry lung in the control group vs. 5.46±1.09 ml/g bloodless dry lung in the NO group; not significant). However, the ‘albumin lung leak index’ was significantly higher in the NO group than in the control group (6.09±1.51 vs. 4.08±1.93, respectively; P<0.05) (Table 3).


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  		Table 3. Effects of inhaled NO on extravascular water right lung content (Qwrl) and albumin lung index (LLI)a

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The aim of this study was to examine the effects of short-term iNO exposure on gas exchange in an experimental model of moderate HMD. Another goal was to establish whether or not iNO can change acute lung injury evaluated by respiratory mechanic and lung vascular permeability measurements. We found that the decrease in oxygen requirement observed in lambs exposed to 20 ppm iNO during the first hour was not sustained. Furthermore, iNO for 5 h increased lung vascular protein leakage in this model. However, this phenomenon was not associated with increased extravascular lung water content or with impaired gas exchange and respiratory mechanics.

These results are quite surprising: we expected that inhaled NO could improve gas exchange in these premature lambs with moderate respiratory failure, as previously demonstrated in severe experimental HMD [2], or in premature newborn infants with severe respiratory failure [3]. iNO did not reduce significantly peak inspiratory pressure, mean airway pressure, and respiratory rate in our experimental model. Only FiO2 and oxygenation index was lower during the first hour of NO exposure. However, oxygenation improvement was transient: no significant difference in oxygen need was observed from 2 h of NO inhalation. Our findings support the hypothesis that the response to iNO is related to the severity of the respiratory failure and that little or no benefit on oxygenation must be expected in moderate respiratory distress syndrome, at least during the first hours of NO inhalation. These results may have several explanations. Firstly, previous reports suggested that the magnitude of iNO response depends on the mechanism of hypoxaemia and on the severity of pulmonary hypertension. In neonates with respiratory distress syndrome, hypoxaemia may result from right-to-left extrapulmonary shunt or from intrapulmonary shunt. Extrapulmonary shunt through ductus arteriosus and/or foramen ovale is the consequence of increased pulmonary artery pressure and increased pulmonary vascular resistance. iNO may reduce intrapulmonary shunt by improving ventilation–perfusion matching [12], and may reduce extrapulmonary shunt by decreasing pulmonary vascular resistance [13]. We contributed to demonstrating that the improvement of systemic oxygenation is greater in hypoxaemic neonates with extrapulmonary shunt than in hypoxaemic neonates with intrapulmonary shunt [13]. Secondly, other investigators demonstrated that patients with the greatest level of pulmonary vascular resistance had the most consistent NO-induced improvement in arterial oxygenation [14,15]. No pulmonary vascular resistance measurements were performed in this study; however, it is likely that hypoxaemia, in our model of moderate HMD, was caused mainly by intrapulmonary shunt. It is also likely that pulmonary vascular resistance was lower than in model with severe respiratory failure. Indeed, persistent pulmonary hypertension and extrapulmonary shunt are considered as a severity criterion in newborn infants with HMD leading to increased mortality [16].

The second objective of the present study was to test the hypothesis that NO inhalation can change lung injury in moderate HMD. Two parameters were measured to evaluate the extend of lung damage: (1) pulmonary vascular permeability and/or filtration rate indices; and (2) respiratory mechanics. Numerous investigators have used them for this purpose [4,5].

Pulmonary vascular permeability and/or filtration rate indices were evaluated by the measurement of extravascular lung water content and of albumin lung leak index. We found that, in spite of similar extravascular lung water content in both groups, albumin lung leak index was increased in the premature lambs with moderate HMD treated by 20 ppm iNO for 5 h. The lung interstitium protein concentration/plasma protein concentration ratio is a function of capillary filtration rate and the permeability–surface area product of the endothelial barrier. Thus, increased LLI may be the result of permeability alterations and/or increased filtration. Conflicting results were found in other in vivo studies [5,17–19]. Kinsella et al. [5] found that 20 ppm of iNO does not alter the albumin lung leak index and the extravascular lung water content in premature lambs with severe hyaline membrane disease: differences in severity of the respiratory failure (severe versus moderate), gestational age of the lambs (115 vs. 130 days), duration of NO exposure (3 vs. 5 h), and FiO2 levels (100 vs. 40%) between both studies may explain the discrepancies in the results. Studies in rats breathing 100% O2 [17] showed that a low dose of iNO (10 or 20 ppm) reduces lung vascular and epithelial permeability; on the other hand, 100 ppm inhaled NO for 40 h increased lung vascular permeability to protein. These results suggest that iNO can either reduce or increase lung vascular protein leakage depending on its concentration. In a previous study, we found that 20 ppm iNO for 76 h combined with hyperoxia increases lung protein leaks in mechanically ventilated full-term newborn piglets [19]. The present findings are consistent with this study and suggest that, in immature lungs, 20 ppm of iNO exceeds the threshold dose above which lung vascular leakage is increased. Because low-dose iNO (1 ppm) was found to be as effective as higher concentrations (5 and 20 ppm) in improving oxygenation in premature newborn infants [3,20], the lowest effective dose should be used in this population.

Pathophysiological mechanisms of increased capillary permeability to plasma protein are uncertain but may include oxidative stress and/or surfactant dysfunction. NO and its metabolites may have the ability to cause significant oxidant damage to lung tissues. NO is a free radical that can be oxidized to nitrogen dioxide in the presence of oxygen. In addition, NO is also able to react with the free radical superoxide (O2-) to form peroxynitrite (ONOO-) and other cytotoxic species such as hydroxyl radical and NO2 [6]. Each of these compounds has the potential to cause significant cellular damage. However, our previous work do not support this hypothesis as, in the same model, iNO did not alter oxidative stress parameters and did not induce lung inflammation [21]. iNO may cause significant surfactant dysfunction. Increased minimum surface tension was seen in piglets mechanically ventilated with 100 ppm NO and 90% O2 for 48 h [22]. Peroxynitrite-induced surfactant phospholipids damage and nitration of surfactant protein A contributed to explain these results [22]. Hallman et al. [23] demonstrated that inhaled NO may promote inhibition of surface activity of the surfactant by oxidizing haemoglobin into methaemoglobin in the alveolar space. As surfactant reduces lung vascular permeability [24], surfactant inactivation in the NO-treated group may have increased transcapillary protein flux. Immature lungs, characterized by a quantitative deficit in endogenous surfactant, may be particularly susceptible to NO-induced impairment of surfactant properties. However, these hypotheses remain purely speculative and we cannot accordingly rule out the assumption that the increased lung leak index in the NO-treated group may result, at least in part, from changes in capillary filtration rate. No thoracotomy for pulmonary vascular catheterization was performed to avoid effects of acute surgery on lung fluid balance as previously reported [9]. However, Kinsella et al. [2] demonstrated that iNO can produce selective pulmonary vasodilatation in premature lambs. Therefore, iNO could have increased capillary filtration coefficient by increasing the pulmonary vascular surface area.

We found that despite increased protein lung leak index, extravascular lung water content was not increased by iNO. This apparent discrepancy was already reported in rabbits exposed to 100% O2 for 3 days [25]. Unchanged extravascular lung water content does not mean that transvascular fluid flow does not change. Lymphatics have the ability to increase the rate of interstitial fluid removal when microvascular fluid filtration increases. Lymph flow may increase sufficiently to balance the increase of capillary filtration. In another way, the increase in albumin lung leak index without change in extravascular lung water content may also reflect injury to components of the interstitial space instead of an increase in vascular permeability [25].

In the present study, we did not find any significant effect of iNO on specific static compliance of the respiratory system and on specific functional residual capacity. Elastic recoil forces of the respiratory system are related, at least in part, to alveolar gas/liquid interface, to elastic properties of lung tissues [25], and to extravascular lung water content. Although Mercer et al. [26] demonstrated that prolonged exposure (9 weeks) to low doses of NO (500 ppb) in adult rat lungs can result in the degeneration of interstitial matrix and connective tissue fiber, to our knowledge no data suggests that short-term NO inhalation can alter the parenchymal structure of the lung. Conversely, several investigators have shown that iNO may cause significant surfactant dysfunction as discussed above [22]. However, NO concentration and FiO2 used in these studies were higher than in the present experiment. We found also that carbon monoxide diffusing capacity decreased progressively in the control animal during the study period and that iNO prevented this decrease. The mechanism of this effect of iNO is unknown but may include higher pulmonary capillary blood flow and/or volume in the iNO-treated lambs [27]. Carbon monoxide diffusing capacity depends on alveolar capillary interface, capillary blood flow and volume, and haemoglobin concentration. As functional residual capacity and haemoglobin concentration did not change, decreased carbon monoxide diffusing capacity was probably related to decreased capillary blood flow. iNO prevented the decrease in carbon monoxide diffusing capacity. Our findings support the hypothesis that short-term exposure to low doses (20 ppm) of iNO does not alter the mechanical properties of lungs.

There are potential limitations in our study. (1) Although the improvement in oxygenation was only significant during the first hour of NO exposure in our model of moderate experimental hyaline membrane disease, we cannot rule out the hypothesis that increased duration to iNO could have prevented late deterioration of lung function. This important issue is currently in evaluation in our laboratory. (2) Although in the present study all premature lambs were ventilated using PEEP between 3.5 and 5 cmH2O and mean airway pressure between 9 and 16 cmH2O, we cannot exclude the possibility that the PEEP and the MAP level may have been insufficient to recruit enough alveoli for a redistribution of blood flow from nonventilated toward ventilated lung units after iNO [28]. A strict protocol, similar to the one used in clinical situation, was used to set MAP, i.e. inspiratory time and PEEP level, according to the oxygen need. PEEP and MAP levels, and FiO2 were similar in both groups. Moreover, inadequate alveolar recruitment would have resulted in high-level pulmonary–venous admixture, and therefore, in high oxygen need. Low mean FiO2 level (35–54%) does not support this hypothesis. (3) Lack of detailed pulmonary vascular haemodynamic measurements does not allow precise understanding of the mechanisms of iNO-induced increase in albumin lung leak index. It remains unclear if iNO results in an increase in pulmonary vascular permeability and/or in an increase in filtration rate indices.

In conclusion, the effects of iNO on gas exchange in an experimental model of moderate HMD was investigated. We found that 20 ppm of inhaled NO for 5 h failed to decrease the ventilatory and oxygen requirements in premature lambs with moderate respiratory distress syndrome except during the first hour of mechanical ventilation. Whether or not iNO can change acute lung injury was also examined: despite similar extravascular lung water content, iNO increased lung vascular protein leakage in this model. However, neither the main respiratory mechanical parameters (static compliance of the respiratory system, functional residual capacity) nor pulmonary diffusing capacity are impaired by iNO. Pathophysiological consequences of this finding remain to be elucidated. Even if iNO does not worsen the deleterious effects of hyperoxia on the main respiratory functions, we suggest using, in premature newborn infants, the lowest effective concentration of inhaled NO to limit NO-induced microvascular protein leakage.


   Acknowledgments
 
This work was supported by grants from the Université de Lille II (Bonus Qualité Recherche 1997) and from the Projet de Recherche Mixte CH & U de Lille (Projet MICRONAT/ARCAS).


   Footnotes
 
Presented at the 7th European Conference on General Thoracic Surgery, Nancy, France, October 21–23, 1999.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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