INTRODUCTION

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The pathogenesis of the acute respiratory distress syndrome (ARDS) involves multiple mechanisms, including inflammatory damage to alveolar wall, surfactant deficiency and dysfunction, as well as altered pulmonary vascular structure and function. This leads to impairment of lung mechanics and gas exchange, and increased vascular-to-alveolar permeability (1- 3). Clinically, ARDS is characterized by progressive lung edema, atelectasis, intrapulmonary shunting, pulmonary hypertension, and arterial hypoxemia. Clinical trials using either exogenous surfactant administration for pediatric and adult patients with ARDS (4, 5), or various concentrations and periods of inhaled nitric oxide (INO) in persistent pulmonary hypertension of the newborn (PPHN) and ARDS (6), have revealed immediate improvement in gas exchange and blood oxygenation, and a reduction in pulmonary vascular resistance. Although these promising results show advantages compared with other currently applied strategies for treatment of ARDS, a substantial number of patients do not respond to either therapy. Therefore, methods to enhance the effect of exogenous surfactant in INO are under extensive investigation.

Combined use of surfactant and INO has not been tested yet in clinical trials, although in some studies surfactant was given before INO treatment (6). In the lungs NO is produced by several cell types, including airway epithelium, macrophages, and intrapulmonary vascular endothelium (12). It acts on vascular smooth muscle cells, reducing vascular tension, increasing pulmonary flow, and may contribute to optimize ventilation- perfusion matching (13). These mechanisms are important in lung physiology as well as in therapeutic strategies for RDS and ARDS. Furthermore, both surfactant and INO have a potential to downregulate cytokine production by inflammatory cells in the lungs (14, 15). We hypothesize that exogenous surfactant may facilitate distribution of NO to collapsed alveoli and that a combined use of surfactant and INO could have a synergistic effect in acutely injured lungs. We designed these experiments to assess whether this combination would be more effective than either method alone. If so, the combination could play a role in treatment regimens for clinical ARDS.

	METHODS

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Surfactant

Surfactant was prepared from fresh pig lungs by bronchoalveolar lavage (BAL), using 0.9% NaCl at room temperature. BAL fluid (BALF) was immediately centrifuged for 15 min at 200 × g and room temperature to remove cell debris. The supernatant was centrifuged for 120 min at 5,000 × g and 4° C, and the pellet containing surfactant was suspended in 0.9% NaCl and laid on a 0.68 M sucrose and saline solution for differential density gradient centrifugation for 30 min at 5,000 × g and 4° C. The material at the interface between saline and sucrose was collected, resuspended in 0.9% NaCl, and centrifuged for 15 min at 10,000 × g and 4° C. This procedure was repeated three times. The final sediment containing natural surfactant from the last high-speed centrifugation was suspended in 0.9% NaCl, extracted with 3 vol of chloroform-methanol (2:1, vol/vol), and the chloroform phase was collected and precipitated with cold acetone at 4° C to remove neutral lipids. After evaporation of chloroform and acetone under nitrogen gas, the dry material was weighed and resuspended in 0.9% NaCl at a concentration of 40 mg phospholipids/ml and stored at 20° C. A single batch of the surfactant preparation was used for the whole experiments, and total phospholipids (TPL) and disaturated phosphatidylcholine (DSPC) in the surfactant preparation were determined as described by Bartlett (16) and Mason and colleagues (17), respectively. In this batch, DSPC was about 55% of TPL, and its surface properties were determined in a modified Wilhelmy balance (see below).

Inhalation of Nitric Oxide

Stock gas (Shanghai BOC, Shangai, China) was obtained as NO 1,000 ppm, balanced in nitrogen (purity > 99.999%) and containing less than 0.5% nitrogen dioxide (NO₂) relative to NO. It was supplied to the afferent limb of a ventilator circuit about 20 cm proximal to the endotracheal tubing connector (Y piece). NO gas flow was regulated by a mass flow controller (Shengye Technological Development, Beijing, China). Concentrations of NO and NO₂ were measured using an NO/ NO₂ analyzer (NOxBOX I; Bedfont Scientific, Kent, UK) with electrochemical sensors and calibrated with 84 ppm NO and 6 ppm NO₂ from a standard calibration gas (AGA, Lidingö, Sweden).

Animal Management

Protocols for animal care and experimental management were approved by the Children's Hospital Scientific Committee (Shanghai Medical University, Shanghai, China). Healthy adult New Zealand White rabbits with a body weight of 2.4-3.2 kg (means ± SD, 2.8 ± 0.2), were sedated with intramuscular 2 mg/kg Diazepam (5 mg/kg; Xudong Haipu Pharmaceutical, Shanghai, China), and anesthetized with intravenous 1% pentobarbital sodium at a dose of 2 ml/kg. Additional intravenous 1% pentobarbital sodium was provided at 0.5 ml/ kg/h to maintain anesthesia. Animals were tracheotomized, intubated, and ventilated mechanically with a pressure-controlled Wave ventilator E-200 (Newport Medical Instruments, Newport Beach, CA). The ventilator was initially set at a peak inspiratory pressure (PIP) of 10- 15 cm H₂O to provide a tidal volume (VT) of 8-10 ml/kg body weight, a frequency of 25/min, an inspiration-to-expiration ratio of 1:2, and a fraction of inspired oxygen (FI_O2) of 0.21. An 18-gauge cannula was inserted into the right femoral artery for collection of blood samples and measurement of blood pressure with a pressure transducer and monitor (Spacelab, Redmond, WA). When the animals had been stabilized from the initial operation, blood was taken for measurement of baseline values of pH, Pa_O2, and Pa_CO2 with a Ciba-Corning 170 automatic blood gas analyzer (Huanqiu Medical Instruments, Shanghai, China), and for determination of nitrite/nitrate and methemoglobin (MetHb) (see below). Baseline values for dynamic compliance (Cdyn) and resistance (Rrs) of the respiratory system were measured with a pneumotachograph GM 250 Navigator (Newport Medical Instruments), using an infant-type differential pressure transducer placed in the Y piece. Cdyn was expressed as ml/cm H₂O · kg and Rrs as cm H₂O/L · s from 10 consecutive breaths. Functional residual capacity (FRC) was determined with the helium dilution method (18), using helium gas (Donghui Medical Gas, Shanghai, China) with a purity of 99.999%, diluted 10 times with pure oxygen when in use. The helium content was measured in a helium analyzer TC-1 (Shanghai No. 4 Medical Instrument Factory, Shanghai, China) and the FRC was expressed as milliliters per kilogram body weight. Physiologic intrapulmonary shunting (S/T) was determined by measuring oxygen content in mixed venous blood (when FI_O2 was temporarily raised to 1.0), taken from the right ventricle of the heart through a thin tube placed in the external jugular vein, and its values were calculated using the standard shunt equation (19), and expressed as a percentage of the total pulmonary blood flow. The animals were then subjected to the experimental protocols.

Experiment Protocols

Oleic acid (Cat. No. 01008; Sigma, St. Louis, MO) was first diluted with 10 vol of 1% bovine serum albumin in saline. A total of 75 µl/kg body weight of this diluted oleic acid, divided into two or three portions, was slowly infused into the pulmonary circulation over a 30-min period, using a syringe connected to an infant feeding tube placed in the right ventricle of the heart. The FI_O2 was raised to 0.6 and kept constant throughout the rest of the experiment. The animals were ventilated with variable PIP and a positive-end expiratory pressure (PEEP) of 4 cm H₂O to maintain VT at 8-10 ml/kg, and a frequency of 30/min, an inspiration-to-expiration ratio of 1:1, to achieve values of blood pH, Pa_CO2, and Pa_O2 in the ranges of 7.30-7.50, 30-40 mm Hg, and 80-100 mm Hg, respectively. Respiratory failure was defined as Cdyn decreasing by > 30% from its baseline level (20), Pa_O2 < 120 mm Hg, and S/T increasing to > 25%, and this moment was regarded as representing treatment time 0 (zero). The animals were then randomly allocated to four treatment groups receiving (1) neither surfactant nor INO (control group); (2) a bolus containing 100 mg/kg of surfactant phospholipids/kg body weight (S group); (3) inhalation of 20 ppm INO (NO group); and (4) 100 mg of surfactant phospholipids/kg body weight and inhalation of 20 ppm NO (SNO group). The surfactant was instilled into animal lungs via an endotracheal tube at a concentration of 40 mg/ml. All the animals were subsequently ventilated for another 6 h and values for Cdyn, Rrs, FRC, and arterial pH, Pa_O2, and Pa_CO2 were measured each hour. S/T was measured after 4 h. At the end of the period of ventilation, animals were killed by an overdose of 5% pentobarbital sodium and the animal lungs were processed (see below). For animals who survived less than 6 h, measured values for physiological parameters recorded before deterioration of heart rate and blood pressure were included in the final analysis, and their lungs were processed immediately after death.

Wet-to-Dry Lung Weight Measurements and Bronchoalveolar Lavage

Immediately after each animal had been sacrificed, an incision was made in the abdominal wall to look for evidence of pneumothorax, and the chest was then opened. In five animals in each group a piece of lung tissue (about 2 g) from the back side of the left lower lobe was cut and its wet weight was determined in an automatic electric balance (AP250D; Ohaus, Florham, NJ). The piece of lung tissue was then put in an oven at 80° C for 48 h and weighed again to obtain its dry weight for calculation of the wet-to-dry weight ratio (W/D). The right lung was lavaged with 0.9% NaCl at 15 ml/kg body weight and room temperature. Each volume of saline was infused three times and then collected. Three additional volumes were provided in an identical manner so that the right lung was lavaged 12 times with 4 vol. More than 85% of the instilled BALF was collected from each animal, and collected BALF was pooled and its total volume recorded. The BALF was immediately centrifuged for 10 min at 200 × g and 4° C to remove cell debris, and the supernatant was stored at 20° C for biochemical analysis. In another four animals in each group both lungs were fixed by vascular perfusion for histologic and morphometric analysis (see below).

Chemical Analysis of Bronchoalveolar Lavage Fluid

Aliquots of BALF were extracted with threefold volumes of chloroform-methanol (2:1, vol/vol) to isolate the phospholipids in the chloroform phase. DSPC was separated from other phospholipids as described by Mason and coworkers (17). Briefly, samples from the chloroform phase were dried under nitrogen gas, oxidized with small volume of osmium tetroxide in carbon tetrachloride for 15 min, and dried again under nitrogen, dissolved in chloroform-methanol (20:1, vol/vol), and passed through a neutral aluminum column. The DSPC fraction was collected by adding to the column a mobile phase of chloroform-methanol-7 M ammonium hydroxide (70:30:2, vol/vol/vol). Amounts of DSPC and TPL were determined according to the methods described by Bartlett (16) and corrected by the total volume of BALF and body weight. Values for TPL are presented as milligrams per kilogram, DSPC as a percentage of the TPL (DSPC/TPL). Total proteins (TP) in BALF were measured according to the method of Lowry and associates (21), using bovine serum albumin as the standard, and corrected by total volume of BALF and body weight; these values are presented as milligrams per kilogram. The DSPC and TP ratio was expressed as micrograms per milligram.

Surface Tension Measurements

Surface tension measurements were performed with a modified Wilhelmy balance (Biegler, Vienna, Austria) according to a method described elsewhere (22). Twenty milliliters of BALF was poured into the trough of the balance system and kept at 37° C. Surface tension of the fluid was recorded continuously during 50% cyclic area compression at a rate of 1 cycle/min, for a total of 120 cycles (i.e., 120 min). Values for minimum and maximum surface tension (_min and _max, respectively) were obtained at minimum and maximum surface area, respectively. The batch of surfactant preparation used in this study was suspended in 20 ml of 0.9% NaCl at a phospholipid concentration of 0.5 mg/ml and cycled for 120 min (23).

Measurements of Nitrite/Nitrate and Methemoglobin

Blood samples representing baseline, treatment time 0 h, and treatment time 4 h were taken for measurement of nitrite and nitrate, using a modified Griess method as described by Shi and coworkers (24), and values are expressed as micromoles per milliliter of serum. MetHb was determined according to the method described by Hegesh and colleagues (25), and expressed as a percentage of total hemoglobin (Hb).

Histologic and Morphometric Examination of Lungs

Lungs from four animals of each group were fixed for histologic examination. The lungs were first inflated with a pressure of 30 cm H₂O for 1 min, and then deflated to 10 cm H₂O. This pressure was maintained while the lungs were perfused for 30 min via the pulmonary arteries with 4% formaldehyde at a pressure of 65 cm H₂O. Representative lung tissue blocks from all lung lobes were embedded in paraffin. Sections stained with hematoxylin and eosin were examined by light microscopy for evidence of lung injury, as described elsewhere (23). Lung expansion was quantified by the point-counting method, and expressed as volume density (VV) of aerated alveolar spaces, using total parenchyma as the reference volume (26). Fifty fields of each lung section were examined from each animal (magnification: ×300), and field-to-field variability was determined by calculating the coefficient of variation of VV [CV(VV)]. A low value of CV(VV) indicates homogeneity of alveolar aeration. This work was performed by a technical staff member who was not aware of the treatment protocol of individual animals.

To compare the lung expansion with the situation before lung injury, four rabbits of similar size were subjected to the protocol and sacrificed when all baseline measurements of the physiological parameters had been completed. These lungs were then processed in the same way as those of experimental animals subjected to lung injury and various treatments as described above, and regarded as normal.

Statistics

Data are presented as means and standard deviation. Survival rate was examined with chi-square and Fisher's exact test. Analysis of variance (ANOVA) was used for parametric data and differences between two groups were further evaluated with the Student-Newman-Keuls post hoc test. Within-group differences were detected with the Wilcoxon signed-rank test. A p value  0.05 was regarded as significant.

	RESULTS

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General Conditions of the Animals

Infusion of oleic acid induced, in all animals, respiratory failure within 4-6 h (mean ± SD, 4.5 ± 0.6), as reflected by marked decreases in Cdyn, FRC, and Pa_O2, and increases in Rrs (Figure 1), S/T (Figure 2), and PIP (an average of 25- 30 cm H₂O). All these parameters had significant changes in mean values compared with corresponding baseline values (p < 0.01). Values for mean systemic arterial pressure at baseline were 73.2 ± 10.7 mm Hg and were maintained at 60-80 mm Hg during the treatment period. Eight of the nine animals in the control group died within 5 h of treatment despite aggressive mechanical ventilation with high PIP, whereas four of nine animals in the NO and S groups, and eight of nine in the SNO group, respectively, survived 6 h of treatment. The survival rate in the SNO group was significantly higher than that in the control group (p < 0.01). The early death occurring among the animals across groups was mainly due to cardiac depression, as indicated by bradycardia and arrhythmia in association with severe hypoxemia, and severe acidosis. The inhaled concentration of NO in the NO and SNO groups was kept at 20 ppm with a variation less than 1.0 ppm, and NO₂ was lower than 1.5 ppm throughout the treatment period.

View larger version (19K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 1.   Lung mechanics and blood gas parameters during the course of the experiment. Values at baseline represent conditions before the induction of lung injury. Labels and group definitions: control (open circles); NO (filled circles); surfactant (open triangles); surfactant and nitric oxide (filled triangles). Bars and ticks are means and SD, respectively. *p < 0.05, **p < 0.01 versus control and NO groups; #p < 0.05, ##p < 0.01 versus S group; +p < 0.05, ++p < 0.01 versus control group.

View larger version (30K): [in this window] [in a new window]   Figure 2.   Intrapulmonary shunting at baseline and at treatment times 0 and 4 h. Values at baseline represent conditions before the induction of lung injury. When measuring S/ T, FIO2 was temporarily raised to 1.0. Group definitions: C = control; NO = nitric oxide, S = surfactant; SNO = surfactant and nitric oxide. Bars and ticks are means and SD. **p < 0.01 versus control group; #p < 0.05 versus NO and S groups; ##p < 0.01 versus the same group at treatment time 0 h by within-group comparison.

Lung Function Measurements

Values for Cdyn, Rrs, FRC, and blood gas parameters at baseline and during the treatment period are shown in Figure 1A-F. During the whole period of observation, VT was generally kept at 8 ml/kg in all the groups when PIP was adjusted to maintain Pa_CO2 within the normal range (Figure 1E). However, pH values in each group were lower than the target range (Figure 1F) despite aggressive management with intravenous biocarbonate sodium, and no attempt was made to overventilate the animal lungs to overcome the acidosis. Values of Cdyn, Rrs, FRC, Pa_O2, Pa_CO2, pH, and S/T were the same across the groups both at baseline and at treatment time 0 h as shown in Figures 1 and 2, but for Cdyn, Rrs, FRC, and S/T, the values are significantly different from corresponding values at baseline. In the control group, there was persistent deterioration of lung function despite the application of vigorous ventilation settings (PIP, PEEP). In the NO group, there were modest improvements in Pa_O2 and S/T, but no substantial improvement in Cdyn, Rrs, and FRC. In contrast, Cdyn, Rrs, FRC, Pa_O2, and S/T were all moderately improved in the S group, compared with the control group. In the SNO group, Cdyn, Rrs, Pa_O2, S/T, and FRC were also improved compared with the control and NO groups, but Pa_O2 and S/T were further improved compared with the S group (for detailed statistical analysis, see captions to Figures 1 and 2).

Chemical Analysis of Bronchoalveolar Lavage Fluid

Values for TPL, DSPC/TPL, TP, and DSPC/TP in BALF are shown in Table 1. TPL and DSPC/TPL values in BALF were significantly higher in the S and SNO groups than in the control and NO groups. The TP value in BALF was about the same as in the control, NO, and S groups, but significantly lower in the SNO group. When DSPC was corrected by TP, it also showed significantly higher values in the S and SNO groups.

Surface Tension Measurements

When the surfactant suspension was analyzed in the Wilhelmy balance, _min and _max reached 1 and 32 mN/m, respectively, within fewer than 10 compression cycles, and remained at these levels for the rest of the cycling period (120 min).

Values for _min and _max in BALF after 120 min of dynamic cycling are shown in Table 1. In the control and NO groups, _min and _max remained > 20 mN/m throughout the period of cycling, whereas in the S and SNO groups only a few cycles were needed to generate a film with _min below 5 mN/m, which was similar to data reported elsewhere for a modified porcine lung surfactant preparation (22).

Measurements of Nitrite/Nitrate and Methemoglobin

Values of nitrite/nitrate and MetHb are presented in Table 2. The baseline level of nitrite/nitrate was about the same in all the groups, and tended to increase during inhalation of NO in the NO and SNO groups. MetHb was kept below 2% of total Hb throughout the inhalation of NO.

Wet-to-Dry Lung Weight Ratio

Values of the wet-to-dry lung weight ratio (W/D) in various groups of experimental animals are shown in Table 3. Low values for W/D were observed in surfactant-treated animals in the S and SNO groups, and the lowest level was found in animals receiving combined treatment with surfactant and INO.

Histological and Morphometric Findings

Findings included prominent atelectasis, hyaline membranes, edema, intraalveolar and interstitial patchy hemorrhage, and infiltration of neutrophils in the lungs of animals in the control and NO groups. In the S and SNO groups, there was improved (but inhomogeneous) aeration of alveoli, and hyaline membranes, edema, hemorrhage, and infiltration of neutrophils were less severe than in the control and NO groups. Results from morphometric analysis are shown in Table 3. Aeration of alveoli was significantly improved in the S and SNO groups as reflected by increased VV and low values for CV(VV), but not as good as that in normal lungs before the induction of injury.

	DISCUSSION

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In this study, we found that intravenous infusion of oleic acid in adult rabbits consistently induced respiratory failure within 4-6 h of mechanical ventilation, with impairment of lung mechanics and gas exchange, and increased intrapulmonary shunting. Both physiological and histological data indicate that the animals had developed acute lung injury and respiratory failure similar to clinical ARDS. Hall and colleagues (27) reported that oleic acid may damage endogenous surfactant as reflected by increased conversion to small phospholipid aggregates and deteriorated surface activity. Putensen and coworkers (28) used a combination of INO and continuous positive airway pressure on adult dogs with acute respiratory failure induced by oleic acid, and found enhanced pulmonary ventilation-perfusion matching and cardiac output due to recruitment of gas exchange units in the lungs. Other studies on experimental acute lung injury revealed that PEEP is required to maintain lung expansion and prevent edema during mechanical ventilation, also after treatment with exogenous surfactant (29). Their findings suggest that surfactant dysfunction and deficiency secondary to lung injury and inactivation by hemorrhagic and proteinatious edema may have a profound impact on the clinical course of ARDS, and that upgrading the pool of surfactant in the lungs, along with adequate ventilator settings, should be beneficial for recruitment of gas exchange units, restoration of lung function, and improvement of blood oxygenation in ARDS. Furthermore, as mentioned above, studies demonstrate that, in addition to pathophysiological mechanisms related to surfactant dysfunction, the release of endogenous NO might be inadequate in ARDS (7). This would suggest that exogenous NO might have a potential as replacement therapy to ensure adequate pulmonary perfusion, thereby potentiating the therapeutic effect of exogenous surfactant.

Effects of INO in oleic acid-induced ARDS may vary between species. Shah and colleagues (30) reported that in an adult pig model of oleic acid-induced acute lung injury, inhalation of 10-80 ppm NO had a beneficial effect on hemodynamic parameters but no effect on gas exchange and intrapulmonary shunting. Leeman and colleagues (31) reported that oleic acid may increase pulmonary arterial pressure and intrapulmonary shunting in dogs, and hypothesized that there is a release of endogenous NO in acute lung injury with intrapulmonary vascular damage. However, inhibition of endogenous NO production had no effect on gas exchange in that experimental model. In contrast, our results clearly show improved Pa_O2 and decreased intrapulmonary shunting in animals receiving INO alone, and these effects were further enhanced by combined treatment with surfactant and INO. However, compared with the values at baseline, i.e., before induction of lung injury, the improvement in lung mechanics, gas exchange, and hemodynamics was modest, and normal lung function was not restored. We speculate that, in this animal model of oleic acid-induced respiratory failure, the pathophysiological mechanisms include both surfactant dysfunction and reduced NO levels, together accounting for the deterioration in lung mechanics and intrapulmonary vasoconstriction.

Timing of treatment and dosage probably determine the therapeutic effect of INO in clinical and experimental ARDS. In our present study, we used 20 ppm NO for inhalation when ARDS was established. This concentration is effective in premature newborn lambs with hyaline membrane disease (15, 19) as well as in experimental models with acute lung injury and ARDS (13, 30), and exceeds the estimated minimum effective concentration for treatment of patients with ARDS (7, 8, 10). It has been suggested that the minimum concentration of NO for reversal of intrapulmonary shunting in hypoxic respiratory failure may be below 20 ppm (32), whereas the concentration required for effective treatment of pulmonary hypertension may be as high as 80 ppm (33). In clinical practice, the minimum effective concentration of INO and required duration of treatment may differ considerably depending on the variability of underlying disease conditions in ARDS. However, current clinical trials and guidelines for clinical use of INO therapy are all based on safety considerations, minimize both the effective concentration of INO and the duration of treatment to avoid overproduction of NO₂ and to reduce risk of methemoglobinemia. Our results indicate that, in accordance with generally accepted concepts, treatment with INO alone, or with INO combined with surfactant, improves oxygenation and reduces intrapulmonary shunting by relaxation of the resistance vasculature of the lungs. Further experiments including hemodynamic measurements are needed to evaluate long-term outcome and possible side effects of this combined therapy in adult animals.

Instillation of surfactant may have an impact on pulmonary blood flow. Yu and colleagues, using a BAL-induced animal RDS model, reported that the effect of exogenous surfactant was associated with activation of endogenous NO production to maintain pulmonary arterial pressure (34). Our present results show that surfactant treatment alone leads to improved lung mechanics and gas exchange, associated with a modest but significantly reduced intrapulmonary shunting. In contrast, animals in the NO group showed only increased Pa_O2 and reduced intrapulmonary shunting, without improvement in lung mechanics. Surfactant-treated animals in both the S and SNO groups had significantly improved lung expansion as demonstrated by moderately increased FRC, high alveolar VV, and low CV(VV). Thus the effects of combined use of surfactant and INO actually reflect both improved diffusion of NO to expanded alveolar spaces and enhanced intrapulmonary blood perfusion.

In conclusion, in this study we found that ARDS induced by intravenous oleic acid and characterized by deterioration of lung mechanics and blood gas exchange, and increased intrapulmonary shunting, can be treated effectively by a combination of exogenous surfactant and INO. The effects of this combined therapy were modest in relation to baseline values but superior to those obtained with either treatment alone. The synergistic effects of exogenous surfactant and INO may have an implication in the treatment of ARDS, and should be further evaluated in clinical trials.