INTRODUCTION

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Acute respiratory distress syndrome (ARDS) may follow a variety of insults. Injury to the alveolar-capillary unit is a hallmark of this condition, with widespread nonuniform lung injury, pulmonary edema, diminished surfactant activity, pulmonary hypertension, and hypoxemia. The incidence of ARDS in adults has been estimated at 1.5 to 8.3 cases/100,000 population, and appears to vary by region (1). Approximately 3% of all pediatric intensive-care-unit (ICU) patients develop ARDS (5). The mortality rate in children with diagnosed ARDS ranges from 35 to 90% (6). Pulmonary hypertension is often present during the course of ARDS, and when persistent is associated with a poor prognosis (9, 10). Systemic intravenous vasodilators such as nitroglycerin, nitroprusside, and prostaglandin E₁ have been used to treat pulmonary hypertension, but their effectiveness is limited because they cause systemic hypotension and ventilation/perfusion mismatch (11). Treatment with inhaled nitric oxide (NO) gas reduces pulmonary vascular resistance, improves oxygenation, and decreases intrapulmonary shunt fraction in animals and humans with diagnosed ARDS (14). Unfortunately, therapy with NO gas in the ventilated patient requires its continuous administration, and its delivery and monitoring remain complex and cumbersome.

Compounds formed by reacting NO with various nucleophiles (NONOates) have vasorelaxant properties and induce vasodilation via release of NO (19). These compounds release NO spontaneously in physiologic solutions, are stable as solids, and are highly soluble in aqueous media (20). We recently showed that intrapulmonary instillation of NONOates results in selective reduction in pulmonary artery pressures and pulmonary vascular resistance without systemic vasodilation or hypotension in a porcine model of thromboxane-induced pulmonary hypertension (21, 22). Intermittent intrapulmonary administration of NONOates, and the avoidance of inhaled NO delivery and monitoring systems, could provide significant clinical advantages over continuously inhaled NO gas. Oleic acid-induced acute lung injury has been used as a model for ARDS in animals (23). In this model, intravenously administered oleic acid produces stable hypoxemia, increased intrapulmonary shunt, pulmonary hypertension, and widespread parenchymal lung injury histologically indistinguishable from human ARDS (14, 15).

We hypothesized that intrapulmonary delivery of a NONOate would improve oxygenation and selectively reduce pulmonary vascular resistance in a porcine model of oleic acid-induced acute lung injury and pulmonary hypertension. We further hypothesized that short-term intrapulmonary use of a NONOate would not cause pulmonary parenchymal or systemic toxicity.

	METHODS

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Animal Model

The study was approved by the Animal Care and Use Committee of the Children's Hospital Research Foundation. The care and handling of animals was done in accordance with National Institutes of Health (NIH) guidelines. Juvenile male purebred Yorkshire pigs (aged 8 to 10 wk, weight 13.4 ± 0.8 kg) were fasted on the day prior to surgery. General anesthesia was induced with ketamine hydrochloride, acepromazine, and atropine. The trachea was intubated and general anesthesia was maintained throughout the study with isoflurane, 70% nitrous oxide, and 30% oxygen. Additional anesthetic medications were provided as needed to maintain analgesia and sedation. Mechanical ventilatory support was provided with a Siemens Servo 900C ventilator (Siemens-Elema AB, Solna Sweden), with a tidal volume (VT) of 15 ml/kg and a positive end-expiratory pressure (PEEP) of 3 cm H₂O. Respiratory rate was adjusted to maintain an arterial PCO₂ of 40 ± 5 mm Hg, and the FI_O2 was fixed at 0.5. Intravenous d-tubocurarine was administered intermittently to maintain muscle relaxation. The animals' core temperature was maintained at 39.0 ± 0.5° C with a heating pad. Femoral venous and arterial catheters (Cook, Inc., Bloomington, IN) were placed by cutdown. During the period of surgical preparation, 5% dextrose (D5) ringer's lactate solution was infused intravenously at 20 ml/kg/h, with the rate reduced during recovery to 5 ml/kg/h. A median sternotomy was performed and catheters were placed in the main pulmonary artery, left atrium, and right atrium. A 16-mm ultrasonic flow probe (Transonics Systems Inc., Ithaca, NY) was placed around the pulmonary artery and connected to a small-animal flowmeter (Transonics Systems). A search was undertaken for a patent ductus arteriosus, and if one was found the ductus was ligated. Following surgery, the animals were allowed to stabilize for 30 min.

Monitoring Parameters

Mean arterial pressure (MAP), mean pulmonary artery pressure (), left atrial pressure (Pla), and right atrial pressure (Pra) were measured with calibrated transducers (Cobe Cardiovascular, Arvada, CO) and an amplifier monitor (Horizon 2000; Mennen Medical, Clarence, NY) with both waveform and digital readout. Cardiac index (CI) was calculated as cardiac output (CO)  animal weight in kg. Pulmonary vascular resistance index (PVRI) was calculated as 79.9 × (  Pla)  CI (dyne · s/cm⁵/kg) and systemic vascular resistance index (SVRI) as 79.9 × (MAP  RAP)  CI (dyne · s/cm⁵/kg). Oxygen saturation (Sa_O2) and heart rate (HR) were monitored with a pulse oximeter (Ohmeda 5250, Louisville, CO). CO was recorded from the digital readout of the flowmeter connected to the pulmonary artery flow probe. Arterial and mixed venous blood gas tensions were determined at baseline and every 10 min throughout the study, using a blood gas analyzer (Model 278; Ciba-Corning Diagnostic, Medfield, MA), and were corrected for core temperature. The alveolar-arterial difference in the partial pressure of oxygen (AaDO₂) was calculated as PA_O2  Pa_O2. Intrapulmonary shunt (%) was calculated as [(CcO₂  CaO₂)/(CcO₂  CvO₂)] · 100, where CcO₂ = pulmonary capillary O₂ content, CaO₂ = arterial O₂ content, and CvO₂ = mixed venous O₂ content. Dead space (%) was calculated as [1  PE_CO2/Pa_CO2] · 100, where PE_CO2 = end-tidal PCO₂, and Pa_CO2 = arterial PCO₂. Pulmonary function measurements and calculations, including respiratory rate (RR), peak and end-expiratory ventilatory pressures, VT, total lung resistance (RTL), and dynamic pulmonary compliance (Cdyn), were made with an in-line Ventrak 1550 pulmonary function monitor (Novametrix Medical Systems, Inc., Wallingford, CT).

Induction of Lung Injury and Pulmonary Hypertension

After baseline hemodynamic, pulmonary function, and gas exchange measurements were made, oleic acid (cis-9-octadecenoic acid; Sigma, Inc., St. Louis, MO) was given intravenously at a dose of 0.09 ml/kg into the femoral vein. Additional doses of oleic acid were given until the pulmonary vascular resistance was two- to threefold greater than its baseline value and Pa_O2 was reduced to 100 to 150 mm Hg. The total amount of oleic acid was administered in all cases over a 30- to 60-min period, and was equivalent in the two study groups of animals, treated with the NONOate 2-(dimethylamino)ethylputreanine/ NO (DMAEP/NO) and those given saline, respectively. Our preliminary investigations indicated that the degree of pulmonary hypertension and hypoxemia specified here was associated with moderate lung injury by histologic examination. Following the induction of lung injury, an additional stabilization period of 30 min was allowed.

NONOate Preparation and Delivery

Several members of the NONOate class of compounds induce dose-dependent vasorelaxation in the isolated rabbit aorta (24). We used the NONOate DMAEP/NO in the present study because of its tertiary-amine and charged cationic end-group characteristics. We believe that these characteristics are associated with reduced transepithelial permeability and limited systemic absorption, and that the NO released from DMAEP/NO therefore enters the pulmonary circulation without the parent compound, minimizing systemic vasodilation. DMAEP/NO (100 mg) was prepared as previously described (21), and was dissolved in 2 ml saline at 3 min before being administered. Solutions of DMAEP/NO or saline (2 ml) were placed in a small-volume nebulizer incorporated in-line along the inspiratory circuit of the ventilator. A single treatment was used, and solutions were nebulized over a 3- to 5-min period at an oxygen flow rate of 10 L/min (FI_O2 = 0.5). Hemodynamic, gas exchange, and pulmonary function measurements were made every 10 min for 1 h following nebulization.

Toxicity Studies

Arterial blood gas samples for methemoglobin and nitrite/nitrate analysis were obtained at baseline and every 30 min throughout all experiments. Methemoglobin concentrations were determined through co-oximetry (Ciba-Corning Diagnostics). All posttreatment methemoglobin values were summed and divided by the total number of measurements to derive a mean posttreatment methemoglobin value. The Griess reaction was used to measure total plasma nitrite concentrations as previously described (25, 26). In this assay, all nitrate was converted to nitrite. Nitrite concentrations were measured spectrophotometrically (Spectramax 250 plate reader; Molecular Devices Inc., Sunnyvale, CA) at an optical density of 550 nm (OD₅₅₀). Nitrite concentrations were calculated by comparison with a standard curve. All posttreatment nitrite values were summed and divided by the total number of measurements to derive a mean posttreatment nitrite/nitrate value. These measurements reflect total nitrite plus nitrate concentration, and are expressed as nitrite/nitrate in µM concentrations. No attempt was made to measure nitrite and nitrate independently of one another.

Animal Euthanasia and Pulmonary Histology

At the conclusion of the experiment, animals were euthanized with an overdose of thiopental. Lung samples from DMAEP/NO- and saline-treated animals were examined through routine histology. Samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 6 µm, and stained with hematoxylin and eosin (H&E). Sections were analyzed by a pathologist blinded to the experimental group from which they came. Two histologic scoring systems were used. The first system utilized a 0- to 3-point grading according to Broccard and colleagues (27). Each section was graded as follows: 0 = normal-appearing lung or atelectasis (atelectasis per se was disregarded and not scored as abnormality); 1 = mild congestion, interstitial edema, and neutrophilic infiltrate, with red blood cells and/or neutrophils seen only occasionally in the alveolar spaces; 2 = moderate congestion and interstitial edema, with neutrophils partially filling the alveolar spaces but without consolidation; and 3 = marked congestion and interstitial edema, with neutrophilic infiltrate nearly or completely filling the alveolar spaces. Inflammation confined to bronchi or in a peribronchial distribution was considered to represent bronchopneumonia and was disregarded. Because Broccard and colleagues' score does not consider the presence or absence of thrombi or hyaline membranes, a second score was calculated, based on the degree of: (1) congestion; (2) edema; (3) inflammation; (4) atelectasis; (5) thrombi; and (6) hyaline membrane formation in each sample. The severity of each of these six factors was estimated on a scale of 1 to 3. The overall histology score was then calculated as the average of the six factors multiplied by the percent of the sample section that was abnormal. Mean Broccard and colleagues' and overall histology scores for each group were then compared.

Statistical Analysis

All data are presented as mean ± SEM. Normally distributed, continuous data were analyzed with the unpaired two-tailed Student's t test. Nonparametric continuous data were analyzed with the Mann-Whitney rank sum test. Serial hemodynamic, gas exchange, and pulmonary function data for the control and DMAEP/NO groups were compared through repeated measures analysis of variance (ANOVA) or repeated measures ANOVA on ranks, as appropriate. Pathologic differences between groups (Broccard and colleagues' score and overall pathology score) were compared through the unpaired two-tailed Student's t test. Significance was attributed to values of p < 0.05.

	RESULTS

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Seventeen animals were randomly assigned to either the saline or DMAEP/NO treatment group prior to the study. Five animals died either during the surgical preparation (n = 2) or during oleic acid infusion (n = 3), leaving 12 animals randomized to the DMAEP/NO (n = 6) or saline (n = 6) treatment group. Gas exchange, pulmonary function, and hemodynamic data after the surgical preparation (baseline) and after oleic acid infusion are shown in Tables 1 and 2, respectively. There was a significant increase in , PVRI, AaDO₂, RTL, and dead space, and a significant reduction in Pa_O2 and Cdyn in both groups after oleic acid infusion. Oleic acid infusion resulted in a reduction in MAP and CI and an increase in intrapulmonary shunt in both groups. SVRI was significantly lower before treatment in the DMAEP/NO group than in the control group. Other than SVRI, there were no significant differences between the two groups in hemodynamic, gas exchange, or pulmonary function variables prior to treatment.

After nebulization treatment, a significant increase in Pa_O2 and decrease in AaDO₂ at 30 and 60 min was observed in the DMAEP/NO group in comparison with saline-treated animals (Figure 1). In addition, DMAEP/NO-treated animals had a reduction in intrapulmonary shunt in comparison with saline-treated animals; however, this result did not reach statistical significance. Animals treated with DMAEP/NO had a significantly greater reduction in than did the control animals (Figure 2). PVRI was reduced to a greater extent in DMAEP/ NO-treated animals then in control animals (from 81 ± 17 dyne · s/cm⁵/kg to 34 ± 8 dyne · s/cm⁵/kg in the DMAEP/NO group; and from 104 ± 16 dyne · s/cm⁵/kg to 64 ± 11 dyne · s/cm⁵/ kg in the saline group at 60 min; p < 0.05). After nebulization, there were no significant changes within each group or between treatment groups in MAP, SVRI, CI, lung compliance, RTL, or pulmonary dead space in comparison with values following oleic acid infusion.

View larger version (28K): [in this window] [in a new window]   Figure 1.   Comparison of effect of nebulized saline and nebulized DMAEP/NO on PaO2, AaDO2, and intrapulmonary shunt after induction of acute lung injury by oleic acid infusion. Dark solid bars represent saline control group, light solid bars represent DMAEP/ NO group. Qs/Qt = intrapulmonary shunt; OA = oleic acid; *p < 0.05 in comparison with control values, as well as with DMAEP/ NO values at the end of the oleic acid infusion.

View larger version (14K): [in this window] [in a new window]   Figure 2.   Comparison of effect of nebulized saline and nebulized DMAEP/NO on mean pulmonary artery pressure (), percent change in mean arterial pressure (MAP), and percent change in cardiac index (CI) after induction of acute lung injury by oleic acid infusion. Diamonds represent saline control group, squares represent DMAEP/NO group; #p < 0.05 in comparison with Ppa at end of oleic acid infusion; *p < 0.05 in comparison with control group.

Mean posttreatment arterial methemoglobin values were similar in the DMAEP/NO- and saline-treated groups (0.27 ± 0.01% versus 0.28 ± 0.01%, p = 0.18). The highest methemoglobin value in the DMAEP/NO-treated animals was 0.5%, whereas the highest value in the saline-treated animals was 0.4%. Serum nitrite/nitrate values before and at 30 min and 60 min after nebulization with DMAEP/NO were 255 ± 43, 261 ± 45, and 242 ± 28 µM, respectively. Serum nitrite/nitrate values before and at 30 min, and 60 min after nebulization with saline were 270 ± 35, 276 ± 31, and 301 ± 40 µM, respectively. There were no significant intra- or intergroup differences in serum nitrite/nitrate.

The histologic findings in both the DMAEP/NO- and saline-treated groups consisted of varying degrees of alveolar congestion, edema, and inflammation, associated with a prominent disturbance in aeration. In addition, microvascular thrombi and hyaline membranes were occasionally seen. The mean Broccard and colleagues' score was 1.7 ± 0.2 in the saline-treated animals and 1.2 ± 0.3 in the DMAEP/NO-treated animals (p = 0.11). The mean overall pathology score was 1.1 ± 0.1 in the saline-treated group and 0.8 ± 0.2 in the DMAEP/NO group (p = 0.19). Therefore, neither scoring system revealed any significant differences between the groups in lung histology.

	DISCUSSION

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NO is a smooth-muscle vasodilator that is rapidly inactivated in the bloodstream upon binding to hemoglobin (28). Inhaled NO gas results in pulmonary vasodilation that is limited to ventilated lung regions, thereby improving intrapulmonary shunt (15, 16). Previous studies of oleic acid-induced acute lung injury in a porcine model demonstrated improvements in oxygenation and intrapulmonary shunt fraction after inhaled NO gas treatment (14). The present study is the first to demonstrate that administration of a soluble NO donor can improve oxygenation and reduce pulmonary hypertension in experimental acute lung injury. This study further supports the concept that soluble NO donors may be an alternative to continuously inhaled NO gas for pulmonary vasodilation. Inhaled NO gas selectively reduces pulmonary hypertension and improves oxygenation in adults and children with ARDS (17, 18). The gas can be delivered with precision, and the response to it is rapid. However, the clinical usefulness of inhaled NO gas is limited by the need for its continuous administration and by complexities of delivery methods and monitoring systems for it, as well as by potential toxicity. Administration of NO gas in the patient-care setting requires NO/nitrogen tanks, in-line electrochemical or chemiluminescence NO and nitrogen dioxide (NO₂) monitors, ventilator adaptations, an additional ventilator source gas/NO blender, and environmental NO and NO₂ gas evacuation systems. Abrupt discontinuation of inhaled NO gas therapy has been associated with life-threatening deterioration in gas exchange and rebound pulmonary hypertension (29).

Soluble NO donor compounds that release NO over periods of hours and to which the alveolar-capillary junction is impermeable hold great promise. The polyamines (e.g., putreanine, spermine, spermidine) are naturally occurring compounds derived from methionine and ornithine, are polar, carry a cationic charge, and can serve as nucleophile carriers of NO. Modification of the polyamine base structure confers variability on the NO release profile. DMAEP/NO is a putreanine-based compound with a half-life for NO release of approximately 135 min.

We have previously shown that DMAEP/NO selectively reduces pulmonary vascular resistance in a porcine model of pulmonary hypertension without acute lung injury (21, 22). In the current study, animals received a moderately severe lung injury characterized by decreased compliance and increased RTL, dead space ventilation, intrapulmonary shunt, and hypoxemia. In this model the histologic appearance of the lungs was notable for interstitial fluid accumulation, proteinaceous alveolar edema, pulmonary capillary obstruction, and microthrombosis. These physiologic and pathologic changes are similar to those observed in humans with ARDS (30, 31). Despite the severity of the injury in our model, nebulized DMAEP/NO was effective in significantly improving oxygenation by over 30% at 30 and 60 min after treatment. In contrast, animals treated with nebulized saline exhibited no significant improvement in oxygenation. The oxygenation effects noted with delivery of nebulized DMAEP/NO were similar to those observed in both animals and human subjects given continuously inhaled NO gas (10, 16, 32). The improvement in oxygenation in the present study was probably due to improvements in intrapulmonary shunt. Confirmation of this explanation requires analysis of the distribution of DMAEP/NO after nebulization, as well as comprehensive and quantitative ventilation/perfusion distribution studies done with multiple inert gas techniques.

We noted posttreatment variability in oxygenation among animals, including one animal with a 12% deterioration in Pa_O2 at 60 min after the administration of DMAEP/NO. There are several possible explanations for this finding. First, NO may have diffused from a well-ventilated lung region to a poorly ventilated one, with subsequent increase in blood flow to the latter region and concomitant worsening of intrapulmonary shunt. Second, delivery of aerosolized DMAEP/NO to the lung in this animal may have been reduced through excessive losses in the ventilatory circuit. A previous study found that nebulization of surfactant may result in delivery to the lung of less than 1% of the amount delivered by instillation (35). Additionally, the underlying disease process in this animal may have been associated with irreversible vasoconstriction and lack of response to DMAEP/NO. Changes in oxygenation with inhaled NO therapy are also variable in humans with acute lung injury (36, 37).

Pulmonary hypertension often occurs in ARDS, and is a result of inflammatory mediator release, alveolar hypoxia, endothelial swelling, and capillary microthrombosis (15). Pulmonary hypertension causes increased right ventricular work, and can ultimately lead to right ventricular failure. Persistent pulmonary hypertension is associated with a poor prognosis in adults and children with ARDS (9, 10). Intravenous administration of vasoldilators may reduce pulmonary vascular resistance, but may also cause hypotension, exacerbate ventilation/ perfusion mismatch, and worsen hypoxemia (11, 12). Animals in the present study that received DMAEP/NO after induction of acute lung injury exhibited a significant reduction in pulmonary artery pressure and PVRI similar to that seen in our two previous studies (21, 22). At the conclusion of the oleic acid infusion, both control and DMAEP/NO-treated animals had a doubling of pulmonary artery pressures to a mean of 25 mm Hg. Values of decreased to 16 mm Hg after 30 min and to 14 mm Hg after 1 h in DMAEP/NO-treated animals. This selective reduction in pulmonary hypertension after DMAEP/NO treatment, unassociated with alterations in MAP, SVRI, or CI, is the most important characteristic of an ideal pulmonary vasodilator.

Aerosolized delivery of soluble NO donors is new, and a comparison between the amount of NO delivered with these compounds and by continuously inhaled NO gas is therefore important. In making this comparison, we made two assumptions. First, we assumed that the amount of NO released from DMAEP/NO in vivo is similar to that released in vitro. Second, because no data are available with which to estimate the percentage of delivered DMAEP/NO or NO gas that actually reaches the lung after losses in the ventilatory system and large airways, we assumed that it was 100% in both cases. Dissolving 100 mg DMAEP/NO in a physiologic solution results in the liberation of 105 µmol of NO gas over the 135-min NO release half-life of this compound as determined with a chemiluminescence meter (data not shown). By convention, NO gas delivery is measured in terms of the concentration of the total inspired gas, which is usually given in ppm. Each ppm of inhaled NO gas is equivalent to 0.0333 µmol NO/L. Animals such as those in the present study, which weighed 13 kg, were ventilated at 20 breaths/min using a VT of 15 ml/kg, and received 5 ppm of inhaled NO gas would receive 88 µmol of NO over a 135-min period. Given these conditions and the assumptions described earlier, aerosolization of 100 mg DMAEP/NO delivers a dose of NO similar to that with NO gas at 5 ppm.

The major sources of potential toxicity of inhaled NO gas are the formation of NO₂, the production of methemoglobin, and the formation of peroxynitrate and other free radicals. NO₂ is produced when NO reacts with O₂ in the delivery system (38, 39). NO₂ has been associated with increased airway reactivity and lung injury (40, 41). Although we did not measure exhaled NO₂ levels, we speculate that given the estimated NO released from DMAEP/NO, along with the short period of nebulization, NO₂ production would be minimal. The conversion of NO to NO₂ depends on the NO concentration, FI_O2, and residence time of NO in the lung (39). Delivery of NO gas in concentrations  20 ppm in the presence of an FI_O2 0.75 has been shown not to produce toxic ( 2 ppm) concentrations of NO₂ (39). This speculation will require confirmation in the laboratory.

Methemoglobin is produced when NO binds with hemoglobin in the circulation. We were unable to measure increased methemoglobin levels in animals treated with DMAEP/NO. The low methemoglobin and low nitrite/nitrate values in the DMAEP/NO-treated group suggests that systemic transit of this compound is insignificant. Furthermore, the findings of decreased methemoglobin values is consistent with that in previous work with acute lung injury in dogs (42). The absence of measureable circulating putreanine in DMAEP/NO-treated animals would provide further confirmation of negligible systemic absorption of DMAEP/NO. This work is currently ongoing in our laboratory. Peroxynitrate, a strong oxidant that catalyzes lipid peroxidation, is formed when superoxide reacts with NO (43). Peroxynitrate free radicals are key mediators in tissue injury, and increased levels of these radicals remain a potential consequence of NO therapy. Evaluation of production of peroxynitrate and other free radicals after treatment with DMAEP/NO awaits further study.

In summary, the present study is the first to demonstrate that a soluble tertiary amine and charged NO donor (NONOate), when administered into the lung, can significantly reduce pulmonary hypertension and improve oxygenation in a porcine model of acute lung injury. It is possible that NONOates will play a role in the treatment of ARDS and other lung diseases associated with pulmonary hypertension and surfactant deficiency. In addition, the ability to deliver NO as an intermittent aerosol with a long duration of activity may be promising in the therapy of chronic lung disease associated with pulmonary hypertension.