Combined Surfactant Therapy and Inhaled Nitric Oxide ... - ATS Journals

Combined Surfactant Therapy and Inhaled Nitric Oxide in Rabbits with Oleic Acid-induced Acute Respiratory Distress Syndrome GUANG FA ZHU, BO SUN, SHAN FU NIU, YING YUN CAI, KE LIN, ROBERT LINDWALL, and BENGT ROBERTSON Children’s Hospital Research Institute; Department of Pulmonology, Zhongshan Hospital, Shanghai Medical University; Institute of Shanghai Shen-Ning, Applied Biochemistry, Shanghai, China; Department of Anesthesiology and Intensive Care, Danderyd Hospital, Danderyd; and Division for Experimental Perinatal Pathology, Karolinska Hospital, Stockholm, Sweden

Intratracheal administration of surfactant and inhaled nitric oxide (INO) have had variable effects in clinical trials on patients with acute respiratory distress syndrome (ARDS). We hypothesized that combined treatment with exogenous surfactant and INO may have effects in experimental ARDS. After intravenous infusion of oleic acid in adult rabbits and 4–6 h of ventilation, there was more than a 40% reduction in both dynamic compliance (Cdyn) of the respiratory system and functional residual capacity (FRC), a 50% increment of respiratory resistance (Rrs), a 70% reduction in PaO2 /FIO2, and an · · increase in intrapulmonary shunting (Q S/Q T) from 4.4 to 33.5%. The animals were then allocated to groups receiving (1) neither surfactant nor INO (control), (2) 100 mg/kg of surfactant (S) administered intratracheally, (3) 20 ppm INO (NO), or (4) 100 mg/kg of surfactant and 20 ppm INO (SNO), and subsequently ventilated for 6 h. After the period of ventilation, the animal lungs were used for analysis of disaturated phosphatidylcholine (DSPC) and total proteins (TP) in bronchoalveolar lavage fluid (BALF), and for determination of alveolar volume density (VV). The animals in the control group had the lowest survival rate, and no improvement in lung mechanics and blood oxygenation, whereas those in the S group had a modest but statistically significant improvement in Cdyn, Rrs, · · PaO2 and FRC, reduced Q S/Q T, lowered minimum surface tension (gmin) of BALF, and increased DSPC/ · · TP and alveolar VV. The NO group had increased PaO2 and reduced Q S/Q T. The SNO group showed improved Cdyn, Rrs, FRC, DSPC/TP, alveolar VV, and gmin of BALF comparable to the S group, but · · there was a further increase in survival rate and PaO2, and additional reduction in Q S/Q T and TP in BALF. These results indicate that, in this animal model of ARDS, a combination of surfactant therapy and INO is more effective than either treatment alone. Zhu GF, Sun B, Niu SF, Cai YY, Lin K, Lindwall R, Robertson B. Combined surfactant therapy and inhaled nitric oxide in rabbits with AM J RESPIR CRIT CARE MED 1998;158:437–443. oleic acid-induced acute respiratory distress syndrome.

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 (Received in original form November 24, 1997 and in revised form February 26, 1998) Supported in part by Grant 95-620 from the China Medical Board of New York, Shanghai Educational Development Foundation (B.S.); Shanghai Medical University Research Funds (S.F.N.) and a Travel Grant for Foreign Expert (R.L.); the Swedish Medical Research Council (Project 3351) and Konung Oscar II:s Jubileumsfond (B.R.). Correspondence and requests for reprints should be addressed to Bo Sun, M.D., Ph.D., Children’s Hospital, Shanghai Medical University, 183 Feng Lin Road, Shanghai 200032, China. E-mail: [email protected] Am J Respir Crit Care Med Vol 158. pp 437–443, 1998 Internet address: www.atsjournals.org

with ARDS (4, 5), or various concentrations and periods of inhaled nitric oxide (INO) in persistent pulmonary hypertension of the newborn (PPHN) and ARDS (6–11), 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

438

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

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 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 3 g and room temperature to remove cell debris. The supernatant was centrifuged for 120 min at 5,000 3 g and 48 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 3 g and 48 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 3 g and 48 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 48 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 2208 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 6 SD, 2.8 6 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 (FIO2) 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, PaO2, and PaCO2 with a Ciba-Corning 170 automatic blood gas analyzer (Huanqiu Medical Instruments, Shanghai, China), and for determination of nitrite/nitrate and methemoglobin

VOL 158

1998

(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 ( Q S/ Q T) was determined by measuring oxygen content in mixed venous blood (when FIO2 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 ml/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 FIO2 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, PaCO2, and PaO2 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), PaO2 , 120 mm Hg, and Q S/ Q 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, and arterial pH, PaO2, and PaCO2 . FRC, . were measured each hour. Q S/ Q 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 808 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 3 g and 48 C to remove cell debris, and the supernatant was stored at 2208 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).

Zhu, Sun, Niu, et al.: Combined Surfactant Therapy and INO in Rabbits 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

439

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 378 C. Surface tension of

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; 1p , 0.05, 11p , 0.01 versus control group.

440


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 Q S/ Q T, FIO2 was temporarily raised to 1.0. Group definitions: C 5 control; NO 5 nitric oxide, S 5 surfactant; SNO 5 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.

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 (gmin and gmax, 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 ar-

VOL 158

1998

teries 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: 3300), 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 General Conditions of the Animals

Infusion of oleic acid induced, in all animals, respiratory failure within 4–6 h (mean 6 SD, 4.5 6 0.6), as reflected by marked decreases in Cdyn, FRC, and PaO2, and increases in · · Rrs (Figure 1), Q S/Q 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 6 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 as-

TABLE 1 TOTAL PHOSPHOLIPIDS, TOTAL PROTEINS, DISATURATED PHOSPHATIDYLCHOLINE IN TOTAL PHOSPHOLIPIDS AND TOTAL PROTEINS IN, AND MINIMUM AND MAXIMUM SURFACE TENSION,* OF BRONCHOALVEOLAR LAVAGE FLUID† Surface tension (mN/m)

Group

TPL (mg/kg)

TP (mg/kg)

DSPC/TPL (%)

DSPC/TP (mg/mg)

gmin

gmax

Control NO S SNO

9.7 6 1.0 10.0 6 2.2 32.3 6 7.3‡ 36.9 6 5.3‡

198 6 16 190 6 32 193 6 34 142 6 17§i

30.6 6 4.3 32.7 6 2.9 44.6 6 4.1‡ 43.8 6 3.5‡

17.1 6 6.0 17.9 6 3.6 73.7 6 25.4‡ 91.1 6 23.9‡

24.3 6 5.2 26.0 6 7.7 9.4 6 7.2‡ 2.3 6 1.6‡

37.0 6 6.2 37.6 6 8.3 29.4 6 2.7 36.1 6 9.1

Definition of abbreviations: TPL 5 total phospholipids; TP 5 total proteins; DSPC 5 disaturated phosphatidylcholine; NO 5 nitric oxide; S 5 surfactant; SNO 5 surfactant and nitric oxide. * gmin and gmax obtained at cycle 120. † Values are means 6 SD (n 5 5). ‡ p , 0.01 versus control and NO groups. § p , 0.05 versus control and NO groups. i p , 0.05 versus S group.

441

Zhu, Sun, Niu, et al.: Combined Surfactant Therapy and INO in Rabbits TABLE 2 BLOOD NITRITE/NITRATE AND METHEMOGLOBIN AT BASELINE AND TREATMENT TIME 0 AND 4 h* MetHb (%)

Nitrite/nitrate (mmol/L) Group

Baseline

0h

4h

Baseline

0h

4h

Control NO S SNO

64.4 6 16.7 58.3 6 9.8 53.2 6 23.0 51.8 6 9.3

78.1 6 12.0 80.5 6 14.2 81.3 6 15.8 66.9 6 9.0

82.9 6 19.4 103.9 6 14.7 74.8 6 32.7‡ 101.2 6 15.5

0.78 6 0.29 0.57 6 0.14 0.60 6 0.15 0.66 6 0.17

0.83 6 0.24 0.59 6 0.14† 0.72 6 0.13 0.73 6 0.16

0.89 6 0.18 1.25 6 0.38 0.78 6 0.13 1.22 6 0.31

Definition of abbreviations: NO 5 nitric oxide; S 5 surfactant; SNO 5 surfactant and nitric oxide; MetHb 5 methemoglobin. * Values are means 6 SD (n 5 8 or 9). † p , 0.01 versus control group. ‡ p , 0.05 versus NO and SNO groups.

sociation 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. 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 PaCO2 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. Val· · ues of Cdyn, Rrs, FRC, PaO2, PaCO2, pH, and Q S/Q 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 · · Q S/Q 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 PaO2 and Q S/Q T, but no substantial improvement in Cdyn, Rrs, and FRC. In contrast, · · Cdyn, Rrs, FRC, PaO2, and Q S/Q T were all moderately improved in the S group, compared with the control group. In · · the SNO group, Cdyn, Rrs, PaO2, Q S/Q T, and FRC were also improved compared with the control and NO groups, but PaO2 · · and Q S/Q T were further improved compared with the S group (for detailed statistical analysis, see captions to Figures 1 and 2).

TABLE 3 WET-TO-DRY LUNG WEIGHT RATIO (n 5 5), VOLUME DENSITY OF ALVEOLAR SPACES (VV), AND COEFFICIENT OF VARIATION OF VV [CV(VV)] (n 5 4)* Group

Wet-to-dry Lung Weight Ratio

VV

CV(VV)

Normal Control NO S SNO

7.93 6 0.14 7.89 6 0.56 7.28 6 0.28‡ 6.93 6 0.41†

0.74 6 0.02† 0.40 6 0.07 0.41 6 0.13 0.67 6 0.02† 0.64 6 0.05†

0.12 6 0.02† 0.41 6 0.08 0.42 6 0.19 0.23 6 0.02‡ 0.23 6 0.08‡

Definition of abbreviations: Normal 5 normal lungs before the induction of injury; NO 5 nitric oxide; S 5 surfactant; SNO 5 surfactant and nitric oxide. * Values represent means 6 SD. † p , 0.01 versus control and NO groups. ‡ p , 0.05 versus control and NO groups.

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, gmin and gmax 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 gmin and gmax in BALF after 120 min of dynamic cycling are shown in Table 1. In the control and NO groups, gmin and gmax 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 gmin 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.

442


DISCUSSION 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 PaO2 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 acidinduced 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

VOL 158

1998

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 PaO2 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. Acknowledgment : The authors are grateful to Dr. Jia Ma for measurement of FRC, Mr. Lie Wei Zhu for morphometric analysis of the lungs, and Mrs. Yue Yin Ding and Feng Fei Xu for technical assistance.

References 1. Hallman, M., R. G., Spragg, J. H. Harrell, K. M. Moser, and L. Gluck. 1982. Evidence of lung surfactant abnormality in respiratory failure: study of bronchoalveolar lavage phospholipids, surface activity, phospholipase activity, and plasma myoinositol. J. Clin. Invest. 70:673–683. 2. Bernard, G. R., A. Artigas, K. L. Brigham, J. Carlet, K. Falke, L. Hudson, M. Lamy, J. R. Legall, A. Morris, and R. Spragg. 1994. The American–European Consensus Conference on ARDS, definition, mechanisms, relevant outcomes, and clinical trial coordination. Am. J. Respir. Crit. Care Med. 149:818–824. 3. Baughman, R. P., K. L. Gunther, M. C. Rashkin, D. A. Keeton, and E. D. Pattishall. 1996. Changes in the inflammatory responses of the lung during acute respiratory distress syndrome: prognostic indicators. Am. J. Respir. Crit. Care Med. 154:76–81. 4. Wilson, D. F., J. H. Jiao, L. A. Bauman, A. Zaritsky, H. Craft, K. Dockery, D. Conrad, and H. Dalton. 1996. Calf’s lung surfactant extract in acute hypoxemic respiratory failure in children. Crit. Care Med. 24:

Zhu, Sun, Niu, et al.: Combined Surfactant Therapy and INO in Rabbits 1316–1322. 5. Gregory, T. J., K. P. Steinberg, R. G. Spragg, J. E. Gadek, T. M. Hyers, W. J. Longmore, M. A., Moxley, G. Z. Cai, R. D. Hite, R. M. Smith, L. D. Hudson, C. Crim, P. Newton, B. R. Mitchell, and A. Gold. 1997. Bovine surfactant therapy for patients with acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 155:1309–1315. 6. The Neonatal Inhaled Nitric Oxide Study Group. 1997. Inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure. N. Engl. J. Med. 336:597–604. 7. Rossaint, R., K. F. Falke, F. Lopez, K. Slama, U. Pison, and W. M. Zapol. 1993. Inhaled nitric oxide for the adult respiratory distress syndrome. N. Engl. J. Med. 328:399–405. 8. Gerlach, H., D. Pappert, K. Lewandowski, R. Rossiant, and K. J. Falke. 1993. Long-term inhalation with evaluated low doses of nitric oxide for selective improvement of oxygenation in patients with adult respiratory distress syndrome. Intensive Care Med. 19:443–449. 9. Zapol, W. M., S. Rimar, N. Gillis, M. Marletta, and C. H. Bosken. 1994. Nitric oxide and the lung. Am. J. Respir. Crit. Care Med. 149:1375– 1380. 10. Krafft, P., P. Fridrich, R. D. Fitzgerald, D. Koc, and H. Steltzer. 1996. Effectiveness of nitric oxide inhalation in septic ARDS. Chest 109: 486–493. 11. Day, R. W., M. Guarin, J. M. Lynch, D. D. Vernon, and J. M. Dean. 1996. Inhaled nitric oxide in children with severe lung disease: results of acute and prolonged therapy with two concentrations. Crit. Care Med. 24:215–221. 12. Gaston, B., J. M. Drazen, J. Loscalzo, and J. S. Stamler. 1994. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med. 149:538–551. 13. Frostell, C. G., M. D. Fratacci, J. C. Wain, R. Jones, and W. M. Zapol. 1991. A selective pulmonary vasodilator reversing hypoxic pulmonary vasoconstriction. Circulation 83:2038–2047. 14. Walti, H., B. S. Polla, and M. Bachelet. 1997. Modified natural porcine surfactant inhibits superoxide anions and proinflammatory mediators released by resting and stimulated human monocytes. Pediatr. Res. 41:114–119. 15. Kinsella, J. P., T. A. Parker, H. Galan, B. C. Sheridan, A. C. Halbower, and S. H. Abman. 1997. Effects of inhaled nitric oxide on pulmonary edema and lung neutrophil accumulation in severe experimental hyaline membrane disease. Pediatr. Res. 41:457–463. 16. Bartlett, G. B. 1959. Phosphorous assay in column chromatography. J. Biol. Chem. 234:466–468. 17. Mason, R. J., J. Nellenbogen, and J. A. Clements. 1976. Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17:281–284. 18. Hall, S. B., A. R. Venkitaraman, R. W. Hyde, and R. H. Notter. 1990. Altered function of pulmonary surfactant in fatty acid lung injury. J. Appl. Physiol. 69:1143–1149. 19. Skimming, J. W., V. G. DeMarco, and S. Cassin. 1995. The effects of nitric oxide inhalation on the pulmonary circulation of preterm lambs.

443 Pediatr. Res. 37:35–40. 20. Sun B., T. Curstedt, and B. Robertson. 1996. Exogenous surfactant improves ventilation efficiency and alveolar expansion in rats with meconium aspiration. Am. J. Respir. Crit. Care Med. 154:764–770. 21. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275. 22. Sun, B., T. Curstedt, and B. Robertson. 1996. Long-term cycling of surfactant films in Wilhelmy balance. Reprod. Fertil. Dev. 8:173–181. 23. Sun, B., E. Herting, T. Curstedt, and B. Robertson. 1994. Exogenous surfactant improves lung compliance and oxygenation in adult rats with meconium aspiration. J. Appl. Physiol. 77:1961–1971. 24. Shi, Y., H. Q. Li, C. K. Shen, J. H. Wang, J. Pan, S. W. Qin, and R. Liu. 1993. Association between protective efficacy of antibodies to tumor necrosis factor and suppression of nitric oxide production in neonatal rats with fatal infection. Pediatr. Res. 34:345–348. 25. Hegesh, E., N. Gruener, S. Cohen, R. Bochkovsky, and H. I. Shuval. 1970. A sensitive micromethod for the determination of methemoglobin in blood. Clin. Chim. Acta 30:679–682. 26. Ennema, J. J., T. Kobayashi, B. Robertson, and T. Curstedt. 1988. Inactivation of exogenous surfactant in experimental respiratory failure induced by hyperoxia. Acta Anaesthesiol. Scand. 32:665–671. 27. Hall, S. B., R. W. Hyde, and R. H. Notter. 1994. Changes in subphase aggregates in rabbits injured by free fatty acid. Am. J. Respir. Crit. Care Med. 149:1099–1106. 28. Putensen, C., J. Rasanen, F. A. Lopez, and J. B. Downs. 1994. Continuous positive airway pressure modulates effect of inhaled nitric oxide on the ventilation–perfusion distributions in canine lung injury. Chest 106:1563–1569. 29. Zucker, A. R., B. A. Holm, G. P. Crawford, K. Ridge, L. D. H. Wood, and I. Sznajder. 1992. PEEP is necessary for exogenous surfactant to reduce pulmonary edema in canine aspiration pneumonitis. J. Appl. Physiol. 73:679–686. 30. Shah, N. S., D. K. Nakayama, T. D. Jacob, I. Nishio, T. Imai, T. R. Billiar, R. Exler, S. A. Yousem, E. K. Motoyama, and A. B. Peitzman. 1997. Efficacy of inhaled nitric oxide in oleic acid-induced acute lung injury. Crit. Care Med. 25:153–158. 31. Leeman, M., V. Z. de Beyl, E. Gilbert, C. Melot, and R. Naeije. 1993. Is nitric oxide released in oleic acid lung injury? J. Appl. Physiol. 74:650– 654. 32. Finer, N. N., P. C. Etches, B. Kamstra, A. J. Tierney, A. Peliowski, and C. A. Ryan. 1994. Inhaled nitric oxide in infants referred for extracorporeal membrane oxygenation: dose response. J. Pediatr. 124:302–308. 33. Roberts, J. D., P. Lang, L. M. Bigatello, G. J. Vlahakes, and W. M. Zapol. 1993. Inhaled nitric oxide in congenital heart disease. Circulation 87:447–453. 34. Yu, X. Q., B. A. Feet, A. Moen, T. Curstedt, and O. D. Saugstad. 1997. Nitric oxide contributes to surfactant-induced vasodilation in surfactant-depleted newborn piglets. Pediatr. Res. 42:151–156.