INTRODUCTION

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Surfactant dysfunction/deficiency has been repeatedly demonstrated in numerous experimental models of acute lung injury as well as in humans with acute respiratory distress syndrome (ARDS) (1). It is therefore perplexing why, other than for neonatal respiratory distress syndrome (RDS) (4), the effectiveness of exogenous surfactant has not been convincingly demonstrated (5, 6). Undoubtedly, factors that may be responsible for surfactant effectiveness are complex and include the type and dose of surfactant, the method of administration, and the etiology and/or severity of the lung injury. Thus, it is still unclear whether it was the surfactant (Exosurf [GlaxoWellcome, Inc., Research Triangle Park, NC], artificial nonprotein containing), the administration technique (aerosol), or the lung injury (sepsis) that was responsible for the demonstrated lack of benefit in the largest published randomized trial of exogenous surfactant in ARDS (5).

Consistent with the results in humans (5), in a neonatal piglet model of ARDS, we were unable to show that Exosurf was effective when administered by bolus instillation using a dose of 67.5 mg phospholipid/kg in a volume of 5 ml/kg (7). In another study, bolus administration of Exosurf at 202 mg phospholipid/kg (volume: 5 ml/kg) was not as effective as Exosurf administered by lung lavage at a calculated retained dose of 136 mg phospholipid/kg (8). The lung lavage procedure consisted of administering Exosurf (13.5 mg phospholipid/ml) using a volume of 35 ml/kg instilled by gravity immediately followed by passive gravity drainage of excess surfactant fluid. Exosurf administered in this manner distributed more evenly than bolus instillation, suggesting that this was one of the reasons for the observed differences in pulmonary function.

Recent data suggest that the saturated phospholipid content of the lung in humans may be as low as 1.9 µmol/kg (9). Thus, much lower doses of exogenous surfactant than previously used should be effective in the treatment of surfactant deficient/dysfunction states, provided that the dose is large enough to overcome the inhibitory effects of foreign protein and lung distribution is uniform. This may not occur when surfactant is administered by either bolus (10) or aerosol (11).

Therefore, in this investigation, we further studied the lung lavage method of surfactant administration in an acute lung injury model. First, we compared the effects of two concentrations (13.5 mg/ml; 4.5 mg/ml) of Exosurf administered by lung lavage. We then compared the effects of several dilute preparations of artificial and natural surfactants.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal Preparation

This study was approved by the Institutional Animal Care and Use Committee of Tripler Army Medical Center and was in compliance with the Animal Welfare Act and the National Research Council's Guide for the Care and Use of Laboratory Animals. These facilities are fully accredited by the American Association for the Accreditation of Laboratory Animal Care. Under pentobarbital anesthesia (Wyeth Laboratories, Philadelphia, PA; initial dose 30 mg/kg, subsequent doses 2.5-5 mg/kg), 4-8 d-old piglets of either sex, weighing 1.8-3.0 kg, were intubated with a 4.0-mm cuffed endotracheal tube. Immediately after intubation, the lungs were inflated with 100 ml of air to reduce pulmonary atelectasis. Mechanical ventilation was initiated with the VIP Bird ventilator (Bird Products Corp., Palm Springs, CA) using a fractional inspired oxygen (FI_O2) of 0.21, ventilator rate of 40, and positive end-expiratory pressure (PEEP) of 2 cm H₂O. The peak inspiratory pressure (PIP) was adjusted to maintain end-tidal CO₂ between 40-45 mm Hg (Hewlett Packard 47210A; Palo Alto, CA). Catheters were placed in the femoral artery, femoral vein, and the right atrium via the external jugular vein. The femoral artery and right atrial catheters were connected to pressure transducers (Hewlett Packard 66#M1176A6) for continuous monitoring of mean arterial blood pressure (MAP) and central venous pressure (CVP), respectively. Infusion of normal saline (100 ml/kg/d) and medications were administered via the femor venous catheter. Body temperature was monitored with a rectal probe and maintained between 37.5-39° C using a warming blanket and heating pads. Hematocrit, serum electrolytes, and osmolality were determined at the beginning and end of the experiment to verify the fluid and electrolyte stability of the animal preparation.

Pulmonary Function

Respiratory flow and airway pressures were monitored continuously using a low dead space pneumotachometer (Hans Rudolph 8313; Kansas City, MO). An 8 French fluid-filled catheter was placed in the lower third of the esophagus for measurement of esophageal pressure (Pes) and its placement validated by occlusion (12). Digitized signals for flow, tidal volume (VT), airway pressure, and Pes were collected on a personal computer-based data acquisition system (AT-CODAS; Dataq Instruments, Akron, OH). Dynamic lung compliance (Cdyn) and lung resistance (RL) were calculated from the previously mentioned data. Functional residual capacity (FRC) was measured by helium dilution (Equilibrated Bio Systems, Inc., Melville, NY). Arterial blood gases and arterial and venous co-oximetry measurements were also obtained. Ventilation efficiency index (VEI) was calculated using the formula:

Hemodynamic Measurements

Hemodynamic measurements consisted of MAP, heart rate (HR), and CVP. The shunt fraction was calculated by the formula = (Cc_O2  Ca_O2)/(Cc_O2  Cv_O2). Ca_O2 and Cv_O2 are the systemic arterial and mixed venous oxygen content, respectively. Cc_O2 is the mean end capillary oxygen content and was calculated as Cc_O2 = (1.39 × total hemoglobin × oxyhemoglobin) + (0.003 × PA_O2).

Surfactant Preparations

Infasurf (ONY Inc., Amherst, NY), an organic solvent extract of natural bovine surfactant isolated by lung lavage with active levels of both apoproteins SP-B and C, was supplied at a concentration of 35 mg phospholipid/ml. This was diluted with 0.9% NaCl to a concentration of 4 mg phospholipid/ml.

KL4-Surfactant (R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ), an artificial surfactant preparation consisting of dipalmitoylphosphatidylcholine (DPPC), palmitoyl-oleoylphosphatidylglycerol, palmitic acid, and a synthetic 21 amino acid peptide containing repeating sequences of one lysine and four leucine, was supplied by the manufacturer at a concentration of 4 mg phospholipid/ml.

Exosurf, a synthetic surfactant consisting of DPPC, cetyl alcohol, and tyloxapol was supplied as a suspension at a concentration of 81 mg phospholipid/ml. 0.45% NaCl was added to this suspension to achieve the two concentrations studied; 13.5 mg phospholipid/ml, which we refer to as undiluted Exosurf because it is the concentration used clinically (14), and 4.5 mg phospholipid/ml.

Experimental Design

After catheter placement, FI_O2 was increased from 0.21 to 1.0 and PEEP from 2 to 6 cm H₂O. PIP was adjusted to maintain the Pa_CO2 between 40-45 mm Hg. Piglets were then paralyzed with 0.6 mg of pancuronium (Astra Pharmaceutical Products, Westborough, MA). Subsequent anesthesia and paralysis were maintained with intermittent doses of pentobarbital (2.5-5 mg/kg) and pancuronium (0.3 mg). After baseline measurements, lung injury was induced by multiple lung lavages with saline, utilizing a modification of the method described by Lachmann (15). Briefly, 35 ml/kg of 0.9% NaCl, warmed to 37° C, was administered through the endotracheal tube from a height 60 cm above the piglet's head. Recovery of saline was accomplished by passive drainage by lowering the connecting tube 60 cm below the piglet's head. This drainage was augmented by gentle, intermittent chest compressions. The average duration of the lavage procedure was 102 ± 1 s. Between lavages (6 min apart), VT was maintained at baseline values by adjusting the PIP. Arterial blood gases were monitored every two to four lavages, and the lavages continued until the Pa_O2 remained below 80 mm Hg for 20 min. The number of saline lung lavages to achieve the above criteria was 13 ± 1. Then the piglets were ventilated for an additional 90 min keeping VT and PEEP constant. Following this, a second series of saline lung lavages were performed (mean duration 96 ± 1 s) to induce further lung injury (7). This series was identical to the first series, including the same end point. The number of saline lung lavages required to meet criteria for this series of lavages was 4 ± 0.

Then hemodynamic and pulmonary function measurements were taken, thereafter referred to as "Sick." Except in the control group, surfactant was administered immediately after "Sick" (about 30 min after the last saline lung lavage). The technique of administration was identical to that described for the saline lung lavage, except that no chest compressions were administered during drainage of the surfactant-mixed fluid. Hemodynamic and pulmonary function parameters were recorded every 30 min during the next 4 h of observation. VT was kept constant by adjusting the PIP.

Experimental Groups

The Control group (n = 6) received no surfactant. The animals were disconnected from the ventilator for 120 s to mimic the period of nonventilation of the surfactant lavage procedure. The other study groups were treated with Infasurf (n = 5), KL4-Surfactant (n = 6), Exosurf (n = 5), and undiluted Exosurf (n = 6). The retained phospholipid (mg/kg) was calculated as: (surfactant concentration × retained volume of surfactant).

The total protein in the surfactant preparation was measured prior to treatment using a Hitachi 911 analyzer (Boehringer-Mannheim Corporation, Indianapolis, IN). Total protein was also measured in the fluid return collected after surfactant lavage.

Bronchoalveolar Lavage (BAL)

BAL samples were collected from the returns of the first lavage of both lavage series and a final lavage performed 4 h postsurfactant administration. One aliquot of BAL fluid was centrifuged at 3,250 rpm for 5 min and analyzed for total protein. Another aliquot was centrifuged at 2,000 rpm for 10 min, frozen at 20° C, and subsequently analyzed for total phospholipid phosphorous content by ascorbic acid reduction (16).

Quasistatic Pressure-Volume Curves

At the end of the 4-h observation period, the animals were euthanized and the ventilator was disconnected for 5 min to allow the lungs to collapse. The lungs were then inflated with room air to 35 cm H₂O pressure in 5 cm H₂O pressure increments and the volume measured after 30 s of equilibration at each pressure. Similar volume measurements were obtained during deflation (35 to 0 cm H₂O).

Surfactant Distribution

Colored microspheres (Interactive Medical Technologies, Los Angeles, CA) were used to evaluate the surfactant distribution as in our previous study (8). Surfactant distribution was determined in seven piglets treated with a surfactant/microsphere mixture followed by 4 h of observation. The study groups were Infasurf (n = 3), Exosurf (n = 2), and undiluted Exosurf (n = 2). Immediately on removal the lungs were frozen in liquid nitrogen and cut into 53 pieces. The microsphere count in each piece was expressed as the ratio of actual/expected (8).

Lung Pathology

Lungs were prepared for pathology as previously described (7). After fixation in formalin, sections were taken from the upper, middle, and lower segments of the lung and stained with hematoxylin and eosin. Lung injury was quantified using a four-point scoring system (0 = no injury, 3 = severe injury) by a pathologist blinded to the treatment groups.

Data Analysis

Statistical analyses were performed using SigmaStat (Jandel Scientific, San Rafael, CA). Data were tested for normality with the Kolmogorov-Smirnov test. Non-normally distributed data were ranked prior to analysis. Data for the undiluted Exosurf group were compared only with the Exosurf group; whereas the Infasurf, KL4-Surfactant, Exosurf, and control groups were compared with each other. Differences within and between groups were determined using two-way ANOVA for repeated measures over time. The Student Neuman-Keuls post hoc test was performed when a significant difference in overall effect was detected. Data for quasi-static pressure-volume measurements were analyzed by one-way ANOVA. Pre- and post-surfactant protein content was analyzed by paired t test. Surfactant distribution data were analyzed for normal distribution through a curve-fitting equation for Gaussian distribution (Graph-Pad Prism; Intuitive Software for Science, San Diego, CA). For all analyses, p < 0.05 was considered significant. Data in all figures and tables are expressed as means ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Surfactant Administration

There were no differences in the duration of surfactant administration among treatment groups; the mean for all groups was 107 ± 3 s. In contrast, the volume of retention was different: Infasurf (4.7 ± 0.4 ml/kg) and KL4-Surfactant (5.8 ± 0.6 ml/ kg) were significantly lower than Exosurf (9.7 ± 1.3 mg/kg) and undiluted Exosurf (10.2 ± 0.9 mg/kg). These correspond to a retained phospholipid dose (mg/kg) of 18.6 ± 1.6, 23.3 ± 3.7, 43.8 ± 5.9, and 137.5 ± 11.9, respectively.

Comparison Between Undiluted Exosurf and Exosurf Groups

The calculated dose of retained phospholipid was significantly lower in the Exosurf group as compared with the undiluted Exosurf group (Student's t test). Despite this, the Pa_O2 was higher in the Exosurf group at all time points, with significance reached at the 150 (330 ± 26 versus 195 ± 24 mm Hg), 180 (353 ± 28 versus 207 ± 23 mm Hg), and 210 (375 ± 24 versus 248 ± 20 mm Hg) min measurements. All pulmonary function parameters except FRC showed greater improvement in the Exosurf group, although these differences were not significant (Table 1, Figure 1).

View larger version (15K): [in this window] [in a new window]   Figure 1.   PaO2 after surfactant lavage in Exosurf and undiluted Exosurf groups. Surfactant was administered immediately after the Sick measurement. Sick measurements taken 20 min after the last saline lung lavage of the second series. *Different from Sick. Different from undiluted Exosurf. p < 0.05.

The baseline BAL total phospholipid level prior to surfactant treatment (Sick) was similar in both groups (Exosurf 0.14 ± 0.06; undiluted Exosurf 0.11 ± 0.02 mg/ml), but this level was significantly higher in the undiluted Exosurf group at the end of the experiment (Exosurf 0.06 ± 0.02; undiluted Exosurf 0.27 ± 0.02 mg/ml).

Comparison Between Infasurf, KL4-Surfactant, Exosurf and Control

The retained phospholipid dose in the Exosurf group was higher than in the Infrasurf and KL4-Surfactant groups.

All surfactant-treated groups demonstrated increases in Pa_O2 within 30 min of administration (Figure 2). The Pa_O2 continued to improve, and by 240 min was significantly higher than at 30 min in the Infasurf (406 ± 28 versus 260 ± 55 mm Hg) and Exosurf groups (364 ± 25 versus 207 ± 46 mm Hg). Finally, the Pa_O2 in all surfactant-treated groups was significantly higher than in the Control group at all time points, although there were no differences between treatment groups.

View larger version (22K): [in this window] [in a new window]   Figure 2.   PaO2 after surfactant lavage in Infasurf, KL4-Surfactant, Exosurf and Control groups. Surfactant was administered immediately after the Sick measurement. Sick measurements taken 20 min after the last saline lung lavage of the second series. *Different from both Sick and Control. Different from 30 min observations. Different from 90 min observation. p < 0.05.

Surfactant-treated groups demonstrated increases in VEI within 30 min of treatment (Infasurf: 0.08 ± 0.02 to 0.13 ± 0.02; Exosurf: 0.07 ± 0.01 to 0.12 ± 0.01; KL4-Surfactant: 0.11 ± 0.02 to 0.16 ± 0.01), and this improvement persisted in all treatment groups (Figure 3). In contrast, Pa_CO2 decreased only in the Infasurf (89 ± 11 to 62 ± 8) and Exosurf (85 ± 3 to 64 ± 5) groups starting at 60 min. Accordingly, pH increased in the Infasurf and Exosurf groups beginning at 60 min (data not shown). Finally, there were no differences between surfactant-treated groups for either VEI, Pa_CO2, or pH.

View larger version (21K): [in this window] [in a new window]   Figure 3.   Ventilation efficiency index (VEI) after surfactant lavage in Infasurf, KL4-Surfactant, Exosurf, and Control groups. Surfactant was administered immediately after the Sick measurement. Sick measurements taken 20 min after the last saline lung lavage of the second series. *All measurements different from Sick. p < 0.05.

Cdyn was higher in all surfactant-treated groups when compared to the Control group. Sustained improvement in Cdyn was noted only in the Infasurf (1.06 ± 0.18 to 1.39 ± 0.15) and Exosurf (1.07 ± 0.02 to 1.46 ± 0.08) groups starting at 30 min. In contrast, Cdyn increased after KL4-Surfactant only at 30 min. FRC increased in all groups by 60 min, although there were no differences found between groups when comparing the percent increase from Sick values.

PIP decreased in all surfactant-treated groups (Infasurf: 27 ± 3 to 23 ± 2 cm H₂O; KL4-Surfactant: 25 ± 2 to 21 ± 1 cm H₂O; Exosurf: 27 ± 2 to 22 ± 1 cm H₂O) within 30 min of surfactant administration, while it did not change in the Control group (32 ± 3 versus 34 ± 3 cm H₂O). This reduction was sustained only in the Infasurf and Exosurf groups. Finally, there were no differences in VT between groups.

No clinically relevant differences were found between groups in any of the hemodynamic measurements (HR, CVP, MAP) or in the pulmonary shunt fraction (data not shown).

Quasistatic Pressure-Volume

There were no significant differences found between groups, although all surfactant-treated groups tended to have higher volumes than the Control group (p = 0.43; power = 0.5). The volume (ml/kg) at 35 cm H₂O pressure was: Infasurf (41.6 ± 3.2); KL4-Surfactant (45.8 ± 4.8); Exosurf (35.1 ± 9.6); undiluted Exosurf (40 ± 1.6); and Control (33.5 ± 7.7) (Figure 4).

View larger version (27K): [in this window] [in a new window]   Figure 4.   Quasistatic pressure-volume curves in Infasurf, KL4-Surfactant, Exosurf and Control groups. Error bars have been omitted for clarity. Please refer to the text for details.

Lung Pathology

Lung injury scores ranged between 1 and 2 in all groups; no differences were found between Control and surfactant-treated groups.

Surfactant Fluid Total Protein

All surfactant preparations had an initial total protein level of less than 3 mg/dl. Total protein levels increased in the surfactant lavage fluid return: Infasurf (13.6 ± 5.1 mg/dl); KL4-Surfactant (18.5 ± 3.1 mg/dl); Exosurf (15.4 ± 8.9); and undiluted Exosurf (25.5 ± 6.1).

BAL Fluid Analysis

As compared with the pretreatment values (BAL fluid from the first wash of the second series of saline lung lavage), BAL protein increased at the end of the 4 h of observation in all study groups, with a trend for higher protein levels in the Control group. The pooled pretreatment BAL protein level (mg/ dl) was 36 ± 4, n = 27), and BAL protein levels at the end of experiment were: Infasurf (91 ± 11, n = 2); KL4-Surfactant (159 ± 31, n = 5); Exosurf (80 ± 8, n = 3); undiluted Exosurf (166 ± 52, n = 4); and Control (289 ± 78, n = 5).

Surfactant Distribution

The average total recovery of microspheres was 91 ± 7%. Data were pooled from all groups. Surfactant distribution was found to be homogeneous based on normally distributed data (Kolmogorov-Smirnov test). Forty-nine percent of lung pieces had microsphere counts that were within 25% of the expected ideal value. The mean for the ratio of actual/expected microsphere count for individual lung pieces was 0.97 ± 0.03. The skewness and kurtosis for the data were 0.341 and 0.330. The r² for the normalized Gaussian distribution curve was 0.83 (Figure 5).

View larger version (17K): [in this window] [in a new window]   Figure 5.   Surfactant distribution as measured by colored microspheres. Data from all animals studied are pooled (Infasurf, n = 3; Exosurf, n = 2; undiluted Exosurf, n = 2). Microsphere count in each lung piece (n = 370) expressed as ratio of actual/expected in increment intervals of 0.2 on the x-axis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our results provide further evidence that the lung lavage method of surfactant administration results in a clinically relevant improvement in pulmonary function after acute lung injury (8, 17). In addition, improvement in pulmonary function can be achieved at much lower doses of surfactant phospholipid than previously utilized in the treatment of ARDS (5, 6, 18). We also showed that this effect can be demonstrated consistently using a variety of artificial and natural surfactant preparations.

Strikingly, we observed a similar degree of improvement in pulmonary function in both Exosurf groups despite the significantly different retained phospholipid doses. Unlike previous experience in both human studies (19, 20) and experimental animal models (21), the beneficial effects of surfactant administered by lung lavage may not be entirely dose dependent. To be sure, the BAL phospholipid content in the undiluted Exosurf group was over four times greater at the end of the experiment. This suggests that not all of the phospholipid delivered into the lung contributes to the immediate improvement in pulmonary function. In fact, the consistently higher oxygenation found in the Exosurf group suggests that the dilute preparation was more effective in delivering surfactant to the alveoli.

Further, the overall improvement in pulmonary function in both Exosurf-treated groups suggests that the lavage method of administration may compensate in part for the lack of surfactant-associated proteins. The absence of protein has been implicated as a possible factor for treatment failure in ARDS (5), and in other experimental models of acute lung injury (7). We believe that the lavage administration technique both facilitates adsorption of surfactant by improving alveolar distribution, as well as removing surfactant inhibitory proteins from the alveolar space, thereby optimizing surfactant function (8).

While other studies have demonstrated beneficial effects from increasing the volume of administration (22), few studies have examined the acute effects of surfactant administered by lavage after acute lung injury. VanDer Bleek (22) administered a large surfactant volume (16 ml/kg) in rabbits after acute lung injury, but only animals placed on cardiopulmonary bypass survived, suggesting that the mortality was due to the detrimental effects of the large volume of administration (23). In contrast, observations from this study suggest that the moderately high volumes of retained surfactant noted in both Exosurf and undiluted Exosurf groups (approximately 10-11 ml/ kg) were well tolerated when administered by lavage. Similar to our results, Eijking and coworkers (17) reported a beneficial effect of lavage administration of surfactant after HCl- induced lung injury in rats, despite the small volume retained. However, they did not report on the duration of the procedure or any associated side effects. In this study, administration was limited to a maximum of 2 min as in our previous study (8) and was generally well tolerated.

Importantly, we have shown that the improvement in pulmonary function in our lung injury model can be achieved at retained doses as low as 19 mg/kg phospholipid. Nevertheless, the lavage technique, as used in the present study, required a much larger administration dose, even in the dilute surfactant groups (140-160 mg/kg). This dose is at least as high as those currently recommended for the treatment of both neonatal RDS and acute RDS. Thus, in terms of resource utilization, the lavage technique offers no advantage to bolus administration.

The lavage technique is the most invasive of the methods of surfactant administration, as it was associated with almost 2 min of apnea (all animals were paralyzed). Thus, strategies aimed at reducing the potential adverse effects while facilitating surfactant distribution need to be investigated. Some of these may include rotation of the animal during instillation and/or drainage, maneuvers to maintain PEEP before and after the procedure, and shortening the duration of administration.

It should be emphasized that the lavage technique simultaneously provides for a method of drug administration and partial removal of airway and alveolar debris. Removing foreign protein, cellular breakdown products, blood, bacteria, and mucus may improve surfactant function and ventilation and reduce lung injury. This concept is supported by the tendency for lower BAL protein levels at the end of the experiment in surfactant-treated piglets. Furthermore, this may in part explain the failure to distinguish between the effects of radically different surfactant preparations in this model of acute lung injury.

Finally, the efficacy of this administration technique needs to be confirmed in a more severe lung injury model. It may only be possible to significantly affect pulmonary function and lung distribution when the injury is nonhomogeneous by physically filling the lung with surfactant. This may avoid the effect of regional differences in lung compliance and eliminate the presence of the air-liquid interface. This concept is supported by the promising experience of Walmrath and colleagues (24) who administered surfactant bronchoscopically into individual lung lobes in patients with severe ARDS. Our results strongly support the need for further experiments to study this method of surfactant administration.