INTRODUCTION

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Perfluorocarbon-associated gas exchange, or partial liquid ventilation (PLV), is a relatively new form of respiratory support in which conventional ventilation is used to treat a lung partially filled with liquid perfluorocarbon (1). PLV has been extensively studied in animal models with normal and injured lungs. In animals with normal lungs, PLV is an effective means of gas exchange that has no deleterious hemodynamic effects (1, 2). In lung-injured animal models, a dose-dependent improvement in oxygenation has been observed during PLV, again with no negative hemodynamic effects (3, 4). Improvements in gas exchange, pulmonary mechanics, and survival have also been demonstrated (5). Use of PLV has recently been reported in human neonates who had failed conventional treatments; similar physiologic improvements in oxygenation, carbon dioxide removal, and lung mechanics occurred (9).

Little information is available regarding the interaction between PLV and exogenous surfactant. Surfactant replacement therapy, an established treatment for premature infants with respiratory distress syndrome (RDS), is not uniformly effective. For reasons that are unclear, some infants do not respond well to exogenous surfactant. Nonresponders have an increased risk of pulmonary and neurodevelopmental morbidity (10). The use of PLV in combination with surfactant may provide a more effective means to both recruit lung volume and stabilize lung units, thereby improving oxygenation and compliance and ultimately reducing lung injury. Tarczy-Hornoch and coworkers recently demonstrated improved lung compliance and reduced inflating pressures in isolated premature lamb lungs treated with surfactant followed by perfluorocarbon as compared with those treated with perfluorocarbon alone (14). Their data suggested that these improvements resulted from reduced interfacial tension after surfactant and perfluorocarbon treatment. A recent in vivo study in premature lambs showed no additional improvement in Pa_O2 or dynamic compliance when a nonprotein containing surfactant and PLV were compared with PLV alone (15). The absence of surfactant-associated proteins in the studied preparation may be important, as protein and nonprotein containing surfactants have different chemical and physiologic properties which may affect how these two agents interact with the lung or other chemicals. However, no similar studies using a protein-containing surfactant with PLV in intact animals have been published.

In this study, we hypothesized that oxygenation, respiratory system compliance, and lung pathology would be improved and hemodynamics unchanged during PLV when used in combination with a protein-containing surfactant as compared with either PLV alone or surfactant alone. Because these two compounds are immiscible, and perfluorocarbon has the greater density, surfactant may be less effectively deposited into the alveolus when given after perfluorocarbon. Therefore, we varied the order of administration of perfluorocarbon and surfactant to assess for possible order effects. In so doing, we tested a secondary hypothesis that the administration of surfactant prior to perfluorocarbon would optimize oxygenation and respiratory system compliance as compared with surfactant following perfluorocarbon.

	METHODS

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This study was approved by the Animal Care and Use Committee of United Hospital, Children's Health Care-St. Paul. The animals were cared for in accordance with the U.S. Department of Agriculture guidelines.

Thirty-two newborn piglets weighing 915 to 2,415 g (1.51 ± 0.38 kg) were randomized into one of four treatment groups: surfactant alone (S; n = 8); partial liquid ventilation alone (PLV-only; n = 8); surfactant followed by partial liquid ventilation (S-PLV; n = 8); and partial liquid ventilation followed by surfactant (PLV-S; n = 8). Piglets were anesthetized with intramuscularly administered ketamine (50 mg/kg/dose) and intubated and ventilated with a time-cycled, pressure-limited ventilator (Dräger Babylog; Dräger Critical Care System, Inc., Chantilly, VA) with an FI_O2 of 1.0, respiratory rate of 30, positive end-expiratory pressure (PEEP) of 4 cm H₂O, peak inspiratory pressure (PIP) to achieve 15 ml/kg tidal volume (VT), and an inspiratory time adequate for end-inspiratory gas flow to return to zero. VT and gas flow were measured at the endotracheal tube using the ventilator's internal hot-wire anemometer system. As the vapor pressure of perflubron is 10.5 mm Hg at 37° C, no volume measurement correction was necessary. Animals were paralyzed with an intramuscular injection of pancuronium bromide (0.2 mg/kg) followed by hourly intravenous injections. Sedation was maintained with alternating hourly doses of ketamine (25 mg/kg) and diazepam (0.05 mg/kg). Catheters were placed in the internal carotid artery and external jugular vein for monitoring arterial gases, intravascular pressure, and administration of fluids and medications. Pressure transducers were calibrated after initial placement at the level of the right atrium. After intubation and stabilization, a tracheotomy was performed and a triple-lumen neonatal "Hi Lo" endotracheal tube (Mallinkrodt Inc., St. Louis, MO) was inserted with the tip positioned proximal to the carina. This was secured with umbilical cord tape to prevent leakage. During the study period, the animals were hydrated with 5% dextrose in 1:4 normal saline with 10 mEq KCl/L at 6 ml/kg/h. A heating blanket was used to achieve normothermia.

Throughout the entire study period, PIP was adjusted to maintain VT of 15 ml/kg. Ventilator rate was adjusted to maintain Pa_CO2 between 35 and 50 mm Hg and pH between 7.35 and 7.50. Sodium bicarbonate was administered for base deficit > 8. Inspiratory time was adjusted based on gas flow as described above. FI_O2, initially 1.0, was adjusted to keep Pa_O2 between 100 and 150 mm Hg.

Oxygenation was continuously monitored using pulse oximetry (Nellcor Inc., Hayward, CA), intravascular sensor (Paratrend 7; Biomedical Sensors, Malvern, PA), and intermittent arterial blood gases (StatPAL II; SenDx Medical, Carlsbad, CA). Peak and mean (Paw) airway pressures were measured at the proximal endotracheal tube by the internal transducer system of the Dräger ventilator. Dynamic respiratory system compliance (Crs) was measured using a computer- assisted measuring system (PeDs; MAS, Inc., Hatfield, PA). A pneumotachometer was interposed between the endotracheal tube and ventilator circuit; pressure and flow measurements were made at 75 Hz. A two-factor least mean squares analysis technique was used to derive values for Crs using the Rohrer equation of motion (16). Arterial blood gases, intravascular pressures, vital signs (521 Neonatal Monitor; Spacelabs Inc., Redmond, WA), ventilator settings, Crs, and measured intratracheal airway pressures were obtained before and after lung injury as well as at 30-min intervals after perfluorocarbon and surfactant instillation. For each set of measurements, we calculated the oxygenation index (OI = (Paw × [FI_O2 × 100])/Pa_O2) and the arterial/alveolar oxygenation ratio (a/A = Pa_O2/[FI_O2 × 700]  [Pa_CO2/0.8]).

After initial measurements, piglets underwent repeated saline lavage to produce lung injury (17). A syringe was used to instill 30 ml/kg amounts of warmed saline into the lungs via a side lumen of the endotracheal tube. After instillation, ventilation was resumed and saline allowed to dwell for 5 to 10 min before suctioning. Multiple lavages were instilled until adequate lung injury was attained. Lung injury was defined as Pa_O2 < 60 mm Hg in FI_O2 = 1.0 with PEEP of 6 cm H₂O and  30% reduction in Crs.

Piglets randomized to receive surfactant were given 100 mg/kg of surfactant (Survanta^®; Ross Laboratories, Columbus, OH) via the distal lumen of the endotracheal tube according to the manufacturer's guidelines. Those randomized to the S-PLV group were mechanically ventilated for 30 min after surfactant administration. Room-temperature, preoxygenated perflubron (LiquiVent^®; Alliance Pharmaceutical Corp., San Diego, CA) was then administered via the pressure monitoring port of the endotracheal tube over 1 to 2 min in an amount that approximated the animal's functional residual capacity. Each animal was rotated through prone and supine positions, and the head was elevated and lowered throughout instillation of the perflubron. When a perflubron meniscus was visible within the endotracheal tube at end-expiration, infusion was stopped. During and immediately after administration of perflubron, PIP was adjusted to maintain VT at 15 ml/kg. Piglets randomized to receive PLV followed by surfactant similarly received perflubron. They were ventilated for 30 min and then received 100 mg/kg of Survanta according to the manufacturer's guidelines. Piglets randomized to receive PLV-only had perflubron administered after lung injury as described above. Data collection resumed 30 min after the final treatment. In addition, data were obtained after the first treatment in the PLV-S and S-PLV groups.

Histologic analysis was performed on lung tissue form all animals, using techniques similar to those previously described for comparison of gas- and liquid-ventilated animals (18). After the animals were killed, the trachea was clamped at end-expiration and lungs were removed. Perflubron was left in situ in animals treated with PLV. Tissues were immediately fixed in 10% formalin. Slides from the cranial dorsal (nondependent) and caudal ventral (dependent) lobes were stained with hematoxylin and eosin and then scored using a semi-quantitative scoring system by a pediatric pathologist (S.C.S.) blinded to treatment group. Variables scored were alveolar and interstitial inflammation, alveolar and interstitial hemorrhage, edema, atelectasis, and necrosis. Each variable was scored using a 0- to 4-point scale, with no injury scored 0, injury in 25% of the field scored 1, injury in 50% of the field scored 2, injury in 75% of the field scored 3, and injury throughout the field scored 4. Maximum possible score was 28.

Descriptive statistics were calculated on all variables and for the percent change observed after lung injury. Data are given as mean ± SD. Significance was set at p < 0.05. A paired t test was used to detect differences before and after lung injury for all animals and for all four groups. Paired t tests were used to assess administration-order differences. A one-factor analysis of variance was used to assess differences between groups at each time point. When significant differences were seen, post hoc analysis was performed with Newman-Keuls multiple range test. Paired t tests were used to assess histologic differences between right and left lobes. When no differences existed, right and left data were pooled. A Kruskal-Wallis analysis was used to assess differences between groups for each histologic variable. When differences were seen, the Wilcoxin rank-sum test was performed, followed by Bonferroni correction.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The weight (1.51 ± 0.38 kg) was not different between groups at randomization. Before lung injury, blood gases, respiratory system mechanics, and hemodynamics did not differ. After saline washout, all animals developed evidence of severe respiratory failure with marked respiratory acidosis and hypoxia compared with pre-injury values. When dynamic respiratory system mechanics were assessed, Crs fell 50%, resulting in doubling of PIP and mean airway pressures to maintain the targeted VT. Heart rate (HR), central venous pressure (CVP), and mean arterial blood pressure (MBP) were similar between groups before and after lung injury. An average of 12.1 ± 5.7 washouts was necessary to produce this level of injury (Table 1).

At 30 min after the final treatment, all piglets had improvements in oxygenation that were independent of treatment method (p < 0.0001; Figure 1). Over time, however, the four groups began to diverge: the S animals experienced a progressive deterioration in their OI, while the three PLV groups maintained their improvements in oxygenation. At 120 min after treatment, the PLV-S group was significantly different than the S-only group (OI: 4.92 ± 0.75 versus 11.70 ± 3.18, p < 0.05). The amount of perflubron administered was 32.8 ± 7.6 ml/kg.

View larger version (17K): [in this window] [in a new window]   Figure 1.   The effect of treatment on percent change in oxygenation index (% change OI). Data obtained after lung injury are shown at time 0; data obtained 30 min after the last treatment (surfactant or perflubron) are shown at time 30 min. All groups show an initial decrease in %  OI in response to treatment (**p < 0.0001 versus time 0 for all four groups). Over time, the effect of surfactant wanes and the three PLV groups continue to improve (*p < 0.05 S versus PLV-S).

All four treatments initially improved Crs and allowed ventilation with the targeted VT at lower PIP (Figure 2). This achieved statistical significance only for the PLV group (33 ± 1.5 cm H₂O after injury versus 24 ± 2.5 cm H₂O after PLV, p < 0.0001). The S group had the smallest percent change in PIP over the 2 h studied. From 60 to 120 min, the percent change in PIP in S-only animals was significantly less than in the groups treated with PLV, with or without surfactant (S-only 1.4 ± 0.1% versus PLV-S 14.4 ± 0.0%; S-PLV 24.3 ± 0.1%; PLV-only 25.6 ± 0.0%; p < 0.05). Although the PLV-only group had the quickest and largest percentage decrease in PIP following treatment, after 30 min it was not statistically different than the other groups treated with the combination of PLV and surfactant.

View larger version (18K): [in this window] [in a new window]   Figure 2.   The effect of treatment on percent change in peak inspiratory pressure (% change PIP). Data obtained after lung injury are shown at time 0; data obtained 30 min after the last treatment (surfactant or perflubron) are shown at time 30 min. The PIP is initially diminished in all four treatment groups but only reaches statistical significance in the PLV-only group (p < 0.0001). Between 60 and 120 min, the surfactant group loses its effect and does not reduce peak pressure as much as the groups treated with PLV (60 min: *p < 0.01 S versus PLV, S-PLV; and PLV-S versus PLV, S-PLV; 90 min: **p < 0.05 S versus all; 120 min: ***p < 0.01 S versus PLV, S-PLV).

Compliance was also initially improved in all the groups following their final treatment (p < 0.01; Table 2). Again, the S group had the smallest percent change in Crs, about 13% over the entire study period. The S-PLV group showed a 73% increase in Crs during the study, the largest change we observed. These two groups were statistically different from each other at each study time point (p < 0.05; Figure 3). The animals treated with PLV-only and PLV-S behaved similarly to each other: at 30 min Crs had increased 30% and 36%, respectively; at 120 min, Crs was increased 45% in both groups. HR, CVP, and MBP did not differ between the groups at any time after treatment.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Effect of treatment on percent change in respiratory system compliance (% change Crs). Data obtained after lung injury are shown at time 0; data obtained 30 min after the last treatment (surfactant or perflubron) are shown at time 30 min. All groups showed an initial improvement in Crs (p < 0.01). At each time point, greater improvements in Crs occurred in the PLV groups as compared with the S group (30 min: *p < 0.05 S versus S-PLV; 60 min: **p < 0.05 S versus all and PLV-S versus S-PLV; 90 min: ***p < 0.05 S versus PLV-S, S-PLV; 120 min: ****p < 0.05 S versus S-PLV).

Administration-order effects are presented in Table 2 and Figures 4 and 5. OI improved after administration of either surfactant (26.76 ± 2.12 after washout versus 5.16 ± 0.91 after surfactant, p < 0.0001) or perflubron (28.34 ± 2.24 after washout versus 5.01 ± 1.06 after perflubron, p < 0.0001). However, OI worsened after administration of surfactant in the PLV-S group (5.01 ± 1.06 after perflubron versus 8.92 ± 1.89 after surfactant, p < 0.03) but did not significantly change after administration of perflubron in the S-PLV group. Crs did not statistically improve after the first treatment in either group but did improve after perflubron in the S-PLV group (0.92 ± 0.13 ml/cm H₂O after surfactant versus 1.12 ± 0.13 ml/cm H₂O after perflubron, p < 0.02). PIP decreased after administration of either surfactant of perflubron (30 ± 5 cm H₂O after washout versus 26 ± 4 cm H₂O after surfactant; 31 ± 3 cm H₂O after washout versus 26 ± 4 cm H₂O after perflubron, p < 0.02). It continued to decrease after administration of perflubron in the S-PLV group (26 ± 4 cm H₂O versus 24 ± 3 cm H₂O, p < 0.004) but increased after surfactant in the PLV-S group (26 ± 4 cm H₂O versus 27 ± 4 cm H₂O, p < 0.02). Paw decreased after both surfactant (12.4 ± 0.8 cm H₂O after washout versus 11.5 ± 0.7 cm H₂O after surfactant, p < 0.02) and perflubron (10.9 ± 0.6 cm H₂O, p < 0.04) in the S-PLV group. In contrast, Paw was unchanged after administration of either surfactant or perflubron in the PLV-S group.

View larger version (15K): [in this window] [in a new window]   Figure 4.   Comparison of the administration-order effects on oxygenation Crs and OI. Both groups improved oxygenation and Crs following the first treatment (p < 0.0001). Oxygenation was maintained when S was given before PLV; it decreased when S was given after PLV (p < 0.03). Crs did not statistically improve following the first treatment in either group. It did improve following perflubron in the S-PLV group (p < 0.02).

View larger version (14K): [in this window] [in a new window]   Figure 5.   Effect of administration order on PIP and mean airway pressure (Paw). PIP decreased following administration of either surfactant or perflubron (p < 0.02). It continued to decrease after administration of perflubron in the S-PLV group (*p < 0.004) but increased following surfactant in the PLV-S group (**p < 0.02). Paw decreased following both surfactant and perflubron in the S-PLV group (p < 0.02 and 0.04, respectively). In contrast, Paw was unchanged after administration of either surfactant or perflubron in the PLV-S group.

The total and individual histologic lung injury scores for both the nondependent and dependent lobes of all animals are shown in Tables 3 and 4. The S animals had the highest total injury scores in both the nondependent and dependent lobes (p < 0.05). In the nondependent lobes, scores were statistically different than those from the S-PLV group; in the dependent lobes, scores were different than those from all groups treated with PLV. There were significant differences between the nondependent and dependent lobe total injury scores in the PLV-only group (nondependent lobes 4.55 versus dependent lobes 2.50, p < 0.004). Individual injury variables did not show any intergroup differences. Side-to-side differences in alveolar inflammation were present in the nondependent lobes of the PLV-S group (right versus left: 1.5 versus 1.0, p < 0.05) and the dependent lobes of the S group (right versus left: 2.0 versus 1.5, p < 0.05). Atelectasis also showed side-to-side variability in the dependent lobes of the PLV group (right versus left: 0.8 versus 1.1, p < 0.05).

	DISCUSSION

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In this study, we compared the effects of PLV with and without surfactant replacement therapy on oxygenation, respiratory system mechanics, hemodynamics, and lung pathology in an animal model of lung injury. Surfactant therapy in combination with PLV improved oxygenation, respiratory system mechanics, and lung pathology to a greater degree than did either surfactant therapy alone or PLV alone, supporting our primary hypothesis. Our secondary hypothesis was only partly substantiated; oxygenation and compliance improved more rapidly when surfactant was administered before PLV, with further increases in compliance when perflubron was administered after surfactant but not when surfactant was administered after perflubron. However, at 2 h, oxygenation was not different between PLV-treated animals, though changes in compliance were greater in the group treated with surfactant followed by perflubron. Finally, evidence of lung injury was least in animals that received surfactant prior to PLV.

Improvements in oxygenation and compliance in all PLV groups compared with surfactant-treated animals suggest greater stabilization of already opened gas-exchanging units and better recruitment of lung volume during PLV. Saline lavage lung injury is known to cause atelectasis by washing out surfactant. Because of its density and spreading characteristics, perflubron opens these atelectatic areas, thus improving ventilation and perfusion matching (19). Placing noncompressible perfluorocarbon in the lung would likewise prevent further collapse. Finally, the presence of oxygenated perflubron in these stabilized gas-exchanging areas of the lung during expiration creates a reservoir of oxygen that continues to participate in gas exchange.

Surfactant therapy is only effective to the extent that it reaches the alveolus and is able to function (20). Saline lavage produces focal and widespread atelectasis, desquamation of bronchial and bronchiolar epithelium, and varying amounts of septal edema, dilated lymphatics, and hemorrhage (17). Surfactant administered into this milieu of damage may neither effectively reach the alveoli nor be fully functional because of proteins in the airways. The addition of perfluorocarbon after surfactant administration likely delivers both compounds more effectively into gas-exchange regions. Debris, edema fluid, and exudates may be moved proximally because of perflubron's higher density. Both effects would improve surfactant function.

After lung injury, all animals required higher PIP to maintain adequate VT, as reflected in their decreased compliance. After treatment, PIP was able to be decreased in all animals. However, the percent decrease in PIP in the S-only group was minimal and transient. By the completion of the study, PIP in this group approached the level immediately following lung injury. Accordingly, compliance changes were minimal in the S-only treatment group. This is consistent with data from humans. In separate studies by Cotton and Goldsmith, compliance values fell in the first hours after surfactant treatment, despite measured increases in functional residual capacity and improvements in Pa_O2 (21, 22). They speculated that these seemingly contradictory findings occurred because of initial stabilization of previously opened lung units, with a much slower recruitment of atelectatic areas. Interestingly, the S-PLV group had the greatest percent change in dynamic compliance. The PLV-S and PLV-only groups had nearly identical trends in their percent change in compliance. They were markedly better than the S-only group and slightly less than the S-PLV group. Because of the small number of animals studied and the large standard deviation, this was statistically significant at only one time point.

Improvements in gas exchange observed during PLV were similar to those seen in previous studies. Premature lambs with respiratory distress syndrome had improved lung mechanics and gas exchange following PLV (6). Houmes and Tütüncü independently demonstrated a dose-dependent increase in Pa_O2 and no deleterious hemodynamic effects using PLV in surfactant-depleted animals (3, 4). Leach and colleagues have shown similar improvements in premature lambs with RDS treated with PLV (6). Other studies, inducing lung injury with oleic acid, showed improved gas exchange, pulmonary mechanics, and survival with PLV (5).

The marked improvements in compliance seen in the S-PLV group is consistent with data recently published by Tarczy-Hornoch and associates. They evaluated in situ interfacial tension in the preterm lamb lung using liquid ventilation with and without surfactant repletion (14). Interfacial tension, as opposed to surface tension, is the force at the liquid/lung interface. In the perfluorocarbon-filled lung, interfacial tensions are not eliminated (23). These investigators showed that treatment with exogenous surfactant prior to liquid ventilation reduces interfacial tension as compared with liquid ventilation alone. This resulted in improved lung compliance and decreased inflation pressures. They inferred that the interfacial tension at the liquid/lung interface was lowered because of increased film concentrations of surfactant. Our findings were similar. In the S-PLV group, the addition of perflubron likely further distributes the previously administered surfactant, resulting in continued decreases in interfacial tensions and improved compliance. Administration of surfactant after PLV may not allow the surfactant to consistently reach the alveoli, as surfactant and perflubron are immiscible and perflubron has a greater density. This, then, might result in surfactant layered above the perflubron/lung interface, with poor adsorption at the alveolar surface. The primary interfacial tension effects in the PLV and PLV-s groups would result from the perflubron, not the surfactant, with the resulting similarities in measured compliances.

In contrast to our findings, Leach and colleagues saw no additive effects of surfactant treatment when combined with PLV (15). In preterm lambs with RDS, they studied lung mechanics, gas exchange, and survival of animals treated conventionally and with PLV techniques, both with and without pretreatment using exogenous surfactant. Animals in the PLV groups had improvements in these key variables, but surfactant treatment produced no additional benefit. This study differs from ours in a number of important respects. They studied a different animal model, the preterm lamb. They used different ventilatory techniques, measuring pressures, not VT, as their targeted variable. We suspect, however, the use of the artificial, non-protein containing surfactant Exosurf (Burroughs-Wellcome, Research Triangle Park, NC) may account for many of these observed differences. The surface tension of Exosurf is reported as 10 to 27 dynes/cm, similar to that of neat perflubron (15, 24). In contrast, the surfactant we studied, and that used in the study by Tarczy-Hornoch and associates, was Survanta. This protein-containing surfactant has an in vitro surface tension of < 8 dynes/cm. Neither surfactant shows a substantial change in surface tension after exposure to perflubron (15). The presence of surfactant apoproteins allows Survanta to adsorb to the air/water interface more rapidly than lipids alone (25, 27). This important physiologic difference may account for our findings.

The histologic results are consistent with our physiologic data: the animals treated with PLV had less lung injury than the animals treated with only surfactant. Other groups have demonstrated objective improvements in pathology following total liquid ventilation (18). A recent study done in our laboratory quantitated pathologic results during PLV. Smith and colleagues evaluated histologic and morphometric parameters following prolonged PLV with conventional and high-frequency techniques (28). As in this study, PLV produced less lung injury, with the greatest effect demonstrated in the lower lobes. Interestingly, in the two groups treated with surfactant immediately after lung injury, we saw the most consistent results between the nondependent and dependent lobes. The S-only group had the highest total histologic injury score, with insignificant variances between nondependent and dependent lobes. The S-PLV group had the lowest scores, again with no differences between nondependent and dependent lobes. When perfluorocarbon was the initial treatment, histologic examination showed pronounced nondependent and dependent lobe differences, with greater nondependent lobe damage, similar to our previous results. This suggests that without the initial adsorption of surfactant at the alveolar surface, layering of perflubron may occur more quickly or spreading may be less consistent. Or, as our own physiologic data and the data of Tarczy-Hornoch and associates suggest, the presence of surfactant allows ventilation with less pressure, producing less secondary injury.

A number of potential problems could have affected our results. This animal model of RDS is created in the laboratory rather than occurring naturally. However, saline lavage-induced lung injury has been extensively studied and is considered a reliable model of severe respiratory distress (17). It has similar pathophysiologic and histologic changes. It responds to similar treatments. Additionally, we demonstrated that all animals had very similar physiologic injury with this technique. Response to surfactant treatment alone was not as great as has been seen in some animal studies and may have been influenced by the mode of delivery (29, 30). Two studies evaluated different surfactant-dosing regimens than the FDA-approved regimen used in this study (30, 31). Although both suggest that greater initial improvements in oxygenation may be seen with an alternative dosing scheme, we chose to study the dosing regimen studied and approved for human use. The response to surfactant we observed is consistent with studies in several animal models and human neonates. (21, 22, 31). In these studies, compliance changed very little during the first few hours after surfactant administration, despite improvements in oxygenation and measured functional residual capacity. Other issues include the duration of the experiment, evaporative losses of perflubron, the technique of ventilation, and the method of lung tissue fixation. It is possible that the time, the differences seen between the groups would disappear or become magnified. This seems unlikely, as in the PLV-treated groups oxygenation and compliance had plateaued and in the S-only group seemed to be worsening. Perhaps with time, the PLV-treated groups would have further distinguished themselves from one another. Evaporative losses were unlikely a factor in this short study. Evaporative losses are roughly 2 to 3 ml/kg/h and would represent only a minimal amount of the total perfluorocarbon administered (9, 28). All animals were ventilated using a VT-targeting strategy in an attempt to prevent either underventilation or overdistention throughout the lung injury and study treatment period. We chose to control for VT rather than peak pressures during this experiment. This may have resulted in less variation in blood gases than a strategy that uses only PIP measurement, since a large measure of the physiologic impact of PIP is in the VT that results. Nevertheless, we still saw significant falls in the PIP required, as have others. Finally, we did not attempt to reinflate the lungs prior to fixation of tissue. This might result in preferential, or even artifactual, preservation of lung volume in the perflubron-filled lungs, if gas were allowed to escape from lungs not treated with perflubron. After animals were killed and prior to tissue fixation, we clamped the airways of all animals at end-expiration, to pathologically demonstrate the retained volume in the lung that resulted from the selected treatment. Since one reason perflubron is a useful respiratory medium relates to its mechanical ability to recruit lung volume, the volume of liquid-filled compartments is not a pathologic artifact but physiologically real. Our injury scores do not suggest differences based primarily on differences in inflation (as reflected by atelectasis); rather, inflammation and necrosis contributed more to the total score.

In conclusion, PLV improved oxygenation, respiratory system mechanics, and lung pathology in this model of acute lung injury. Administration of surfactant before perflubron produced initial and sustained increases in compliance, but only transient improvements in oxygenation when compared with perflubron alone. Surfactant administered after perflubron had no effect on compliance and initially worsened oxygenation. Pathologic lung injury was reduced in the lower lobes of all PLV-treated animals and reduced throughout the lungs in animals treated with S followed by PLV. Taken together, our findings suggest potential physiologic and pathologic benefits to the combined use of surfactant and perflubron, especially early use of perflubron after surfactant administration.