INTRODUCTION

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Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), are defined by hypoxemia, decreased pulmonary compliance, and bilateral infiltrates on chest radiograph (1). ARDS continues to be associated with a relatively high mortality (2, 3). Although artificial ventilation remains the primary supportive therapy for this group of patients, this intervention has also been shown to contribute to the progression of the existing lung injury (4). Overinflation of some areas of the lung due to the use of high peak inspiratory pressures and/or large tidal volumes can injure lung tissue, as can shear forces created between adjacent regions of inflated and collapsed alveoli (6, 8). Since patients with ALI and ARDS are known to have relatively nonuniform patterns of lung injury, instituting ventilatory strategies that would accentuate regional differences in lung compliance and/or augment the shear forces within the lung would result in further lung dysfunction.

Alterations to the pulmonary surfactant system can occur with some forms of mechanical ventilation (9, 10). In a rabbit model of acute lung injury, ventilatory modes utilizing increasing tidal volumes (VT) correlated significantly with an increased conversion of functionally active, large surfactant aggregates (LA) into inferior functioning small aggregate (SA) forms (9). This ventilation-mediated LA conversion, in turn, resulted in a significant decrease in LA surfactant pool sizes, which contributed to alveolar instability and lung dysfunction. Based on these results, it was postulated that ventilatory strategies aimed at minimizing changes in LA pool sizes would decrease the progressive lung dysfunction typically observed in patients with ALI when standard ventilation strategies are used.

High-frequency oscillation (HFO) is a ventilation strategy employing very high respiratory frequencies with extremely small VTs (11). Moreover, the application of mean airway pressures sufficient to prevent alveolar collapse during HFO minimizes regional differences in alveolar distention. Although HFO has been demonstrated to have benefits over conventional mechanical ventilation (CMV) both in neonatal models of lung injury and in the neonatal patient population, experience with this mode of ventilation in adult models of injury and patients with ARDS is limited (12, 13). In addition, studies addressing the mechanisms responsible for the effects of this ventilation strategy on the endogenous pulmonary surfactant system of injured lungs are unknown.

The purpose of this in vivo study was to evaluate and compare the effects of HFO and CMV on lung physiology and the surfactant system in adult rabbits with acute lung injury. The evaluation of both the physiological response to changes in ventilatory strategies and the surfactant kinetics evaluated in this study will provide important insight into the mechanisms responsible for the difference in lung function observed following a period of ventilatory support with these two modes of mechanical ventilation.

	METHODS

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Animal Preparation

Adult New Zealand White rabbits (2.2 ± 0.1 kg) had lung injury induced with N-nitroso-n-methylurethane (NNMU) (King's Laboratories, Inc., Blythewood, SC), and were anesthetized, ventilated with a pressure-limited infant ventilator (model IV-100B; Sechrist, Anaheim, CA), and instrumented as previously described (9).

Ventilation Strategies, Experimental Groups, and Protocol

Ten minutes after initiation of ventilation and recording of baseline parameters, animals were assigned to one of seven experimental groups (Time 0), involving either conventional mechanical ventilation (CMV) or high-frequency oscillation (HFO). Group 1, HFO for 120 min; Group 2, CMV for 120 min; Group 3, euthanized at Time 0; Group 4, CMV for 60 min followed by HFO for 60 min; Group 5, HFO for 60 min followed by CMV for 60 min; Group 6, CMV for 60 min; and Group 7, CMV for 60 min with low VT and increasing positive end-expiratory pressure (PEEP).

During CMV, the fraction of inspired oxygen (FI_O2) was 1.0, the inspiratory:expiratory ratio was 1:1, and the VT was 10 ml/kg for all groups except Group 7 in which a VT of 5 ml/kg was delivered. The respiratory rate and PEEP were set at 30 breaths/min and 3.5 cm H₂O, respectively, except in Group 7 where the rate was increased to 60/min and the PEEP was increased in 3 to 5 cm H₂O increments every 15 min.

The oscillator settings included an FI_O2 of 1.0, a frequency of 15 Hz, and a 33% inspiratory time. Amplitude was adjusted throughout the experimental period with the goal of maintaining Pa_CO2 values less than 50 mm Hg. Mean airway pressure was initially set 7 cm H₂O below the PIP setting recorded during CMV immediately prior to the switch to HFO. Airway pressure (Paw) was subsequently increased in 1 to 2 cm H₂O increments every 15 min if Pa_O2 values remained below 200 mm Hg.

Ventilatory parameters, mean arterial blood pressure (MABP) values, and arterial blood gas (ABG) analyses were recorded at 5-15 min intervals for all animals throughout the experimental period. If an animal died during the ventilatory period, the time of death was noted and a gross postmortem examination was performed. At the end of the ventilatory period (T = 60 or 120 min), all surviving animals were euthanized with an overdose of thiopental.

Surfactant Analyses

Immediately following euthanasia of animals, a whole lung lavage was performed to recover alveolar lavage and surfactant for analysis as previously described (10, 14, 15). Separate animals were randomized to Groups 1 and 2 for in vivo surfactant aggregate conversion analyses over a 1-h time period (T = 60-120 min) as previously described (10). To minimize differences in delivery efficiency and distribution of these radiolabeled LA, all animals were ventilated during and for 1 min following the instillation procedure with CMV using a VT of 10 ml/kg and 3.5 cm H₂O PEEP prior to transferring them back to their assigned ventilatory strategy. At the end of the ventilatory period, these animals underwent lung lavage and the lung tissue was processed as previously described (10).

Statistical Analysis

Statistical analyses of the physiological data measured over time and single comparisons between groups were analyzed as previously described (16).

	RESULTS

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A total of 65 animals received NNMU and were anesthetized and instrumented. Of these, 53 met the oxygenation inclusion criteria and were subsequently assigned to one of the seven experimental groups. Thirty-eight of the 53 animals were randomized to the first six experimental groups for evaluation of their physiological responses to the ventilation strategies and these animals all survived the experimental period and had their endogenous surfactant system analyzed after euthanasia. Ten of the animals were randomized to either Group 1 (CMV-2 h) or Group 2 (HFO-2 h) for in vivo evaluation of surfactant aggregate conversion, and five animals were assigned to Group 7 (CMV-PEEP) for analysis of their physiological responses to conventional ventilatory strategies involving a lower VT and increasing levels of PEEP. There were no significant differences in body weight or baseline Pa_O2, Pa_CO2, pH, MABP and peak inspiratory pressure (PIP) values among the various groups. There were also no significant differences among the groups with respect to the mean time interval between injection of NNMU and the start of the experimental period (44 ± 2 h).

Experiment 1

Mean Pa_O2 and Pa_CO2 values for Group 1 (CMV-2 h) and Group 2 (HFO-2 h) during the period of ventilation are shown in Figure 1. Mean Pa_O2 values of animals in Group 2 (HFO-2 h) were significantly higher than their respective baseline values starting at 5 min and continued to increase over the remaining period of ventilation (p < 0.05). Mean Pa_O2 values of animals in Group 1 (CMV-2 h) were significantly lower from 30 to 240 min compared with their respective baseline values (p < 0.05) (Figure 1A). These changes resulted in significantly higher mean Pa_O2 values in Group 2 (HFO-2 h) compared with values in animals in Group 1 (CMV-2 h) from 5 to 120 min (p < 0.05).

View larger version (18K): [in this window] [in a new window]   Figure 1.   (A, B) Effect of CMV and HFO ventilation strategies on PaO2 and PaCO2 in NNMU-injured adult rabbits. Values are means ± SEM. *Significantly different from value at baseline (T = 10 min), p < 0.05. aSignificantly different from CMV-2 h, p < 0.05.

In animals maintained on CMV, mean Pa_CO2 values remained unchanged from baseline values over the 2-h period. For animals ventilated with HFO for 2 h, Pa_CO2 values tended to decrease over time (Figure 1B). Statistical comparisons between groups showed that animals in Group 2 (HFO-2 h) had significantly lower mean Pa_CO2 values compared with animals in Group 1 (CMV-2 h) from 15 to 120 min (p < 0.05).

MABP values increased significantly in Group 2 (HFO-2 h) from 75 to 120 min compared with baseline values (p < 0.05). Comparisons of MABP between groups, however, showed no significant differences at any time point (data not shown).

Mean (± SE) PIP values recorded at baseline prior to randomization into Group 1 (CMV-2 h), Group 2 (HFO-2 h), and Group 3 (Baseline) were 30 (± 1) cm H₂O, 30 (± 2) cm H₂O, and 30 (± 2) cm H₂O, respectively. Five minutes after transfer to HFO, the mean Paw in animals in Group 2 (HFO- 2 h) was 24 (± 1.0) cm H₂O and did not significantly change over the ventilatory period. There was also no significant change in the amplitude of oscillations settings over the experimental period in this group, with values ranging from 36 to 42 cm H₂O. Mean PIP values for animals in Group 1 (CMV-2 h) increased significantly to a mean value of 33 (± 1) cm H₂O at 120 min (p < 0.05 versus baseline).

Total alveolar surfactant pool sizes, LA pool sizes, and the percentage of the total recovered alveolar surfactant pool present in LA forms are shown in Figure 2A. The percentages of radiolabel intratracheally instilled LA converted into SA in vivo over the final 60-min period of ventilation in Groups 1 and 2 are shown in Figure 2B. There were no significant differences in the total alveolar surfactant pools or LA surfactant pool sizes among the different experimental groups. However, the percentage of surfactant recovered as LA (shown in parentheses) was significantly greater in Group 2 (HFO-2 h) and in Group 3 (Baseline), compared with values obtained for Group 1 (CMV-2 h) (p < 0.05) (Figure 2A). Comparison of in vivo LA conversion between groups (Figure 2B) revealed that animals ventilated for 2 h with HFO had significantly less LA conversion into SA forms compared with animals ventilated with CMV for 2 h (p < 0.05). There were no significant differences between groups in the total radiolabel recovery (Group 1 = 47.0 ± 0.6%, Group 2 = 54.6 ± 4.2%) or in the percentage tissue association of the administered radiolabeled LA (Group 1 = 47.0 ± 2.7%, Group 2 = 43.4 ± 5.0%).

View larger version (17K): [in this window] [in a new window]   Figure 2.   (A, B) Effect of CMV and HFO ventilation strategies on alveolar surfactant CAW pool size, LA pool size, the relative percentage of surfactant in LA form, and surfactant aggregate conversion in NNMU-injured adult rabbits. Values are means ± SEM. aSignificantly different from CMV-2 h, p < 0.05.

Total protein recovery in the crude alveolar wash (CAW) revealed no significant differences among groups with an average of 469 ± 33 mg/kg for all animals. Previous studies have demonstrated that normal adult rabbits have approximately 15 mg/kg of total protein in the CAW as recovered by lung lavage (17).

Experiment 2

Mean Pa_O2 and Pa_CO2 values for groups undergoing a ventilatory change after 60 min are shown in Figure 3. In animals switched from CMV to HFO, mean Pa_O2 values were significantly lower than their respective baseline values during the CMV portion of ventilation from 5 to 60 min (p < 0.05), but then increased when switched to HFO to values not significantly different from baseline values at subsequent time points. In animals starting with HFO and changed to CMV, mean Pa_O2 values were significantly greater than baseline values during the HFO portion of ventilation from 5 to 60 min (similar to HFO-2 h, Figure 1A) but fell when switched to CMV to levels significantly lower than baseline values from 65 to 120 min after the onset of ventilation (p < 0.05). Animals in Group 6 (CMV-1 h) had oxygenation responses similar to the first 60 min of ventilation for animals in Group 4 (CMV- HFO), with Pa_O2 values significantly lower than baseline from 15 to 60 min (p < 0.05). Similar to results observed in Experiment 1, oxygenation of animals during ventilation with HFO was generally superior to oxygenation values recorded in animals during CMV.

View larger version (21K): [in this window] [in a new window]   Figure 3.   (A, B) Effect of altering ventilation strategies on PaO2 and PaCO2 in NNMU-injured adult rabbits. Values are means ± SEM. *Significantly different from value at baseline (T = 10 min), p < 0.05. aSignificantly different from HFO-CMV, p < 0.05.

Mean Pa_CO2 values for these groups are shown in Figure 3B. In animals ventilated with CMV initially, (CMV-HFO; 5 to 60 min), values were similar to baseline, however these values decreased over time when these animals were switched to HFO, and reached values significantly less than baseline at 105 and 120 min (p < 0.05). Mean Pa_CO2 values for animals in Group 5 (HFO-CMV) were significantly lower than baseline from 5 to 120 min (Figure 3B) (p < 0.05). Statistical comparisons between groups showed that animals in Group 4 (CMV-HFO) had significantly lower mean Pa_CO2 values than animals in Group 5 (HFO-CMV) at 105 and 120 min (p < 0.05) (Figure 3B).

Similar to results shown in Experiment 1, MABP increased from baseline at 65 to 120 min in animals in Group 4 (CMV- HFO) (p < 0.05), although there were no significant differences noted among groups at any time point. There were also no significant changes in PIP values or in the Paw and the amplitude of oscillation settings over time during a given ventilation strategy in any of the groups in this experiment.

Results of surfactant analyses are shown in Figure 4. There were no significant differences in the total alveolar surfactant pool sizes, LA surfactant pool sizes, or percentage of surfactant recovered as LA among the experimental groups. Total protein analysis of the alveolar lavage also revealed no significant differences in protein recovery among Groups 4, 5, and 6 with an average of 447 ± 34 mg/kg for all animals.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Effect of altering ventilation strategies on alveolar surfactant CAW pool size, LA pool size, and the relative percentage of surfactant in LA form in NNMU-injured adult rabbits. Values are means ± SEM.

Experiment 3

Figure 5 shows gas exchange values of the individual animals ventilated with a lower VT and higher PEEP levels (CMV- PEEP) compared with the animals ventilated with CMV. By 30 min all animals assigned to the CMV ventilatory strategy had Pa_O2 values that were decreased relative to baseline and all animals died before the end of the 120-min ventilatory period (Figure 5A). During the experimental period, the PEEP settings were increased to maximum values of 15 to 18 cm H₂O. As the VT was maintained at 5 ml/kg throughout the ventilatory period, the resulting PIP values ranged from 42 to 50 cm H₂O. Figure 5B shows that these animals also had marked increases in Pa_CO2 levels, both when compared with baseline values and with animals described in the other experiments in this study. MABP values for these animals revealed a downward trend over time, consistent with hemodynamic compromise associated with the increasing PEEP values. These findings suggest that hypotension was the likely cause of death in four of the five animals in this group. The fifth animal had a pneumothorax on postmortem examination and barotrauma likely contributed to its death.

View larger version (14K): [in this window] [in a new window]   Figure 5.   (A, B) Effect of increasing PEEP levels during CMV on PaO2 and PaCO2 in NNMU-injured adult rabbits. Individual animal data are shown.

	DISCUSSION

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Numerous investigations have been directed at determining the mechanisms responsible for the deterioration in lung function associated with mechanical ventilation-induced lung injury (5, 18). Ventilatory strategies resulting in alveolar overdistention and/or those permitting repeated opening and collapse of portions of the lung are believed to be important factors responsible for this injury. Based on this information, it was hypothesized that strategies aimed at mitigating these factors in patients with ARDS requiring ventilatory support would potentially impact the high mortality associated with this disease. Indeed, ventilation utilizing low tidal volumes or a "low stretch" approach (VT = 6 ml/kg) has recently been shown to significantly decrease the mortality of patients with ARDS compared with strategies using higher tidal volumes (VT = 12 ml/kg) (19).

HFO is a unique ventilatory technique that also has the potential to minimize the above described deleterious effects of mechanical ventilation. For example, when instituted appropriately, HFO is capable of recruiting atelectatic regions of lung while preventing the cyclic opening and closing of lung units. The results of the present study demonstrated that in addition to improving gas exchange in lung-injured animals, HFO also minimized the deterioration in alveolar surfactant aggregate forms compared with animals ventilated using more standard ventilatory approaches. Although the tidal volume utilized in our CMV animals (10 ml/kg) was somewhat higher than those recommended by the recent NIH study (6 ml/kg), it is likely that a significant number of patients would still be subjected to this range of tidal volume ventilation at some point over the course of their disease. To specifically address this issue, we included a group of animals ventilated with a lower VT (Group 7). Unfortunately a poor physiological outcome was obtained and animals died at various time points over the experimental period precluding meaningful surfactant analyses.

The results of the present study were consistent with previous studies demonstrating improved gas exchange when HFO was implemented compared with CMV in models characteristic of neonatal respiratory distress syndrome (20). Using the whole lung saline lavage model in adult rabbits, McCulloch and colleagues demonstrated superior oxygenation and less morphological lung damage in animals managed with HFO compared with CMV (20). Similarly, premature baboons ventilated with HFO had a less severe degree of hyaline membrane formation when lungs were examined postmortem compared with animals ventilated conventionally (22). These data, as well as the knowledge that HFO has successfully been used clinically in neonates with respiratory distress syndrome (RDS), provide a good rationale for testing this strategy in adult patients with acute lung injury. Although HFO has not been thoroughly evaluated in models of ARDS with alterations of the endogenous surfactant system reflecting the clinical situation, it was tested in adult patients with ARDS in the early 1980s. The results of these studies were disappointing however, presumably due to the inappropriate application of HFO in these patients (24, 25). Specifically, relatively low mean airway pressures were utilized when initiating HFO in order to minimize peak inspiratory pressure values. At the time, high airway pressures generated by the mechanical ventilator were felt to be the major contributor to the lung damage induced by mechanical ventilation. It is now felt that higher tidal volumes, which may be associated with higher airway pressures, are the most important factor in causing lung damage. When low mean airway pressures are utilized with the very small tidal volumes associated with HFO, the lung will not be adequately recruited thereby resulting in suboptimal gas exchange. Recently, a preliminary study evaluating HFO in patients with ARDS reported that the application of significantly higher mean airway pressures resulted in superior oxygenation levels and improved outcomes with no increase in the incidence of barotrauma (26). Despite these promising results, however, further studies are required to elucidate the specific effects of HFO on the injured lung, and the mechanisms responsible for these effects. As alveolar surfactant alterations have been shown to contribute to ventilator-induced lung injury, we focused the present study on characterizing the effects of HFO on the pulmonary surfactant system in a model with surfactant changes typical of those observed in patients with ARDS (17).

Alveolar surfactant lowers surface tension at the alveolar air-liquid interface, thereby preventing alveolar collapse. Surfactant removed from the lung via saline lavage procedures can be separated into LA and SA fractions based on their buoyant density (27). LA surfactant forms can reduce surface tension to low values when tested in vitro and in vivo, and have been shown to be the metabolic precursor of the SA fraction (28). Because SA are functionally inferior to LA, alveoli containing a lower percentage of surfactant in LA forms are likely to have a higher surface tension at the air-liquid interface.

In vitro and in vivo studies have shown that certain aspects of mechanical ventilation can lead to deterioration in the alveolar surfactant environment. Specifically, ventilation strategies utilizing larger tidal volumes resulted in a greater formation of SA over a specific period of time compared with strategies using lower VTs (10). In vitro experiments have confirmed that a combination of the phasic changes in surface area associated with tidal volume ventilation, together with the presence of a serine-dependent protease, now known to be a carboxyesterase, is primarily responsible for the conversion of surfactant LA into SA forms (29). Because HFO is associated with minimal changes in surface area of the lung due to the extremely small tidal volumes utilized, the contact area for potential proteases within the injured lung capable of converting LA into SA forms is minimized. Our results confirmed that surfactant LA forms were preserved when HFO was utilized compared with conventional ventilation strategies.

In the present study, animals had evidence of lung injury as well as surfactant abnormalities when ventilation was initiated. These abnormalities included a reduced proportion of LA forms recovered in the lung lavage compared with typical noninjured, normal rabbits. At the start of the experimental period (T = 0), approximately 32% of the alveolar surfactant in the NNMU-injured animals was present in LA forms (Baseline) compared with approximately 50% of alveolar surfactant in LA forms in normal, mature adult rabbits (17, 30). This finding was consistent with previous studies utilizing this model of lung injury and is also reflective of the changes in endogenous surfactant aggregate forms reported in patients with ARDS (31, 32). Over a subsequent 2-h period of conventional mechanical ventilation (CMV-2 h), the percentage of LA further decreased to 21%. In contrast, animals managed with HFO (HFO-2 h) had a preservation of alveolar surfactant in LA forms, which was not significantly different from those animals euthanized at baseline (Baseline). Subsequent experiments evaluating the in vivo conversion of LA suggested that these differences in percentage LA recovery between groups was due to differences in the conversion of LA into SA in vivo as a result of the different ventilation strategies.

In the second series of experiments, we investigated the effects of switching ventilatory strategies from HFO to CMV and vice versa. Although oxygenation could be improved with HFO after a relatively short period of CMV, alterations to the alveolar surfactant environment were not reversible. Specifically, CMV resulted in a decreased percentage LA forms in animals ventilated for 1 h (CMV-1 h; 26% LA) compared with animals ventilated with HFO only (HFO-2 h; 40% LA). When animals were subsequently switched from CMV to HFO (CMV-HFO; 29% LA) further conversion of LA into SA was mitigated, but the relative quantity of surfactant present in LA forms at euthanasia was still reduced compared with animals ventilated with HFO for the entire duration. It should be noted, however, that the duration of ventilatory periods in this study was relatively short, and that longer periods of CMV prior to switching to HFO, as may be experienced clinically, may have resulted in more marked changes in aggregate forms and therefore suboptimal improvements in oxygenation upon switching to HFO. The changes in aggregate forms even after 60 min of CMV suggest that instituting optimal ventilatory modes as soon as possible after the onset of mechanical ventilation may be preferable in patients with acute lung injury, at least from the perspective of surfactant changes.

Based on numerous experimental results and the recently reported NIH-sponsored clinical trial, recommendations for managing patients with ARDS requiring mechanical ventilation involve using relatively low tidal volumes and adequate levels of PEEP in order to prevent alveolar collapse (19, 33). Although such strategies were attempted in the present study, it was not possible to achieve sustained improvements in oxygenation comparable with the HFO group without hemodynamic compromise and/or barotrauma in these animals (CMV- PEEP). It should be noted, however, that in the present study, the low VT, high PEEP strategies were implemented with the main objective of achieving oxygenation responses similar to those observed in animals managed with HFO. It is possible that achieving lower oxygenation values using the lower VT strategy would prove to be adequate in the clinical setting. Given the results of the surfactant analyses, however, it appears that HFO ventilation may offer the advantage of not only minimizing the harmful mechanical stresses and hemodynamic consequences of conventional modes of ventilation, it may also preserve the status of the alveolar surfactant system in its more functionally active forms. Further studies are required to confirm the potential long-term benefits of HFO over CMV in the clinical setting.