INTRODUCTION

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Partial liquid ventilation (PLV), in which conventional gas ventilation is superimposed on lungs partly filled with liquid perfluorocarbon, is an emerging adjunctive strategy during mechanical ventilatory support of pediatric (1) and adult patients (2, 3) with severe lung injury. Several uncontrolled, nonstandardized Phase I and II clinical trials have suggested improvements in gas exchange and pulmonary mechanics with PLV (1, 2). Multiple short-term studies in a variety of animal models of acute lung injury have shown similar improvements in gas exchange (4), concomitant reductions in intrapulmonary shunt fraction (5, 6), improvements in pulmonary compliance (5, 6), increases in end-expiratory lung volumes (8), and reduced morphometric and histologic evidence of lung injury (7). Improvements in oxygenation appear to be dose-dependent (4). However, an optimal dose of perfluorocarbon has not yet been identified in the clinical setting.

Different mechanisms of benefit have been offered for the physiologic effects of PLV. For instance, recruitment of consolidated and atelectatic dependent alveoli would account for both early improvements in oxygenation (by reducing shunt and improving ventilation-perfusion matching) and improvements in compliance. PLV may also alter lung perfusion patterns (10, 11). Certain physical characteristics of some perfluorocarbons could make "tamponade" of injured alveoli possible, and thereby attenuate increases in pulmonary capillary permeability (12, 13) and lung water accumulation (10). Additionally, some studies have reported reduced markers of inflammation in PLV-treated lungs (3, 6, 14, 15).

Since increased pulmonary capillary permeability is central to the pathophysiologic definition of acute respiratory distress syndrome (ARDS) (16), we hypothesized that PLV with the perfluorocarbon LiquiVent (perflubron; Alliance Pharmaceutical Corp., San Diego, CA) would produce a dose-dependent reduction in pulmonary capillary permeability to protein in an experimental model of lung injury, mediated directly or through reductions in pulmonary neutrophils, as compared with gas ventilation. Lung water changes should parallel these changes in permeability. We conducted the following studies to test these hypotheses.

	METHODS

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Animal Preparation

The studies were approved by the Washington University School of Medicine Animal Studies Committee. Fourteen healthy mongrel dogs (weight range: 18.0 to 25.6 kg; mean: 20.1 ± 1.9 kg) were anesthetized with sodium pentobarbital (35 mg/kg) administered via a forelimb peripheral vein, intubated with a size 9 cuffed endotracheal tube (Mallinckrodt, St. Louis MO), and ventilated with a Harvard pump respirator (Harvard Apparatus Co., South Natick, MA) or a Puritan Bennett MA-1 mechanical ventilator (Bennett Respiration Products, Santa Monica, CA), with the following settings: fraction of inspired oxygen (FI_O2) = 1.0, tidal volume (VT) = 15 ml/kg, and respiratory rate adjusted to yield a normal Pa_CO2 at baseline. Peak airway pressure averaged 22 cm H₂O, with only one animal showing a pressure transiently exceeding 30 cm H₂O. A positive end-expiratory pressure (PEEP) of 7.5 cm H₂O was added only after lung injury was induced.

Instrumentation was performed in a sterile fashion, with animals fixed in the supine position. After percutaneous insertion of bilateral femoral size 8.5 French introducer sheaths (Baxter Heathcare Corp., Irvine, CA), a size 7.5 French balloon-tipped pulmonary artery catheter (Baxter) and a 110-cm size 7.0 French pigtail catheter (Cook, Bloomington, IN) were positioned in the pulmonary artery under fluoroscopic guidance for hemodynamic monitoring and blood withdrawal, respectively. A size 6.0 French introducer (Cook) was inserted percutaneously into the external jugular vein and radionuclides were administered via a 5-cm length of infant feeding tube placed in this introducer sheath. A 20-gauge arterial catheter (Arrow International, Reading, PA) was inserted percutaneously into a femoral artery, using a Seldinger technique for continuous blood pressure monitoring and blood sampling. Catheter patency was maintained through intermittent flushes with heparinized saline.

Cardiac output (CO) was measured with the thermodilution technique (difference of two successive measurements < 10%) using a CO computer (American Edwards Laboratories, Irvine, CA). Pressure transducers (Baxter) were calibrated to the center of the lateral chest and connected to a monitor (Model 742; Mennen, Clarence, NY) for monitoring of systemic and pulmonary arterial pressures and periodic pulmonary capillary wedge pressure (PCWP) measurement. Continuous systemic and pulmonary arterial pressures (Psa and Ppa) were recorded with a portable computer (Macintosh 165B; Apple, Inc., Cupertino, CA) with Acknowledge 2.0 software (BIOPAC Systems, Goleta, CA). Blood gas tensions were measured with a blood gas analyzer (Model 1306; Instrumentation Laboratories, Lexington, MA). A transurethral bladder catheter was placed in all animals.

Experimental Protocols

The experimental time line of the study is summarized in Figure 1. All dogs underwent baseline measurement of hemodynamics and oxygenation. These measurements were repeated at approximately hourly intervals throughout the study. A 500-ml bolus of normal saline was administered after insertion of intravenous access lines, and was followed by a continuous infusion of normal saline at 50 ml/h that was continued for the duration of the study. Lung injury was induced with oleic acid (OA) at 0.08 ml/kg (Sigma Chemicals, St. Louis, MO), mixed with 70% alcohol to a volume of 3 ml and administered in three 1-ml aliquots separated by 5-ml saline flushes over a total period of 2 min, via the proximal port of the pulmonary artery catheter. At 75 min after administration of OA, a PEEP of 5 cm H₂O was applied, and was increased to 7.5 cm H₂O at 115 min after injury.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Experimental timeline with time intervals shown as mean values for all animals studied. The bold arrow indicates OA-induced lung injury, the shaded arrow indicates timing of perflubron (LQV) administration for PLV, and open arrows indicate the beginning of the series of PET scans in hours after perflubron administration. The early PET scans were performed approximately 5 h after injury, and the late scans were performed about 21 h after injury. MPO = myeloperoxidase.

Neuromuscular paralysis was induced in all animals at 120 min, using a 2-mg loading dose of pancuronium bromide (Organon Inc., West Orange, NJ) followed by hourly pentobarbital in a dose of 65 mg and pancuronium at 0.5 to 1 mg given intravenously for the duration of the study to maintain anesthesia and paralysis. Arterial pressure was supported with norepinephrine as needed in six of 14 animals (two control, one high-dose, and three low-dose animals).

Animals were divided into three groups: high-dose PLV (n = 4), low-dose PLV (n = 5), or CMV control (n = 5). Aside from administration of perfluorocarbon in the PLV groups, all animals were treated in the same fashion. Following perfluorocarbon loading in the appropriate study groups, all animals were transported to a positron-emission tomography (PET) scanner while being ventilated with an ambubag having an attached PEEP valve and 8 L of supplemental oxygen. PET measurements of pulmonary capillary permeability, regional lung water concentration (LWC), and fractional pulmonary blood flow (PBF) were made at a mean of 5.4 ± 1.2 h and again at a mean of 21.3 ± 1.3 h after OA-induced lung injury (Figure 1). Importantly, no dogs were suctioned at any time. Animals were maintained in the scanner overnight, until completion of the PET scanning protocols, when they were transported back to the animal facility for lung tissue sampling and euthanasia.

Administration of Perflubron

In the PLV groups, loading with perflubron at a low dose (10 ml/kg; n = 5) or high dose (30 ml/kg; n = 4) was done 120 min after lung injury, via a side-port adapter (Glaxo Wellcome Inc., Research Triangle Park, NC) placed between the endotracheal tube and the ventilator circuit to allow dosing without interrupting mechanical ventilation. The adapters were removed between doses of perflubron. Peak airway pressures were kept below 35 cm H₂O during perflubron administration, and the rate of administration was adjusted according to peak pressures. Supplemental dosing of perflubron (1.2 ml/kg/h) was given every 3 h to both the high- and low-dose groups undergoing PLV.

PET Techniques

PET was used to measure the pulmonary transcapillary escape rate (PTCER) of ⁶⁸Ga-labeled transferrin (an index of pulmonary capillary permeability), regional LWC, and PBF, with H₂¹⁵O used for the latter two measurements. Measurements were made with an in-house designed Super-PETT 3000-E in eight animals (two controls; three high-dose animals, and three low-dose animals). Performance characteristics of this system have been described previously (17). PET measurements on the remaining animals were made with a Siemens/CTI ECAT EXACT HR+ scanner (Siemens/CTI, Knoxville, TN). Methods and validation procedures for the techniques used to measure PTCER (18), LWC, and PBF (21) with PET have been previously reported.

After a blank (background) scan was performed, animals were placed in the scanner in the supine position with the most caudal PET slice 1 to 2 cm below the level of the dome of the diaphragm (identified and marked earlier by fluoroscopy). Positioning was confirmed through a short transmission scan. A 900-s transmission scan was then performed to correct for tissue attenuation during subsequent emission scans. No supplemental perflubron dosing was given from the time of transmission scan acquisition to completion of the PET protocol. Before the PET study, pilot studies were performed with both scanners and using phantoms to prove that despite the high density of perflubron, attenuation correction on the transmission scan remained accurate.

To measure PBF and LWC, we injected 60 mCi of H₂¹⁵O into the central circulation of each animal over a period of 60 s, during which 20 3-s scans were obtained. After equilibration of the injected radionuclide with tissue water (4 min), a 300-s equilibrium water scan was obtained and used to calculate LWC and the apparent regional partition coefficient for the tracer used in the calculation of PBF. During the equilibrium scan, 0.5-ml blood samples were drawn every 30 s, and the radioactivity of each sample was measured in a calibrated well counter. Thereafter, 6 mCi of ⁶⁸Ga-citrate were injected for the measurement of PTCER. The ⁶⁸Ga rapidly dissociates from its citrate and binds avidly to endogenous transferrin. Each animal was scanned for 44 min beginning at time of ⁶⁸Ga administration: 4 × 300s scans then 16 × 90s scans.

Lung Tissue Sampling

Further sedation with pentobarbital was given to animals in the animal facility before lung tissue sampling. Midline thoracotomy was performed and bucket-handle incisions were made. A size 8.5 French introducer (Baxter) was placed in the inferior vena cava below the diaphragm, and the vena cava distal to the introducer was clamped. At least 2 L of saline were used to perfuse the lungs in situ. While this was being done, a second size 8.5 French introducer (Baxter) was placed in the intrathoracic portion of the descending aorta, and blood was allowed to drain through it by gravity. Each lung lobe was removed serially, after clamping of the bronchus and vascular supply during inflation. Ventilator tidal volumes were reduced accordingly to avoid overinflation. Dogs were euthanized with excess sodium pentobarbital and potassium chloride immediately before removal of the last lobe of the lung.

Two 2 cm-by-2 cm matching lung samples were taken from each of three sites, consisting of a nondependent area of the upper lobe, a nondependent area of the lower lobe, and a dependent area of the lower lobe of each lung. Samples of lung tissue for biochemical analysis were placed in individually labeled containers filled with saline, and were stored on ice until 200-mg pieces could be cut from the respective samples in duplicate. These smaller pieces were then frozen in phosphate-buffered saline (PBS) at 70° C for later analysis of myeloperoxidase (MPO) activity.

Image Analysis

Images were reconstructed to produce seven transverse slices through the lung. Regions-of-interest in the lung and heart were defined and refined from the transmission, blood flow, equilibrium water, and ⁶⁸Ga scans as described previously (24). These regions were kept in computer memory and designated as "hemislice" regions representing one lung on each side. From four to seven evaluable slices, encompassing the most caudal lobes, were analyzed for PTCER and LWC. For PTCER (expressed as k₁/blood volume in units of 10⁴/min) and LWC (expressed as ml water/100 ml lung) determinations, hemislice regions were further subdivided into upper and lower regions by a line from the lateral chest wall to the main pulmonary artery or to the base of the heart (depending on which could be visualized on any given slice). In the PLV group, this approximated the level of high-density perflubron seen on the transmission scan, yielding predominantly perflubron-filled areas and gas-filled areas. Both hemislice and upper/lower regions (Figure 2) were analyzed separately for LWC and PTCER for all evaluable tomographic slices. Results within groups were then averaged.

View larger version (128K): [in this window] [in a new window]   Figure 2.   Transmission scan on which hemislice upper and lower regions are shown for a representative dog (low-dose perflubon group), demonstrating anatomic landmarks used to define detailed regions of interest. Opaque, dependent perflubron is seen in the dorsal regions of the lungs.

For each set of PET scans, PBF was analyzed on three consecutive tomographic slices containing the most lung tissue (as determined visually), using the same hemislice regions (as those used for PTCER calculation) from computer memory. To normalize the regional PBF data for differences in CO, PBF in each picture element (pixel) was expressed as a fraction of the total blood flow to the region. To evaluate the relationship of PBF to anatomic position within a region, the x and y coordinates for each pixel, along with the respective fractional PBF values for each pixel, were recorded. The pixel data were then sorted, first by their y coordinates. Next, within each value for y, the data were sorted again by their x coordinates. The result was a listing of pixels by location, beginning in the most ventro-medial portion of a particular region and ending with the most dorsolateral portion of the same region. Each region contained ~ 400 to 500 pixels. Arbitrarily, the data were divided into 20 "bins," which were stacked vertically in the ventral-to-dorsal direction, with the result that each bin contained 20 to 25 pixels, which could then be averaged. By keeping the number of bins per region and the number of tomographic slices per dog constant, bin values could be averaged across dogs, allowing comparisons of experimental groups.

Tissue Biochemical Analysis

For the lung MPO assay, tissue stored in PBS at 70° C was allowed to thaw. Assays were performed according to the methods of Goldblum and colleagues (25). Briefly, 200-mg lung samples were blotted dry and homogenized (Tissue Tearor, Model 985-370; Biospec Products [Fisher Scientific], Pittsburgh, PA) on ice in 1 ml 0.5% hexadecyltrimethylammonium bromide (HTAB; Sigma) buffered to pH of 6 using 50 mM phosphate buffer, and then sonicated (Sonic Dismembrator, Model 300; Fisher Scientific) on ice for 40 s. The sample was then transferred to a microfuge tube and spun (Model 5415C centrifuge; Eppendorf, Westbury, NY) at 14,000 rpm for 15 min at 4° C. Supernatant was transferred to new tubes and the process was repeated to obtain a total of three supernatants. MPO in the supernatant was measured with a standard spectrophotometric assay (Model UV160U spectrophotometer; Shimadzu, Kyoto, Japan). For the blank scan, 50 µl of HTAB solution was added to 1.45 ml of test substrate (4.175 mg o-dianisidinehydrochloride with 416.5 µl of 1:1,000 H₂O₂ mixed in 25 ml of sodium phosphate buffer, pH 6.0 [Sigma]). The absorbance change (A) at 460 nm of test substrate mixed by inversion, for an aliquot of each supernatant, was measured every 5 s for 3 min, with the readings recorded on a strip chart recorder. Data were plotted on a curve and reported as the linear slope over 1 min (A/min/mg lung 10⁴). Perflubron per se has no direct inhibitory effect on the MPO assay (15).

Statistical Analysis

All data are presented as mean ± SD. For hemodynamic and oxygenation variables as well as PTCER and LWC, statistical significance was determined with a repeated measures analysis of variance (ANOVA), using the general linear models procedure of the Statistical Analysis System (SAS Institute, Cary, NC). Comparison of regional fractional PBF in different groups was done with the SAS ANOVA procedure. MPO data in different groups were compared through a t test and by Kruskal-Wallis one way ANOVA (SigmaStat for Windows; Jandel Scientific, Chicago, IL). Statistical significance was set at p  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

There were no significant differences or trends for PTCER or LWC at any time for the upper versus lower lung subregions or for the high- versus low-dose groups. Therefore, all data presented in the following sections are limited to measurements for an entire hemislice (regions both with and without perflubron) and for both dosage groups combined (n = 9 animals in the PLV group).

Hemodynamics and Oxygenation

Baseline values were not significantly different for any of the variables considered, and no significant differences between the PLV group and the control group over time were found for mean PCWP, Ppa, Psa, CO, or pH (Table 1). In all groups, pH fell from its baseline value, but there were no significant differences between groups.

Baseline Pa_O2 (Figure 3) was not different in the control and experimental (PLV) groups. After injury, but before the application of PEEP, Pa_O2 fell in both the control and PLV groups. The decrease was greater in the control group (Figure 3), although the difference was not statistically significant. After the perflubron loading dose was administered (or after the equivalent time period in the control group), Pa_O2 in the two groups was similar (p = 0.19). Oxygenation then deteriorated in the PLV group, and Pa_O2 was significantly lower at the time of both the first and the second set of PET scans (p = 0.002 and p = 0.0001, respectively). Reductions in oxygenation were not associated with differences in CO, PCWP, mean Ppa, or Psa.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Oxygenation over the course of the study. In both the control (solid circles) and the PLV (open circles) groups, oxygenation deteriorated after OA-induced lung injury (nadir PaO2 before the institution of PEEP). Although the difference was not statistically significant, PaO2 was lower in the control group. Over time, with the institution of PEEP, oxygenation recovered and after perflubron loading, PaO2 was not different in the control and PLV groups (p = 0.19). PaO2 decreased over time in the PLV group, and was significantly lower than in the control group at both 5 h (*p = 0.002) and 21 h (* p = 0.0001).

LWC

The LWC, although increased by comparison with that of uninjured historical controls (26), was not different in the different study groups (Figure 4).

View larger version (24K): [in this window] [in a new window]   Figure 4.   Mean values for LWC in the control (CMV) and experimental (PLV) groups (bold and shaded bars, respectively). Reference normal values for LWC, were obtained by analyzing baseline (uninjured) data from 30 consecutively recently studied dogs receiving PEEP at 10 cm H2O (26). The crosshatched box represents the 25th to 75th percentiles. Values of LWC are statistically unchanged over time, but remain higher than normal values.

PBF

The pattern of regional PBF in the control group (Figure 5) was similar to that observed in previous studies of uninjured lungs. This is the expected finding when PEEP is used in this lung injury model (26).

View larger version (22K): [in this window] [in a new window]   Figure 5.   Average ventral-dorsal distribution of fractional PBF for both control and PLV experimental groups at each timepoint, as compared with normal animals on zero positive end-expiratory pressure (solid line). Bin numbers represent equal collections of picture elements on multiple PET images of PBF, with lower bin numbers situated in ventral regions and higher bin numbers situated in dorsal regions of the lungs. Each symbol represents the mean value for all dogs in each experimental group at the timepoints studied. Values for the control group (open and shaded circles) are similar over time, and are close to normal values (solid line). Values in the PLV (open and shaded triangles) group were also similar over time, but modest redistribution of PBF from dorsally located to ventrally located bins is shown. The difference between control and PLV groups approached statistical significance. p = 0.09.

Regional PBF in the PLV group showed a trend toward redistribution of blood flow away from the dorsal lung regions (Figure 5). These were regions with the highest concentration of perflubron. The fractional PBF in image bins 15 through 20 (dorsal lung regions) at 5 h and 21 h was 6% lower in the PLV group than in the control group (p = 0.09, ANOVA).

The perfusion pattern did not change over time in either group.

PTCER

Average PTCER was increased, indicating lung injury after OA administration, in both the control and PLV groups as compared with historical normal values without PEEP (40 ± 10 10⁴/min) (27) (Figure 6). There was no significant change in PTCER over time in either of the study groups.

View larger version (22K): [in this window] [in a new window]   Figure 6.   Mean values for PTCER (index of pulmonary capillary permeability) in the control and experimental (PLV) groups (bold and shaded bars, respectively) are compared with historical normal values for uninjured animals on zero positive end-expiratory pressure (27) (shaded box represents PTCER mean ± 1 SD). Values are essentially unchanged over time, but remain higher than normal values.

MPO

A lower value for MPO activity (A/min/mg lung) was seen for all regions sampled in the PLV animals as compared with control animals (overall 36% reduction in MPO activity for the PLV group compared with the control group). Similar regional reductions in MPO activity were noted in the PLV group when compared with the control group for all areas sampled (reductions of 37% for the upper lobe nondependent region, 29% for the lower lobe nondependent region, and 39% for the lower lobe dependent region). When only the upper lobe regions were considered (not directly affected by the presence of perflubron), mean MPO values were significantly lower in the PLV group (5.10 ± 2.94) than in the control group (A = 8.12 ± 2.79/min/mg lung 10⁴) (p = 0.016) (Figure 7).

View larger version (17K): [in this window] [in a new window]   Figure 7.   Mean values for MPO levels (measure of neutrophil activity) in upper, nondependent lung areas (essentially free of perflubron) are shown for the control and PLV groups (bold and shaded bars, respectively). MPO activity was significantly lower in the PLV group. *p = 0.016.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study was performed to assess the effect of PLV on pulmonary capillary permeability and lung water accumulation during a 21-h period of observation after acute lung injury. Our hypothesis was that both of these indices of injury would improve over time in PLV-treated animals. We compared the effects of conventional gas ventilation with those of two doses (10 ml/kg and 30 ml/kg) of intratracheally administered perflubron. Our data represent the only intermediate-term (21-h) data on permeability and lung water accumulation in a large animal model of acute lung injury treated with PLV, and are therefore particularly relevant to the potential clinical use of PLV.

Effects on Gas Exchange

Oxygenation in the control group deteriorated after injection of OA, as evidenced by the nadir Pa_O2 (lowest recorded Pa_O2 before the institution of PEEP). Five hours of after injury, oxygenation with a PEEP of 7.5 cm H₂O had recovered to baseline values, and remained stable over the next 15 h of measurement (Figure 3). This recovery of oxygenation suggests that the level of PEEP chosen for this study was optimal, and that most areas of atelectatic or consolidated lung had been successfully recruiteda result consistent with previous work by our group with this model of lung injury (26). In the group of dogs treated with PLV, oxygenation deteriorated over time and was lower at 5 and 21 h than in the CMV group, despite a higher nadir Pa_O2. Although other investigators have shown significant dose-dependent improvements in oxygenation with PLV (4), such improvements in oxygenation have not been reported uniformly (12, 15, 28). An additional study suggests that initial improvements may not be sustained over time (28).

Fractional PBF distribution in the control group was similar to that in normal, uninjured controls (Figure 5) (i.e., animals failed to show as much redistribution of PBF as is usually seen in this model [29]). Previous studies with this model (26) have shown that this is the expected effect of using PEEP. In contrast, the PLV group showed a modest though not quite statistically significant amount of perfusion redistribution as compared with the control group (Figure 4), which is consistent with previous findings in uninjured (11) and injured lungs (10).

With respect to LWC, and in contrast to the findings in the present study, Gauger and colleagues (10) reported that PLV treatment in the OA-induced lung injury model was associated with significant reductions in LWC. However, their measurements were made at 135 min after OA administration, the dose of OA that they used was significantly higher than in our study (leading to greater injury and edema accumulation), and they performed suctioning on these animals before perflubron dosing.

Because edema fluid has a lower density than perflubron, small-airway and alveolar fluid tends to be displaced centrally by this perfluorocarbon. Thus, suctioning may be effective for reducing LWC during PLV. Since we were primarily interested in determining whether perflubron reduced lung water accumulation by a direct effect, no suctioning was allowed in our protocol at any time.

Oxygenation in the OA-induced model of lung injury is a composite result of extravascular lung water accumulation, dorsal atelectasis, and regional PBF (30). Since oxygenation was preserved in the control group despite increases in LWC and normal measured fractional pulmonary blood flow, it can be inferred that dorsal atelectasis was reversed by the application of PEEP (26). Under these circumstances the addition of perflubron (which carries approximately 50% less oxygen than gas with a fractional O₂ of 1.0) to these dorsal lung units actively participating in gas exchange would be expected to result in less effective overall gas exchange, other factors being constant.

Effects on Lung Injury

PTCER in the OA-induced model of lung injury has previously been shown to be stable over approximately 24 to 28 h with zero end-expiratory pressure (27). In the control group of the present study, absolute values for PTCER were similar to those previously reported. The degree of injury produced, as assessed by measurements of PTCER, is moderate as compared with that observed in ARDS patients through use of the same measurements (19).

We found no difference in capillary permeability in the control group as compared with the PLV group at either 5 or 21 h after injury, whether the data were analyzed by region (i.e., containing or not containing perflubron), by dose, by time, or collectively (Figure 6). Although Colton and colleagues (13) reported early reductions in permeability in a rodent lung injury model treated with PLV, others have reported little or no change in permeability (12, 31). Since PTCER has been shown to correlate with histologic evidence of injury in previous studies (20), our finding of ongoing pulmonary capillary leak are unexpected, given reported reductions in histologic evidence of lung injury in previous (6, 7, 15, 28) but not all (9) studies.

MPO (used as a marker of pulmonary neutrophil number) has been consistently reduced with PLV in a variety of small-animal models of acute lung injury (6, 12, 14, 15). Although the mechanisms for a reduction in MPO have not been elucidated, they do correlate with reduced tissue neutrophil numbers (14, 25) as well as with improved histologic and morphometric indices of injury (6, 14). Since perflubron is nearly twice as dense as normal lung tissue, tissue regions containing this perfluorocarbon will, when normalized for weight, show a bias in favor of the PLV group (less lung tissue per gram of weight, and therefore lower MPO values). However, we found lower values for MPO in the PLV than in the CMV groups (Figure 2) even when nondependent lung regions that were free of perflubron (based on density comparisons, as in Figure 2) were analyzed (Figure 7).

We measured MPO in anticipation of finding reduced PTCER (decreased permeability) with PLV. The reduction in MPO, and reports by others of histologic improvement (despite a lack of effect on permeability) as a marker of lung injury, raise the issue of what might be the underlying mechanism for these observations. Recent work has suggested a possible role for immune modulation of the pulmonary inflammatory response (3, 32, 33), including the previously noted reduction in pulmonary neutrophils (6, 14, 15).

Despite a short-term survival advantage (over 3 to 8 h) for PLV over conventional ventilation in porcine models of lung injury (6, 34), failure of PLV to reduce PTCER or LWC over a period of 20 h in our study suggests that direct tamponade of the injured alveolus is not the primary mechanism of benefit of PLV. Rather, benefits of PLV may be mediated by a modification of the immune response to the initial injury or to subsequent lung injury sustained as a result of the effects of mechanical ventilation on inflamed lung tissue, as evidenced by the reduction in MPO activity that we observed even in lung regions in which there was no direct tissue contact with perflubron (35). In other words, PLV, when applied under the conditions used in the present study, may be a more "lung friendly" approach to the mechanical ventilation of injured, inflamed lungs. Whether PLV will provide benefits beyond improvements obtained with the open lung approach (36) in patients with acute lung injury or ARDS remains to be determined by the outcome of ongoing clinical trials.

Conclusion

In the OA-induced model of pulmonary edema and lung injury in dogs, in which the PEEP level can fully reverse deterioration in gas exchange, the addition of PLV to CMV did not improve oxygenation. No dose-dependent attenuation of pulmonary capillary leak or lung water accumulation attributable to PLV could be demonstrated over approximately 21 h of observation. There was a significant reduction in pulmonary neutrophil activity, suggesting that the benefits of PLV may be due to modifications in the inflammatory response rather than to a direct mechanical sealant effect on injured alveoli.