Effect of lung-protective ventilation on severe Pseudomonas aeruginosa pneumonia and sepsis in rats

Kiyoyasu Kurahashi,1 Shuhei Ota,1 Kyota Nakamura,1 Yoji Nagashima,2 Takuya Yazawa,3 Minako Satoh,1 Asako Fujita,1 Ritsuko Kamiya,1 Eri Fujita,1 Yasuko Baba,1 Kanji Uchida,1 Naoto Morimura,1 Tomio Andoh,1 and Yoshitsugu Yamada1

Departments of 1Anesthesiology and Critical Care Medicine, 2Molecular Pathology and Oncology, and 3Cellular Pathobiology, Yokohama City University Graduate School of Medicine, Yokohama, Kanagawa, Japan 236-0004

Submitted 5 December 2003 ; accepted in final form 19 April 2004


    ABSTRACT
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 ABSTRACT
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Pneumonia caused by Pseudomonas aeruginosa carries a high rate of morbidity and mortality. A lung-protective strategy using low tidal volume (VT) ventilation for acute lung injury improves patient outcomes. The goal of this study was to determine whether low VT ventilation has similar utility in severe P. aeruginosa infection. A cytotoxic P. aeruginosa strain, PA103, was instilled into the left lung of rats anesthetized with pentobarbital. The lung-protective effect of low VT (6 ml/kg) with or without high positive end-expiratory pressure (PEEP, 10 or 3 cmH2O) was then compared with high VT with low PEEP ventilation (VT 12 ml/kg, PEEP 3 cmH2O). Severe lung injury and septic shock was induced. Although ventilatory mode had little effect on the involved lung or septic physiology, injury to noninvolved regions was attenuated by low VT ventilation as indicated by the wet-to-dry weight ratio (W/D; 6.13 ± 0.78 vs. 3.78 ± 0.26, respectively) and confirmed by histopathological examinations. High PEEP did not yield a significant protective effect (W/D, 4.03 ± 0.32) but, rather, caused overdistension of noninvolved lungs. Bronchoalveolar lavage revealed higher concentrations of TNF-{alpha} in the fluid of noninvolved lung undergoing high VT ventilation compared with those animals receiving low VT. We conclude that low VT ventilation is protective in noninvolved regions and that the application of high PEEP attenuated the beneficial effects of low VT ventilation, at least short term. Furthermore, low VT ventilation cannot protect the involved lung, and high PEEP did not significantly alter lung injury over a short time course.

acute lung injury; ventilator-induced lung injury; lung protective strategy; cytokines; bacterial toxins


DESPITE ONGOING IMPROVEMENT in antimicrobial therapies, pneumonia still represents a life-threatening condition with high mortality and morbidity, especially for critically ill patients (10, 26). In fact, severe pneumonia and associated sepsis are the most common cause of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Although oxygen supplementation and mechanical ventilation are a common mode of support in these patients, ventilation itself has the potential to cause systemic spread of infection (4, 20) or lung injury (29). Randomized controlled trials involving ALI patients have shown that a lung-protective strategy with low tidal volume (VT) ventilation (VT of 6 ml/kg) (1) and application of positive end-expiratory pressure (PEEP) (2) reduced patient mortality when compared with conventional mechanical ventilation (VT of 12 ml/kg).

Pneumonia is a common cause of ALI and usually presents inhomogenously, yielding discrete areas of infection and leaving other regions of the lung relatively spared. Therefore, we designed a study to investigate whether VT and PEEP maintained their beneficial effects on various areas of the lung in the context of severe pneumonia. We modified an animal model of pneumonia that we described previously (17, 18, 3032) to mimic the course of unilateral pneumonia. Pseudomonas aeruginosa was utilized to induce infection, as it is a common causative pathogen of nosocomial pneumonia (6, 7, 27, 35). In addition, P. aeruginosa pneumonia, particularly when induced with the PA103 strain, frequently leads to bacteremia and sepsis (9, 11, 12, 17, 18, 24, 30).

Thus the first objective of this study was to test the hypothesis that low VT ventilation would reduce lung injury of the affected lungs and nonaffected lungs following instillation of cytotoxic bacterial pathogen. Furthermore, we investigated whether this reduced lung injury would diminish the severity of sepsis and tested the hypothesis that application of high PEEP would further protect the lungs from ventilator-induced lung injury.


    METHODS
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Pathogen and animals. Bacterial suspensions of P. aeruginosa strain PA103, which is a cytotoxic strain that produces the type III secreted toxins, ExoU and ExoT (11, 14), were prepared as previously described (32). Briefly, PA103 was subcultured on Vogel-Bonner minimal medium plates and inoculated into trypticase soy broth (Becton Dickinson, Sparks, MD) containing 10 mM nitrilotriacetic acid (Dojin, Kumamoto, Japan) and incubated overnight. The bacterial pellet was washed twice with lactated Ringer solution (L/R; Otsuka, Tokyo, Japan) and diluted into the appropriate number of colony-forming units (CFU) per milliliter in L/R as determined by spectrophotometry. The number of bacteria in the solution was confirmed by serial dilution followed by culture on agar plates.

All protocols for animal experiments were approved by the Animal Research Committee of Yokohama City University. Specific pathogen-free male Sprague-Dawley rats, weighing 310–400 g (9–11 wk) (Japan SLC, Shizuoka, Japan), were used for all animal experiments.

Experimental groups. Sixty-three rats were used to evaluate lung injury and systemic responses of acutely infected animals. After initial anesthesia was given, rats were randomized to receive one of three ventilation settings: high VT + low PEEP (VT 12 ml/kg, PEEP 3 cmH2O), low VT + low PEEP (VT 6 ml/kg, PEEP 3 cmH2O), or low VT + high PEEP (VT 6 ml/kg, PEEP 10 cmH2O). Thirty rats (control group) received an air space instillate that did not contain bacteria and followed the same procedure.

Animal preparation. Anesthesia was induced by injection of 25 mg of pentobarbital sodium into the peritoneal cavity. A tracheotomy was performed, and a 14-gauge plastic cannula (SR-OT1451C; Terumo, Tokyo, Japan) was inserted into the trachea to serve as an endotracheal tube. Mechanical ventilation was maintained by a constant volume pump (SN-480-7; Shinano, Tokyo, Japan) with an inspired oxygen fraction of 1.0 at a VT of either 12 or 6 ml/kg. The VT delivered by the ventilator was calibrated volumetrically by collection of expired gas. In brief, the ventilator circuit was connected to a test lung that would generate intracircuital pressure of ~20 cmH2O while expiratory gas collected from the expiration port. VT was adjusted accordingly. A PEEP of either 3 or 10 cmH2O was applied. Ventilation frequency was adjusted to maintain the arterial carbon dioxide tension between 35 and 50 Torr. A curved polyethylene tube with an inner diameter of 0.58 mm (PE50, Becton Dickinson) was carefully inserted through the endotracheal tube into the left lung for subsequent instillation of bacterial suspension. The right carotid artery was catheterized with a 24-gauge plastic cannula (SR-OT 2419C, Terumo) for measurement of blood pressure and sampling of arterial blood. After completion of these preparations, rats were placed in the left lateral decubitus position on a warming device and were observed for at least 30 min until stabilization of respiration and circulation.

Next, instillation was performed over a 10- to 15-min period (time 0). Arterial and airway pressures were monitored continuously on a hemodynamic monitor (Life Scope 12; Nihon Kohden, Tokyo, Japan). A rectal probe (ME-PDK061, Terumo) was used for continuous temperature monitoring (CTM-303, Terumo), and body temperature was maintained between 36 and 38°C via a warming device. Blood (700 µl) was sampled every hour for blood gas analysis, measurement of the efflux of the air space protein tracer into the circulation, and measurement of cytokines. Blood gas and acid-base analyses were performed with a critical care analyzer (OPTI3; AVL Scientific, Roswell, GA). The volume of withdrawn blood was replaced by equivolume intra-arterial administration of L/R, just after the blood sampling. In addition, 1 ml of L/R was given intra-arterially at an interval of 10, 5, or 2 min when systolic blood pressure decreased to 85, 75, or 65 mmHg, respectively. Six hours after infection, rats were deeply anesthetized and euthanized.

Instillation of P. aeruginosa and alveolar protein tracers. The instillate (3 ml/kg) contained PA103 (4 x 107 CFU/ml), 0.5 µCi of 131I-labeled human albumin (Daiichi Radioisotope, Tokyo, Japan) as the air space protein tracer, and 5% bovine serum albumin (Wako, Osaka, Japan) in L/R to evaluate transpulmonary protein leakage. The instillate also contained 10 µg of Evans blue (Sigma) to visualize the instillation on autopsy. Animals in which the instillate was not limited to one lung were excluded from study. The instillates were administered by a published method, and calculations were performed as described (38).

Bronchoalveolar lavage. A set of rats in each group underwent collection of bronchoalveolar lavage (BAL) fluid 6 h after the instillation. Five BALs were performed in each lung via instillation of 1.5 ml of PBS containing 0.1% EDTA per BAL. Approximately 100 µl of BAL fluid were set aside for bacterial cultures. The remaining volume was centrifuged at 3,400 g at 4°C for 20 min to obtain the supernatant, which was stored at –80°C until use.

Measurement of lung injury. Lung injury was estimated in four different ways: 1) the efflux of intra-alveolar 131I-labeled albumin into the circulation, 2) the wet-to-dry weight ratio (W/D) of the lungs, 3) the concentration of lactate dehydrogenase (LDH) in BAL fluid, and 4) the lung histology.

1) We calculated the efflux of 131I-labeled albumin into the circulation by multiplying the counts measured in the blood sample by the systemic blood volume (body wt x 0.07), as previously reported (16, 37). Efflux was used as an indication of alveolar epithelial barrier destruction.

2) A set of rats in each group underwent measurement of W/D. Each lung was harvested 6 h after the infection, individually homogenized, placed in a preweighed aluminum pan, and dried to constant weight in an oven at 80°C for 3 days. The W/D of the lungs was calculated in the established manner (34, 37) and taken as a reflection of lung edema.

3) The concentration of released LDH in the BAL fluid was measured according to the manufacturer's protocol (CytoTox 96; Promega, Madison, WI). The values were compared with the serial dilution of LDH-positive control (lysed L929 fibroblast cells) as standards. One unit of LDH in this study was the same level of the enzyme found in 1,000 lysed L929 fibroblast cells.

4) Lungs of the last set of rats were used for histological and immunohistochemical examination. The lungs were fixed with 4% formalin in PBS overnight. They were then infused with 10% formalin and sectioned and processed for paraffin-embedded sections. Four-micrometer-thick lung tissue sections were stained with hematoxylin and eosin (H-E) for routine histological examination.

Bacterial cultures. To assess bacteremia, we cultured arterial blood (100 µl) on tryptic soy agar plates. For the quantification of bacteria in the lungs, BAL fluid was diluted with sterile PBS and streaked onto agar plates. Bacterial colonies were counted after incubation at 37°C for 12 h, and we determined the quantity of bacteria by multiplying counted colonies by the dilution ratio.

Leukocyte count. The leukocyte count in the systemic circulation was quantified microscopically. Arterial blood (25 µl) obtained 6 h after the infection was added to 475 µl of 2% acetic acid containing 0.01% Gentian violet, and cell counts were multiplied by the dilution factor to obtain the number of leukocytes in the sample.

Assay for cytokines in BAL fluid and blood. A biological TNF-{alpha} assay was performed with mouse sarcoma cells, WEHI-13VAR (CRL2148; American Type Cell Culture, Manassas, VA), as previously reported (8, 18). We calculated the TNF-{alpha} activity of each sample by comparing absorbance to that of a standard curve made from dilutions of rat TNF-{alpha} (PharMingen, San Diego, CA) between 1.2 and 1,250 pg/ml. The lower limit of detection for this assay was 1.2 pg/ml. One of the most potent CXC chemokines, growth-related oncogene (GRO)/cytokine-induced neutrophil chemoattractants (CINC)-1, was measured by enzyme-linked immunosorbent assay (ELISA) with a commercial ELISA kit following the manufacturer's protocols (RPN2730; Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK). The lower limit of detection was 4.7 pg/ml.

Immunohistochemistry of lungs. For immunohistochemistry, sections were immersed in 0.3% hydrogen peroxide to inactivate intrinsic peroxidase activity. After antigen retrieval by microwave treatment in a citrate buffer and blocking with 10% normal rabbit serum-PBS, sections were treated with the primary antibody against rat TNF-{alpha} (R&D Systems, Minneapolis, MN; diluted 1:200) overnight at 4°C. After the labeled antigens were visualized with a catalyzed signal amplification system kit (Dako, Carpinteria, CA) and diaminobenzidine, the nuclei were counterstained with hematoxylin. Each sample was examined at five randomly selected fields, and the alveolar epithelial cells were scored 3–0 in a blinded fashion according to their intensity of staining: score 3 indicated intense staining, score 2 was moderate staining, score 1 was mild staining, and score 0 was faint or no staining. The medians of these scores were used to determine TNF-{alpha} positivity.

Permeability of lungs to systemic TNF-{alpha}. A separate group of rats (n = 12) was used to determine the permeability of lungs to systemic TNF-{alpha}. The protocol for this experiment was the same as the general protocol except that 131I-albumin was not administered in this experiment. Two hours after instillation of bacteria, 125I-labeled TNF-{alpha} (IM206, Amersham Pharmacia) was administered intravenously. Arterial blood samples were collected 10 and 20 min after the injection, and radioactivity was measured as counts per min (CPM)/g of plasma. Total CPM injected was calculated via a modification of an established method (17, 37): we multiplied CPM/g of plasma by estimated plasma volume [body wt (g) x 0.07 x (1 – hematocrit/100)]. Rats were killed at 6 h, and BAL samples of both lungs were collected separately by the protocol outlined above. A portion of recovered BAL was weighed and measured for CPM/g, and we calculated total CPM in the air space by multiplying the CPM/g with the total volume of the liquid instilled to obtain BAL (7.5 ml). The percentage of TNF-{alpha} in the air space was calculated from total CPM in the air space divided by total CPM injected. This series of experiments also included a set of spontaneously breathing control rats without instillation that were anesthetized and placed in the left lateral decubitus position for 6 h without mechanical ventilation. Muscle relaxants were not administered to these spontaneously breathing control rats, and anesthesia was maintained with intraperitoneal pentobarbital.

Statistical analysis. The mean values of measurements made only once during the protocol were compared by unpaired t-tests. Measurements made more than once per animal were compared by repeated-measures ANOVA. Pairwise comparisons were made by one-factor ANOVA followed by Scheffé's post hoc analysis. Scattered data were analyzed with Mann-Whitney U-test. Significance was set at P <0.05. Values are reported as means ± SE or as median with 25th and 75th percentiles.


    RESULTS
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 ABSTRACT
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Exclusion of animals. Two animals in the high VT + low PEEP group and one animal in the low VT + high PEEP group were excluded from the study, because they died before completion of the experiment. These deaths were attributed to intra-abdominal hemorrhage secondary to intraperitoneal pentobarbital injection and from exsanguination from a neck incision. One animal in the high VT + low PEEP group, two animals in the low VT + low PEEP group, and one animal in the low VT + high PEEP group were excluded from the study because of instillation of the right lung or both lungs. This was confirmed by multiple methods after completion of the study, including bacterial counts in BAL fluid, staining of lungs with dye in the instillate, and examination of the placement of an instillation catheter.

Respiration and airway status. Ventilation frequency required to maintain partial pressure of arterial carbon dioxide (PaCO2) in the desired range was highest in the low VT + high PEEP group followed by the low VT + low PEEP group (Fig. 1A). Peak airway pressure increased after the instillation except the low VT + high PEEP group, and the value was significantly lower in the low VT + low PEEP group at the end of the experiment (Fig. 1B).



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Fig. 1. Ventilation frequency and peak airway pressure. A: ventilation frequency was adjusted to maintain partial pressure of arterial carbon dioxide (PaCO2) between 35 and 50 Torr. B: peak airway pressure was continuously measured. Bacterial suspension instillation was performed at time 0. VT, tidal volume; PEEP, positive end-expiratory pressure. Values are means ± SE. *P < 0.05 vs. high VT + low PEEP group. {dagger}P < 0.05 vs. low VT + low PEEP group; ¶P < 0.05 vs. baseline value. Here and throughout figures, numbers in parentheses refer to numbers of samples.

 
Injury in the bacteria-instilled lungs. After instillation of bacteria, the presence of alveolar protein tracer in the circulation increased in a time-dependent manner, indicating injury to the alveolar epithelial cells (Fig. 2A). The tracer levels were significantly less in the low VT + low PEEP group when compared with those in the high VT + low PEEP group and the low VT + high PEEP group. In control animals, the alveolar protein tracer did not leak into the circulation with any of the ventilation settings, indicating no alveolar epithelial injury (Fig. 2B). Two other indexes for the injury of the instilled lung, LDH concentration in the BAL fluid and W/D of the lung, were high in all groups (Fig. 3, A and B), and we found no differences when comparing the groups.



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Fig. 2. Air space protein tracer in blood. A: infected animals. B: uninfected control animals. The quantity of 131I-albumin that entered the circulation via the lungs was calculated and is shown as a percentage of the initial dose. Instillation was performed at time 0. Values are means ± SE. ¶P < 0.05 vs. baseline value; {dagger}P < 0.05 vs. low VT + low PEEP group.

 


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Fig. 3. Assessment of lung injury. Unless otherwise mentioned, data are shown from rats that underwent instillation of bacterial suspension into the left lung, and lungs were harvested 6 h after the instillation. A: concentration of lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid. BAL of both lungs was performed separately 6 h after the instillation. B: lung wet-to-dry weight ratio (W/D). C: W/D of rats instilled with vehicle solution instead of the bacterial suspension (control). D: concentration of tumor necrosis factor (TNF)-{alpha} in the BAL fluid. E: concentration of growth related oncogene/cytokine-induced neutrophil chemoattractants-1 (GRO/CINC-1) in the BAL fluid. Means ± SE. *P < 0.05 vs. high VT + low PEEP group.

 
Concentrations of TNF-{alpha} and GRO/CINC-1 in the BAL fluid of the instilled lungs were elevated in all three groups, and we found no differences in TNF-{alpha} and GRO/CINC-1 concentrations when comparing the groups (Fig. 3, D and E).

Histopathological study of the instilled lungs revealed severe lung injury, i.e., intra-alveolar hemorrhage, protein precipitation, and leukocyte infiltration into the alveoli, and edematous thickening of perivascular space in the instilled lungs (Fig. 4A). We found no difference in histopathology when comparing the three groups.



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Fig. 4. Representative micrographs of lung tissue stained with hematoxylin and eosin. A: instilled lung in the high VT + low PEEP group. B: noninstilled lung of uninfected rats ventilated with high VT + low PEEP for 6 h: no injury of lung components or inflammatory cells were seen.

 
Bacteremia and systemic responses. All rats were bacteremic at the end of the experiment, and there was no difference in the quantity of bacterial colonies among the groups (Table 1).


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Table 1. Leukocyte and bacterial counts, TNF-{alpha} concentration, and fluid supplementation in experimental groups

 
Mean arterial pressure decreased in a time-dependent fashion and was significantly lower than the baseline values in all groups (Fig. 5A). There was no difference in the mean arterial pressure among the three groups. The volume of extra fluid given to the animals to maintain systolic blood pressure averaged 11–13 ml, with no difference in the volume of administered fluid among the groups (Table 1). Blood base excess decreased continuously after infection was induced, and the animals developed severe acidosis 6 h after the instillation (Fig. 5B). There was no significant difference in the degree of base excess among the groups.



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Fig. 5. Mean arterial pressure (MAP), base excess, and alveolar-arterial oxygen difference (A-aDO2) for 6 h. Instillation was performed at time 0. A: calculated MAP as an indicator of hemodynamics. B: base excess as an indicator of acid-base status. C: A-aDO2 as an indicator of oxygenation. Values are means ± SE. ¶P < 0.05 vs. baseline value; {ddagger}P < 0.05 vs. low VT + high PEEP group.

 
Oxygenation, calculated as alveolar-arterial oxygen difference (A-aDO2), deteriorated after bacterial instillation. A-aDO2 increased in a time-dependent manner and was significantly higher at 6 h when compared with baseline values in the high VT + low PEEP group and in the low VT + low PEEP group (Fig. 5C). In contrast, oxygenation was well maintained in the low VT + high PEEP group, even after the instillation of bacteria. Accordingly, A-aDO2 was significantly lower in the low VT + high PEEP group compared with the other two groups.

The leukocyte count in the circulation at 6 h after the instillation of bacteria was low (Table 1). We found no significant difference in the leukocyte count in the circulation when comparing the groups.

The concentration of TNF-{alpha} in the circulation measured at 6 h after the instillation was elevated in all groups, but there was no statistical difference among the groups (Table 1).

Effect of ventilation on the contralateral lung. A negligible amount of LDH was found in the BAL fluid of noninstilled lungs (Fig. 3A). The W/D of the noninstilled lungs in the high VT + low PEEP group was elevated (Fig. 3B). The W/D of the noninstilled lungs in the low VT + low PEEP group and in the low VT + high PEEP group were significantly lower than those in the high VT + low PEEP group, which were comparable to the values of control, noninfected animals (Fig. 3C). Histopathological study of the noninstilled lungs showed an enlargement of the perivascular space in the high VT + low PEEP group (Fig. 6A). Overdistension of alveoli was also observed in the high VT + low PEEP group and in the low VT + high PEEP group (Fig. 6, A and C). These abnormal findings were not seen in the low VT + low PEEP group (Fig. 6B).



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Fig. 6. Representative micrographs of lung tissue stained with hematoxylin and eosin (A–C) or immunohistochemistry of TNF-{alpha} (D–F). A, D: noninstilled lung in the high VT + low PEEP group: enlargement of the perivascular space (arrows) and overdistension of alveoli (*) were seen, and intense staining for TNF-{alpha} was observed in alveolar epithelial cells, bronchioepithelial cells, and alveolar macrophages. B, E: noninstilled lung in the low VT group. C, F: noninstilled lung in the low VT + high PEEP group: overdistension of alveoli (*) were seen.

 
The concentration of TNF-{alpha} in the BAL fluid of noninstilled lung in the high VT + low PEEP group was significantly higher than that in the other two groups (Fig. 3D). The concentration of GRO/CINC-1 in BAL fluid of noninstilled lungs was elevated above baseline but was still lower than concentrations seen in the BAL of instilled lungs (Fig. 3E). Immunohistochemical analysis showed positive labeling for TNF-{alpha} in the alveolar epithelial cells of the noninstilled lungs in the high VT + low PEEP group (Fig. 6D). In contrast, the signal intensity of these cells in the low VT + low PEEP group and in the low VT + high PEEP group was low (Fig. 6, E and F). The positivity score of TNF-{alpha} in the high VT + low PEEP group (median was 3, and 25th and 75th percentiles were 1 and 3) was significantly higher when compared with that in the low VT + low PEEP group (median was 1, and 25th and 75th percentiles were 1 and 2) or in the low VT + high PEEP group (median was 2, and 25th and 75th percentiles were 1 and 2). The fraction of TNF-{alpha} in the BAL fluid from noninstilled lungs that was attributed to entry from the systemic circulation in any infected groups was comparable to that in noninfected, unventilated control rats (Fig. 7).



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Fig. 7. The fraction of 125I-labeled TNF-{alpha} in the air space over total TNF-{alpha} injected into the circulation. 125I-TNF-{alpha} was injected 2 h after the instillation of Pseudomonas aeruginosa. Lungs were harvested 6 h after the instillation. 125I-TNF-{alpha} in the air space of instilled lungs and noninstilled lungs was calculated as a fraction of the amount injected. As a reference, the value for the noninfected unventilated control rats is presented.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study demonstrated that the lung-protective ventilatory strategy could not ameliorate damage in the affected lung during the acute phase of severe pneumonia and concomitant sepsis induced by a cytotoxic strain of P. aeruginosa. Although lung edema and inflammation also occurred in the noninvolved lungs when we used the relatively higher VT of 12 ml/kg, which is still clinically acceptable, the use of low VT ventilation protected noninvolved lungs from ventilator-induced lung injury. Physiological changes in the noninvolved lung were confirmed morphologically (Fig. 6, A–C), demonstrating that the use of low VT ventilation prevented development of perivascular fluid accumulation that was observed in the high VT + low PEEP group.

To investigate mechanisms of lung edema in the noninvolved lung, we assessed TNF-{alpha} concentration in the BAL fluid. The increase in TNF-{alpha} seen with conventional ventilation was attenuated by the use of low VT ventilation with or without PEEP, which was also consistent with the degree of pulmonary edema seen in each group. Hamacher and colleagues (15) investigated the BAL fluid of early-phase ARDS patients and found high concentrations of TNF-{alpha} in their BAL fluid. Furthermore, incubation of human lung microvascular endothelial cells with the BAL fluid from early-phase ARDS was cytotoxic to the endothelial cells, and neutralization of TNF-{alpha} inhibited the cytotoxic activity of the BAL fluid. Therefore, TNF-{alpha} detected in the BAL fluid of the noninfected lungs may be responsible for at least a portion of the lung endothelial cell damage and associated pulmonary edema in the noninfected lungs of the high VT + low PEEP group.

The influx of TNF-{alpha} in the noninstilled lungs from the circulation was negligible in all experimental groups, and immunohistochemical findings confirmed production of TNF-{alpha} in alveolar macrophages and in bronchial and alveolar epithelial cells with conventional ventilation. Furthermore, the trend in the positivity score of TNF-{alpha} of the cells in the noninstilled lungs correlated well with the concentration of TNF-{alpha} in the BAL fluid of noninstilled lungs (Fig. 3D). These observations suggest that pulmonary epithelial cells and alveolar macrophages of noninfected lungs release TNF-{alpha} in the alveolar space when lungs are ventilated with high-stretch, high VT ventilation in the presence of sepsis physiology and/or with infection of other part of lungs. This idea is supported by a report from Pugin and coworkers (22), which demonstrated marked elevation of TNF-{alpha} in the supernatant of the culture medium when alveolar macrophages were stretched in the presence of the gram-negative bacterial toxin lipopolysaccharide (LPS). In an ex vivo study performed by Ricard and colleagues (25), isolated nonperfused rat lungs were ventilated for 2 h with a VT of 42 ml/kg. When LPS was given intravenously 50 min before lungs were removed, TNF-{alpha} concentration in BAL fluid was elevated in the ventilated lungs.

Histological study revealed only the sparse presence of inflammatory cells in noninstilled lungs. Although the chemoattractant molecule GRO/CINC-1 was present in the BAL fluid of noninstilled lungs, the concentration was relatively low in all three experimental groups. In a model of intraperitoneal inflammation, Call and associates (3) found that the ratio of local to systemic chemokine concentration was a significant factor for local neutrophil recruitment to the peritoneal cavity. Thus the relatively low concentration of GRO/CINC-1 in the unaffected lung compared with that of the affected lung may account for the absence of infiltrates in the noninstilled lungs. Furthermore, Wilson and associates (39) determined that the BAL fluid of lungs ventilated with high VT was associated with increased TNF-{alpha} at the early stage of lung injury but not in the latter stages. Thus the high concentration of TNF-{alpha}, low concentration of chemokine, and absence of inflammatory cells in the noninvolved lungs in the present study may represent an early stage of ventilator-induced lung injury, and TNF-{alpha}-mediated inflammation may be evident at later stages of high VT + low PEEP ventilation.

In the present study, all animals were bacteremic by the end of the experiment, and there was no difference in the degree of bacteremia among groups. The PA103 strain used in the present study produces a type III secreted toxin, ExoU (11), which is cytotoxic to lung epithelial cells and alveolar macrophages in vitro (11, 18). Instillation of PA103 into the air spaces of animals produced severe lung injury (30, 32, 33) and septic shock (18), which is consistent with the results of the present study. This injury likely limits the utility of lung-protective ventilation in the affected lung in this P. aeruginosa pneumonia model.

Careful attention was given to the determination of VT in this experiment. Using the P. aeruginosa pneumonia model, Savel and colleagues (29) demonstrated that low VT ventilation correlated with decreased alveolar permeability; however, they failed to show the difference in alveolar protein permeability indicated by a percentage of instilled 125I-albumin in blood. Furthermore, they demonstrated that large VT, 15 ml/kg, in the absence of pneumonia resulted in lung injury (e.g., increased epithelial protein permeability and alveolar infiltrates), indicating that ventilation with large VT per se caused lung injury. In another study by Frank and coworkers (13), mechanical ventilation of rats with a VT of 12 ml/kg did not result in lung injury. In the present study, a VT of 12 ml/kg did not result in lung injury in rats that were not infected as demonstrated by alveolar protein tracer analysis (Fig. 2B), lung W/D (Fig. 3C), and light microscopic analysis of H-E-stained lung section (Fig. 4B).

As ventilation frequency was adjusted to control PaCO2 within a certain range, high frequency, e.g., >120/min, was necessary in the low VT groups. This extreme of ventilation frequency may produce some error in the determination of VT actually delivered to the lung; when short inspiratory time is used, actual VT delivered to the lung is highly dependent on the flow rates generated by the ventilator and the corresponding compressible losses in the ventilator tubing. To estimate actual lung delivered VT, we measured compressible volume in the ventilator tubing. The compressible volume was 0.03 ml/cmH2O; therefore, the compressible losses are relatively small compared with VT and can therefore be regarded as negligible. It is still possible that a short inspiratory time without an end-inspiratory pause may result in some difference in an inspiratory volume distribution within the lungs; short expiratory time might have generated intrinsic PEEP, resulting in increased total PEEP above externally applied PEEP. Although a concomitant decrease in VT was likely to limit the generation of intrinsic PEEP in the low VT groups, this could exaggerate the overdistending effect of PEEP in the nonaffected lung.

High PEEP (10 cmH2O) was utilized in the present study to protect lungs from ventilator-induced lung injury, in accordance with previous reports (13, 36). However, the present study showed that an increased PEEP had no benefit in terms of the W/D of lungs, LDH concentration in BAL fluid, or cytokine concentrations in BAL fluid. Although high PEEP resulted in better oxygenation, likely due to recruitment of dependent lungs, it also resulted in a translocation of alveolar protein tracer from the infected lungs into the circulation, possibly secondary to a higher peak airway pressure or larger lung volume. Furthermore, high PEEP resulted in overdistension of alveoli in the nonaffected lung, which is consistent with previous studies (23, 28). Rouby et al. (28) warned that lung recruitment and overinflation occurred simultaneously after an increase in intrathoracic pressure and that selection of the optimal PEEP level should focus on limiting lung overinflation. Accordingly, when lung injury is inhomogeneous, as in the present experiment, application of high PEEP may not be justified. However, the long-term effects of PEEP were not assessed in the present study. Further, these data do not exclude a beneficial effect of high PEEP in more homogenous lung disease, i.e., early stage of RDS in the infant or early stage ALI/ARDS in secondary ARDS.

Although particular care was made to avoid bacterial instillation in the right lung, some contamination was unavoidable, and animals were excluded from study if right lung BAL bacterial counts were >3 x 103 CFU/ml. Of the remaining rats, the highest bacterial count in the right lung BAL was 630 CFU/ml (Table 2). Because previous studies demonstrated a threshold of 105 CFU/ml of bacteria in 100 ml of BAL fluid of intubated patients to qualify for a diagnosis of ventilator-associated pneumonia (19), the right lungs in these animals were considered as noninvolved lungs for the purposes of experimentation.


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Table 2. Bacterial counts in the BAL fluid from instilled and noninstilled lungs

 
Plasma and BAL fluid TNF-{alpha} levels were measured by a bioassay, which has several advantages over an immunoassay (5, 21). First, the bioassay corrects for the presence of soluble receptors for TNF, which may buffer TNF-{alpha} activity. Second, the immunoassay cannot distinguish biologically active TNF-{alpha} from biologically inactive precursors of TNF-{alpha} or protein-bound TNF-{alpha}. Although the bioassay may possess lower sensitivity when compared with the immunoassay, this problem can be overcome through the use of cells with increased sensitivity to TNF-{alpha} (8).

The present study possesses some limitations. First, there are likely pathophysiological differences between pneumonia in humans and rodents. Second, pneumonia involves a process of inoculation, incubation, and subsequent appearance of clinical symptoms of infection. In contrast, the experimental animals in the present study received abrupt inoculation of high-dose bacteria in the air spaces. Third, animals were observed for only 6 h following infection. Thus this study cannot be generalized to conditions of ventilation for longer durations. Last, the precise relationship between TNF-{alpha} and lung edema was not explored in the present study. Further experiments are warranted to clarify these issues.

We conclude that ventilatory mode cannot protect the involved lung in the context of severe pseudomonal pneumonia, but low VT ventilation is protective in noninvolved regions. Furthermore, the application of 10 cmH2O of PEEP attenuated the beneficial effects of low VT ventilation over a short-term period.


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 ABSTRACT
 METHODS
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 DISCUSSION
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This study was supported in part by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research nos. 12671495 (to K. Kurahashi), 13670184 (to Y. Nagashima), and 13671565 (to Y. Yamada); by a Yokohama Foundation for Advancement of Medical Science grant (to K. Kurahashi); by a Grant in Support of Promotion of Research at Yokohama City University (to K. Kurahashi); by the Yokohama Academic Foundation (to K. Kurahashi); and by a 2003 Young Scientist Award from the Yokohama City University 21 COE program (to K. Kurahashi).


    ACKNOWLEDGMENTS
 
The authors thank Drs. Thomas R. Martin and Yoshinori Kamiya for invaluable comments regarding this manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. Kurahashi, 3-9, Fukuura, Kanazawa-ku, Yokohama, Japan, 236-0004 (E-mail: kiyok{at}med.yokohama-cu.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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