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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
acute lung injury; ventilator-induced lung injury; lung protective strategy; cytokines; bacterial toxins
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All protocols for animal experiments were approved by the Animal Research Committee of Yokohama City University. Specific pathogen-free male Sprague-Dawley rats, weighing 310400 g (911 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- 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-
activity of each sample by comparing absorbance to that of a standard curve made from dilutions of rat TNF-
(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- (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 30 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-
positivity.
Permeability of lungs to systemic TNF-.
A separate group of rats (n = 12) was used to determine the permeability of lungs to systemic TNF-
. 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-
(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-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
|
|
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.
|
|
|
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- 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).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To investigate mechanisms of lung edema in the noninvolved lung, we assessed TNF- concentration in the BAL fluid. The increase in TNF-
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-
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-
inhibited the cytotoxic activity of the BAL fluid. Therefore, TNF-
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- in the noninstilled lungs from the circulation was negligible in all experimental groups, and immunohistochemical findings confirmed production of TNF-
in alveolar macrophages and in bronchial and alveolar epithelial cells with conventional ventilation. Furthermore, the trend in the positivity score of TNF-
of the cells in the noninstilled lungs correlated well with the concentration of TNF-
in the BAL fluid of noninstilled lungs (Fig. 3D). These observations suggest that pulmonary epithelial cells and alveolar macrophages of noninfected lungs release TNF-
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-
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-
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- at the early stage of lung injury but not in the latter stages. Thus the high concentration of TNF-
, 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-
-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.
|
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- 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.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|