INTRODUCTION

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In recent years there has been an increased appreciation that some mechanical ventilation strategies can cause or augment lung injury, which is characterized by intense cellular infiltration, pulmonary edema, hyaline membranes, and surfactant dysfunction (1, 2). In addition to this pathological damage, we and others have recently demonstrated that these injurious patterns of mechanical ventilation can also lead to biotrauma (3) with initiation of an inflammatory response, and the release of many cytokines into the lung (4), and that ventilatory strategies that minimize end-inspiratory lung volumes and decrease end-expiratory collapse attenuate this cytokine response (4). Unfortunately, in the clinical setting it is not always possible to adopt a strategy that will minimize ventilator-induced injury/inflammation because of the need to maintain adequate gas exchange.

For the past few years our laboratory has been studying a natural cytoprotective mechanism present in virtually all cellsthe heat shock response or the stress response. The stress response, when triggered prior to or concurrent with an otherwise lethal injury, has been shown to be protective in a number of experimental models. For example, the induction of the stress response by a brief period of hyperthermia (heat stress) or by nonthermal means (sodium arsenite injection) protected rats against the lethal effects of experimental sepsis (7) and intratracheal instillation of phospholipase A₂ (PLA₂) (11). One of the mechanisms of protection may be related to the actions of a set of proteins produced during the stress responsethe heat shock or stress proteins (HSP) (12). The mechanisms by which HSPs may be protective have not been fully elucidated, but we have proposed that one possibility may be the binding of HSPs to cytokines, preventing their release from inflammatory cells (13).

Given the observation that injurious forms of ventilation can induce a pulmonary cytokine response, and given the effect of the stress response on cytokine dynamics in other models, in the present study we set out to examine the hypothesis that the exposure of animals to a brief period of heat stress would attenuate some of the detrimental consequences induced by injurious patterns of mechanical ventilation. To examine this hypothesis, we used an ex vivo rat lung model that allowed us to isolate the effects of ventilation, independent of changes in pulmonary hemodynamics and influx of cytokines from the systemic circulation.

	METHODS

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Animals and Heating Protocol

Seventy-six Sprague-Dawley rats (360 ± 50 g) were anesthetized with an intraperitoneal injection of pentobarbital (37 mg/kg) and were randomly allocated to receive either sham treatment (control group, C) or were exposed to heat stress (study group, H). The heating protocol consisted of placing the animals into a preheated (41° C) neonatal incubator until their rectal temperature reached 41° C for 15 min. This protocol was used based on our previous studies that indicated that it reliably led to the induction of the heat shock response as assessed by the induction of HSP72 (protein of 72 kD molecular weight) and was associated with no mortality (10). After the heating protocol, the rats were returned to their cages at room temperature for recovery. The remainder of the experimental protocol was carried out with the investigator performing the surgery, the physiology, and the biochemistry blinded as to allocation of rats to heated or sham groups.

Experimental Protocol (n = 76)

Eighteen hours after sham treatment or heat stress, the rats were anesthetized with xylazine (2.5 mg/kg) and ketamine (75 mg/kg) intraperitoneally. They were then tracheostomized and the trachea was cannulated with a 14-gauge needle sutured in place with a silk tie. Mechanical ventilation was started (Harvard Rodent Ventilator 683, South Natick, MA) at a respiratory rate (RR) of 40 beats/min, and a tidal volume (VT) of 7 ml/kg.

To harvest the lungs, a midline laparotomy was followed by systemic heparinization (500 IU/kg) through the inferior vena cava (IVC). A median sternotomy was performed, the thymus was resected, and the great vessels were exposed. At this point, the abdominal aorta and inferior vena cava were transected allowing free flow of blood from the lungs while the lungs were still ventilated. The lungs and heart block were removed, with the lungs inflated at the end- inspiratory volume, and placed into a warm (37° C), humidified chamber (Isotemp Waterbath; Fisher Scientific, Chicago, IL). The lungs were slowly inflated twice to total lung capacity (TLC), defined as the lung volume at a transpulmonary pressure (Ptp) of 25 cm H₂O, and were then allowed to deflate passively. Pressures were measured with a Validyne HP 45 transducer (Validyne Engineering Corp., Northridge, CA) and recorded on a Gould ES 1000 recorder (Gould Inc., Cleveland, OH).

To ensure stability of the model, we did not proceed with the protocol unless the following criteria were met: prompt anesthesia (less than 10 min), absence of major bleeding during the tracheal cannulation procedure, correct surgical procedure, and stability of the airway pressure for more than 20 s with the lung inflated to TLC. Lungs from 68 animals fulfilled these criteria; these lungs were then mechanically ventilated with the following high volume-zero (positive end-expiratory pressure (PEEP) strategy that we have previously shown to be injurious, and to produce an increase in lung lavage cytokine levels: tidal volume (VT) = 40 ml/kg, RR = 40 beats/min, PEEP = 0. Peak inspiratory pressure was monitored while the lungs were ventilated for 120 min. Pre- and postventilation measurements of pressure-volume curves were made. Subsequently, lungs were analyzed for the presence of HSP72 from lung homogenates, for surfactant pools sizes and activity, and for cytokine determinations (tumor necrosis factor (TNF)-, interleukin [IL]-1, and macrophage inflammatory protein [MIP]-2) as described below.

Pressure-Volume Curves (n = 28 Lungs)

Pressure volume (P-V) curves (pre- and postventilation) were determined (n = 14/group) as previously described (14) by inflating the lungs with 0.5- to 1-ml aliquots of air to a maximal transpulmonary pressure (Ptp) of 25 cm H₂O. A deflation P-V curve was then obtained in a similar fashion by withdrawing 1-ml aliquots. Because all animals did not reach the same volume at 25 cm H₂O, we summarized the data by averaging the volume data at 5 cm H₂O intervals of pressure (0-25 cm H₂O). The volume data at each of these points for each animal were obtained by linear interpolation.

Detection of HSP72 (n = 20 Left Lungs)

To examine the effect of the heating protocol and the period of ventilation on the induction of HSP72, lungs from a different group of animals were removed and immediately frozen in liquid nitrogen before and after mechanical ventilation (preventilation, n = 5/group [control and heated], left lungs; postventilation, n = 5/group [control and heated], left lungs). HSP72 was detected by Western immunoblotting as previously described (13). Briefly, lungs were thawed, homogenized in cold phosphate-buffered saline (PBS) and centrifuged at 10,000 × g for 15 min. The supernatants were collected and protein concentration was measured with a colorimetric reaction using bicinchonic acid protein assay reagent (Pierce Chemical Co., Rockford, IL). The samples were then suspended in sodium dodecyl sulfate- glycerol sample buffer. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with 50 µg total protein loaded per lane. After gel electrophoresis, proteins were transferred to nitrocellulose membrane and labeled with primary monoclonal antibody, mouse anti-human immunoglobulin G (IgG), specific against the inducible form HSP72 (Stressgen Biotechnologies Corp., Victoria, BC, Canada), 1:1000 concentration. After secondary labeling with goat anti-mouse IgG conjugated with horseradish peroxidase-linked, 1:5000 concentration (Amersham, Richmond, CA), protein was visualized using enhanced chemiluminescence (ECL; Amersham) by autoradiography.

Cytokines (n = 20 Right Lungs)

Before ventilation (n = 5/group; right lungs) and at the end of the ventilation period (n = 5/group; right lungs) the lungs were submitted to three consecutive lung lavages, using normal saline. Each lavage was performed as follows: 3 ml of saline was slowly injected into the lung, then withdrawn slowly a few seconds later. Cell free supernatants were collected by centrifugation at 1,200 rpm, 4° C, for 10 min (GPR; Beckman Instruments, Inc., Palo Alto, CA). Aliquoted samples were stored at 80° C. After all the samples were collected, TNF-, IL-1, and MIP-2 concentrations from the lavage samples were measured in duplicate in a blinded fashion, using specific rat enzyme-linked immunoabsorbent system (ELISA; Genzyme, Boston, MA). Results were analyzed spectrophotometrically (wavelength 450 nm) using a microplate reader (Thermomax; Molecular Devices Corporation, Sunnyvale, CA) and changes in absorbance were converted to picograms per milliliter (pg/ml) based on the standard cure.

Surfactant (n = 20 Lungs)

A separate set of animals was used for surfactant analysis. We used 5 lavages of 12 ml each; 12-ml aliquots were introduced and withdrawn three times. Surfactant was isolated from lung lavage material as previously reported (15) and a different set of lungs was used for pre- (n = 5/group) and postventilation (n = 5/group) determinations. Briefly, an aliquot of the lavage was centrifuged at 150 × g for 10 min to obtain a cellular pellet. The supernatant was used to separate the large surfactant aggregates (LA) from the small surfactant aggregates (SA) by centrifugation at 40,000 × g for 15 min. The large aggregates, obtained from the 40,000 × g pellet, were resuspended in a small volume (approximately 2 ml) of saline and aliquots were frozen until further use. The small aggregates, in the 40,000 × g supernatant, were also aliquoted until further analysis. Total surfactant pool sizes were determined by phospholipid-phosphorus analysis on the various isolated subfractions. Aliquots of the crude lavage material, the cellular pellet from the 150 × g spin, as well as the large aggregates and small aggregates were extracted by the method of Bligh and Dyer (16). The phosphorus in the different aliquots was then determined as described by Duck-Chong (17). Total protein in the lavage material was measured using the method described by Lowry and coworkers using bovine serum albumin as a standard (18). Surface tension reducing activity of the large aggregate fraction was examined on a pulsating bubble surfactometer as described by Enhorning (19). For this procedure, the large aggregates fractions were recentrifuged and resuspended in 150 mm NaCl, 1.5 mm CaCl₂ at a concentration of 2.5 mg phospholipid/ml. Samples were incubated at 37° C for at least 90 min prior to being analyzed. Each large aggregate sample was pulsated for 3 min at 20 pulsations/min and the surface tension at minimum bubble size was expressed after 10 s of adsorption as well as after 50 pulsations.

Statistics

All the values reported in the text and in the figures are expressed as mean ± standard deviation of the mean, unless stated otherwise. The Kolmogorov-Smirnov test was used to assess the normality of the group's distributions. One-way analysis of variance (ANOVA) was performed for multiple group comparisons. Pairwise multiple comparisons were performed using the Student-Newman-Keuls when equal variance was found. A p level of < 0.05 was accepted as significant. SigmaStat for Windows (Jandel Corporation, CA) was used as statistical software.

The protocol was approved by the Animal Care Committee of the University of Toronto.

	RESULTS

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A total of 68 animals out of 76 fulfilled the inclusion criteria described in METHODS. Figure 1 is a representative Western blot using antibodies against HSP72. Whereas in control animals there was no significant accumulation of HSP72 pre- or 2 h postventilation, in lungs from heated animals there was a significant accumulation of this protein indicating that the heating protocol was sufficient to initiate a stress response (Figure 2).

View larger version (17K): [in this window] [in a new window]   Figure 1.   HSP72 detection in the lungs. Lungs were homogenized and subjected to Western blot analysis using antibodies directed to HSP72. Lanes 1 and 2: lungs from animals from the control group pre- and postmechanical ventilation, respectively. Lanes 3 and 4: lungs from heated animals pre- and postventilation, respectively.

View larger version (27K): [in this window] [in a new window]   Figure 2.   Summary of quantitative densitometry of Western blots. Autoradiographs of Western blots measuring HSP72 in the lungs from five animals/group were analyzed by quantitative densitometry of appropriately exposed films. There was a significant increase in HSP72 in the heated animals, but there was no significant change in HSP72 after mechanical ventilation. The intensity is expressed as arbitrary densitometric units, absorbency units (A) by millimeter (mm) wide.

There was no significant difference between the preventilation P-V curves of lungs from control and heated animals (Figure 3). However, comparing preventilation with postventilation lung volumes measured at a Ptp of 25 cm H₂O, there was a greater decrease in chord compliance (47.5%) in the control group compared with the 17.2% decrease in the heated group (Figure 4) (p < 0.001).

View larger version (23K): [in this window] [in a new window]   Figure 3.   Pressure-volume curves of lungs prior to mechanical ventilation (circles) and after mechanical ventilation (squares). The two panels represent mean values of P-V curves from lungs pre- and postventilation (left: control; right: heated). The P-V curves of both groups developed an inflection point and were shifted downward; the changes in chord compliance ([volume at a pressure of 25 cm H2O]/[25 cm H2O]) were less marked in the heated group.

View larger version (38K): [in this window] [in a new window]   Figure 4.   Lung volumes at transpulmonary pressure of 25 cm H2O. After 2 h of mechanical ventilation there was a 47% decrease in lung compliance in control lungs, compared to a 17% decrease in the previously heated animals (p < 0.005).

Before the 2 h of injurious ventilation, both control and heated groups had low lavage concentrations of TNF-, IL-1, and MIP-2. However, after the period of injurious mechanical ventilation lung lavage from control animals demonstrated higher cytokine concentrations in the lung lavage (TNF- 722 ± 306 pg/ml, IL-1 902 ± 322 pg/ml, and MIP-2 363 ± 104 pg/ml), compared with the heat-stressed rats (TNF- 233 ± 119 pg/ml; IL-1 124 ± 53 pg/ml; MIP-2 73 ± 52 pg/ml; p < 0.05) (Figure 5). There was no overlap in the postventilation cytokine concentrations (control versus heat stress) for any of the three different cytokines.

View larger version (24K): [in this window] [in a new window]   Figure 5.   Cytokine data from lung lavage. There were no significant differences in cytokine concentrations before the 2 h of mechanical ventilation, but after ventilation, cytokine levels were markedly decreased in the heated group.

Results of surfactant pool sizes and total protein measurements are shown in Table 1. The total amount of surfactant recovered in the lavage was lower in the two groups that were not mechanically ventilated compared with the two ventilated groups. However, there were no significant differences in total amount of surfactant between lavages obtained from the heated or control animals. Prior to mechanical ventilation, the percentage of large aggregates (% LA) in total alveolar surfactant was 56.2 ± 9.3% in control animals and 52.8 ± 5.2% in heated animals (p > 0.05) with no significant difference between the heated and control group (Table 1). In contrast, after mechanical ventilation there was a significant difference between these two groups, as the control animals had 54.6 ± 3.3% of large aggregates remaining in the lavage as compared with heated lungs, which demonstrated a significant increase in large aggregates percentage (65.2 ± 5.5%; p < 0.05) (Table 1). Measurements of the total amount of protein in the lung lavage revealed a significant increase in the ventilated lungs compared with the nonventilated lungs; however, there were no significant differences between lavages obtained from the heated lungs compared with control lungs (Table 1).

Isolated large aggregates were analyzed for both the ability to form a surface film (adsorption) and surface tension reduction during dynamic compression and expansion. The results, shown in Table 1, revealed that after 10 s adsorption, the large aggregates obtained from nonventilated lungs had reached a significantly lower surface tension than large aggregates isolated from ventilated lungs. There were no significant differences between large aggregates isolated from heated or control lungs. Similarly, large aggregates isolated from nonventilated lungs were able to reduce the surface tension during 2.5 min of pulsation (50 pulsations) to significantly lower values than large aggregates obtained from ventilated lungs (p < 0.05). These differences were observed at all time points during the pulsation period (not shown). Again, there was no significant difference between heated and control samples.

	DISCUSSION

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The major finding of this study is that the stress response was associated with attenuation of the ventilator-induced decrease in compliance, with a reduction in the production of inflammatory cytokines, and with an increase in the percentage of the surfactant large aggregates. These data are the first to demonstrate that the stress response may be protective against a mechanically induced injury, and also suggest that nonventilatory approaches have the potential of decreasing ventilator- induced lung injury.

It has become evident in recent years that certain patterns of mechanical ventilation (excessive end-inspiratory stretch and/or recruitment-derecruitment) can lead to lung injury (1, 2, 14, 20). Using an ex vivo, nonperfused lung, we previously demonstrated that mechanical ventilation can cause a marked decrease in compliance (14) and an increase in lung lavage cytokine levels (4). As in the present study, we used large tidal volumes (40 ml/kg)values that are much greater than those usually observed in the clinical setting. Although these values are quite large, they produce regional stretch that is likely comparable (in a relative sense) to that observed in some patients with the acute respiratory distress syndrome (ARDS) as these patients have marked heterogeneity of their lung disease and may experience high regional lung stretch in nondependent areas of the lung even when relatively small tidal volumes are used (23). We and others have shown that the use of high levels of PEEP may protect lungs from this iatrogenic lung injury (24). However, in a number of patients it may not be possible to use such "protective" ventilatory strategies because of the difficulty of maintaining adequate gas exchange. Thus, the purpose of the present study was to assess the feasibility of using a nonventilatory strategy to mitigate the injury that can be induced by mechanical ventilation.

As our nonventilatory strategy, we used the stress response, a natural defense mechanism that is present in all nucleated cells, from plants to animals, and triggered by a number of different stimuli (8, 12). In laboratory settings, the most common strategy used to initiate the stress response is the exposure of animals or cells to a brief and controlled period of heat. The cells exposed to this protocol temporarily suspend the production of most polypeptides and produce a set of intracellular proteins called the stress proteins or heat shock proteins (HSP) (27). In previous studies, we demonstrated that HSP72, the most inducible of the stress proteins and used in most studies as a marker to determine the up-regulation of HSPs, may be present in the lungs as soon as 2 h after exposure to heat, peaks between 12 and 24 h, and remains elevated for up to about 72 h (11). Also, we have shown that rats exposed to heat stress prior to (13) or immediately after (7) lipopolysaccharide (LPS) stimulation had lower plasma levels of TNF- and IL-1 when compared with controls not submitted to the stress response. In the present study, we confirm further that HSP72 is present in the lungs 18 h after exposure to heat stress and that this protein is markedly reduced in the lungs of control animals (Figures 1 and 2) even after 2 h of mechanical ventilation. These findings do not exclude the possibility that a mechanical stimulus can induce the stress response; it is possible that the stimulus used in this study was not sufficiently intense or sufficiently prolonged to induce this response.

In the current study we observed the marked decrease in compliance that we had previously demonstrated using a high volume/low PEEP strategy, but we also found that this decrease in compliance was significantly attenuated in animals that had been exposed to heat stress 18 h before initiation of the injurious ventilatory strategy (Figures 3 and 4). There are a number of possible explanations for the stress-induced improvement in compliance in response to an injurious ventilatory strategy. Because the primary role of surfactant is to confer mechanical stability to the lung by reducing the surface tension at the air-liquid interface, we postulated that there may have been some effect of the stress response on the surfactant system to explain the improvement in compliance. The biophysical function of surfactant is accomplished by the large surfactant aggregates (LA), one of its two main subfractions (28). The other subfraction, composed of small aggregates (SA), is not surface active (28). Surfactant dysfunction, in particular a relative decrease in the proportion of the surface active large aggregates within the airspace, has been shown to contribute to lung injury (29). We investigated the effect of heat shock on the surfactant pool sizes and function of surfactant isolated from ventilated and nonventilated rats. Prior to the injurious ventilation strategy there were no significant differences between heated and control animals in any of the surfactant parameters measured (Table 1). The surfactant obtained from these lungs contained approximately 50% large aggregates, which were capable of reducing the surface tension to below 10 mN/m. After mechanical ventilation there was a significant increase in the total amount of surfactant in both the heated and control lungs as compared with the nonventilated groups. However, the lungs from heated animals had significantly more of their surfactant in the surface-active, large aggregate form compared with the lungs from the controls. The increase in total surfactant was likely due to increased secretion of large aggregates due to the stretch associated with ventilation. We speculate that a slower conversion of these secreted large aggregates into the small aggregates in the heated animals may have resulted in the increased percentage of LA observed in these lungs. The surface activity measurements revealed no significant differences between samples obtained from heated or control lungs before mechanical ventilation, but after ventilation the surface tension-reducing activity of the large aggregates was significantly impaired. This inhibition in surface activity after mechanical ventilation was likely due to protein inhibition (30). Because this inhibitory effect is concentration dependent, we suggest that the reduced activity of the surfactant large aggregates may have contributed to the decrease in compliance in both groups and that the higher concentration of large aggregates present within the airspace of heated lungs after ventilation contributed to the improved compliance of these lungs compared with the nonheated control lungs.

Another possible explanation for the protective effect of the stress response relates to our finding that lung lavage from animals exposed to heat stress contained significantly lower concentrations of TNF-, IL-1, and MIP-2 2 h after mechanical ventilation compared with control lungs (Figure 4). These results are in accord with our previous in vivo studies (13) in which we found that exposing rats to heat stress prior to LPS stimulation produced lower levels of inflammatory cytokines. Similarly, heat stress of alveolar macrophages reduced the concentration of TNF- in response to LPS. HSP72 is a molecular chaperone that binds to proteins and is thought to be important for the proper three-dimensional configuration of proteins. In the previous study, we found that HSP72 formed an intracellular complex with TNF-, thus preventing release of TNF- from the cell. The precise mechanisms by which the stress response might provide cytoprotection are not known. In the studies cited above and in the current study, the injury may have been inflammatory in nature as we have detected increased levels of cytokines in lung lavage after injurious ventilation. Experimental data provide evidence that excessive levels of cytokines can be deleterious to organ function, although proinflammatory cytokines play protective roles in several models of infection. Since cytokines are important mediators of inflammation, the decreased levels of cytokines after ventilation in heated lungs could explain our findings. Also, recent in vitro and in vivo studies have shown that TNF- can reduce levels of surfactant protein (SP)-B and -C mRNA (31, 32), and SP-A and SP-B protein (33), as well as inhibiting the synthesis of the main surfactant phospholipid phosphatidylcholine (34, 35). Conversely, in vitro studies have shown that components of the surfactant system were able to decrease the secretion of inflammatory cytokines by alveolar monocytes (36). Further studies are required to determine if the stress response affected both the surfactant system and the cytokine network independently or whether the two observations are interrelated.

The results presented in this study, in combination with other studies demonstrating that the stress response can markedly decrease mortality rates and attenuate plasma cytokines in experimental models of acute lung injury and sepsis (13, 37, 38), may also have therapeutic implications. It has been suggested that the injurious ventilatory strategies may also lead to the development of multisystem organ failure by causing release of cytokines into the systemic circulation (3, 4, 39, 40). Current approaches to minimizing ventilator-induced lung injury are based on the concept that appropriate changes in the ventilatory pattern will attenuate the lung damage. Our findings may lead to a paradigm shift in which novel therapy for ventilator-induced injury is aimed not only at decreasing pressures or volumes in the lung, but also directed at development of interventions that are aimed directly at preventing the initiation of the inflammatory response.