Published ahead of print on October 2, 2003, doi:10.1164/rccm.200304-544OC
© 2004 American Thoracic Society Alveolar Instability Causes Early Ventilator-induced Lung Injury Independent of NeutrophilsDepartment of Surgery, SUNY Upstate Medical University, Syracuse; Department of Biology, SUNY Cortland, Cortland; Department of Oral Biology and Pathology, SUNY Stonybrook, Stonybrook, New York; and Department of Surgery, Mayo Medical School, Rochester, Minnesota Correspondence and requests for reprints should be addressed to Jay M. Steinberg, D.O., Department of Surgery, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, New York, NY 13210. E-mail: steinbja{at}upstate.edu
Intratracheal instillation of Tween causes a heterogeneous surfactant deactivation in the lung, with areas of unstable alveoli directly adjacent to normal stable alveoli. We employed in vivo video microscopy to directly assess alveolar stability in normal and surfactant-deactivated lung and tested our hypothesis that alveolar instability causes a mechanical injury, initiating an inflammatory response that results in a secondary neutrophil-mediated proteolytic injury. Pigs were mechanically ventilated (VT 10 cc/kg, positive end-expiratory pressure [PEEP] 3 cm H20), randomized to into three groups, and followed for 4 hours: Control group (n = 3) surgery only; Tween group (n = 4) subjected to intratracheal Tween (surfactant deactivator causing alveolar instability); and Tween + PEEP group (n = 4) subjected to Tween with increased PEEP (15 cm H20) to stabilize alveoli. The magnitude of alveolar instability was quantified by computer image analysis. Surfactant-deactivated lungs developed significant histopathology only in lung areas with unstable alveoli without an increase in neutrophil-derived proteases. PEEP stabilized alveoli and significantly reduced histologic evidence of lung injury. Thus, in this model, alveolar instability can independently cause ventilator-induced lung injury. To our knowledge, this is the first study to directly confirm that unstable alveoli are subjected to ventilator-induced lung injury whereas stable alveoli are not.
Key Words: ventilator-induced lung injury • cytokine • in vivo microscopy • alveolar mechanics It is well established that mechanical ventilation can exacerbate the pulmonary injury associated with the acute respiratory distress syndrome (ARDS) by direct mechanical damage. More recently, it has been suggested that ventilator-induced inflammatory injury may also play a role. Mechanical ventilation has been shown to cause cytokine upregulation and release into the systemic circulation (1, 2) as well as local release into the alveolus (bronchoalveolar lavage fluid [BAL]) (2, 3). However, a recent study was unable to confirm that injurious ventilation caused cytokine release (4). Furthermore, the possibility exists that the elaboration of cytokines occurs exclusively in the pulmonary parenchyma after ventilator-induced lung injury, without systemic release, with locally expressed cytokines acting in a paracrine fashion to promote tissue injury. Evidence for local upregulation of cytokines was illustrated by Andrejko and Deutschman, who demonstrated an increase in tumor necrosis factor (TNF) in the hepatic parenchyma after cecal ligation and puncture without a concomitant increase in serum TNF (5). They postulated that the paracrine effect of TNF might be the reason for the failures of clinical trials using anti-TNF agents in patients with sepsis. Similar findings have been obtained in other organ systems such as the lung where immunohistochemical analysis has demonstrated upregulation of TNF in inflamed human lung parenchyma (6–8) as well as epithelial cell culture (9). A potential mechanism for the upregulation of cytokines either locally or systemically during mechanical ventilation of the acutely injured lung is alteration in alveolar mechanics (i.e., the dynamic change in alveolar size and shape during ventilation), specifically alveolar instability and recruitment/derecruitment (R/D). With this in mind, we used a unique methodology that allowed us to directly visualize subpleural alveoli. Previous studies demonstrated that normal alveoli are extremely stable with minimal movement during ventilation (10–15). Surfactant deactivation causes a continuum of altered unstable alveolar mechanics, which we defined as repetitive alveolar collapse and expansion (10). In these studies we determined if the alveoli in the area of interest were normal and stable or abnormal and unstable and subsequently analyzed these specific areas for histologic evidence of injury and immunohistochemical evidence of cytokine upregulation. Using this protocol we could correlate alveolar instability with lung injury and cytokine upregulation. We hypothesized that alveolar instability would cause an initial mechanical injury, which would serve as an early "trigger" for an inflammatory response causing neutrophil recruitment specifically into areas with unstable alveoli. Neutrophil-derived proteolytic tissue damage would exacerbate the mechanical injury, resulting in severe pulmonary damage consistent with ventilator-induced lung injury. Furthermore, we postulated that stabilizing alveoli with elevated positive end-expiratory pressure (PEEP) would decrease shear stress–induced lung damage and subsequent release of cytokines. Some of the results of these studies have been previously reported in abstract form.
Surgical Preparation Anesthetized pigs (n = 11) weighing between 15 and 20 kg were placed on mechanical ventilation. Left carotid artery and right internal jugular vein catheters were placed for homodynamic monitoring and blood sampling and the right femoral Swan–Ganz catheter was placed for cardiac output (  ) and filling pressures.
Protocol
Alveolar mechanics.
Necropsy. The lung tissue directly beneath the microscope coverslip was harvested at necropsy, and thus we had a record of the alveolar mechanics of each of the histologic samples. Tissue samples were prepared for hematoxylin and eosin staining, immunohistochemical studies, and lung water determination. In addition, BAL was collected from the left lower lobe for subsequent biochemical analysis.
Histology.
Immunohistochemistry.
BAL fluid protein.
Elastase and collagenase activity.
Serum/BAL fluid TNF and IL-6.
Detailed methodology.
Hemodynamics and Lung Function Tween instillation caused a profound pulmonary dysfunction as evidenced by a significant decrease in arterial PO2 (Figure 2) as well as increase in S/T (Figure 3)
and PCO2 (Table 1)
. Furthermore, a significant increase in mean, peak, and plateau airway pressures with a concomitant decrease in lung compliance was observed (Table 2) . The addition of PEEP in the Tween + PEEP group significantly improved oxygenation (Figure 2), S/T (Figure 3), PCO2 (Table 1), and lung compliance (Table 2). However, PEEP also increased mean, peak, and plateau airway pressures as compared with both Control and Tween groups (Table 2). Both the Tween and Tween + PEEP groups developed slight respiratory acidosis as compared with the Control group (Table 1). Cardiac output () was significantly (p < 0.05) decreased 30 minutes after Tween instillation and was not improved by increased PEEP in the Tween + PEEP group (Control group = 4.9 ± 0.5, Tween group = 2.6 ± 0.2, Tween + PEEP group = 2.4 ± 0.5 L/minute). Systemic mean arterial pressure was significantly (p < 0.05) decreased 240 minutes after Tween instillation in both Tween groups (Control group = 101 ± 15, Tween group = 54 ± 19, Tween + PEEP group = 59 ± 9 mm Hg) as compared with the Control group. Only minor changes occurred among groups in pulmonary artery and wedge pressures and central venous pressure.
In Vivo Alveolar Mechanics In the Control group, alveoli did not change size during tidal ventilation and manifested a low I – E  as displayed in Figure 4
. Tween instillation caused abnormal alveolar mechanics with marked instability (high I – E, Figure 4). With the addition of PEEP in the Tween + PEEP group, alveoli were stabilized, with I – E values similar to Control levels (Figure 4).
Histology Lung tissue from the Control group displayed thin alveolar walls and no intraalveolar edema fluid typical of normal lungs (Figure 5A) . In contrast, lung tissue from the Tween group demonstrated thickened and congested alveolar walls and intraalveolar edema fluid consistent with acute lung injury (Figure 5B). These pathologic changes were ameliorated by the addition of PEEP (Figure 5C), with reduced alveolar wall thickening and intraalveolar edema (Table 3) . There were no significant differences in the number of neutrophils sequestered in the lung among any of the groups (Table 3).
Immunohistochemistry Five high-power photomicrographs were randomly analyzed for color intensity by image analysis. Lung tissue from both the Tween group and the Tween + PEEP group demonstrated no significant increase in TNF expression as compared with the Control group (Control group = 3.8 ± 0.4, Tween group = 3.9 ± 0.5, Tween + PEEP group = 3.9 ± 0.5% of square micrometers occupied by the dye/total square micrometers of the slide). In contrast, IL-6 expression was significantly (p < 0.05) elevated in the Tween group as compared with the Control group, and the addition of PEEP after Tween caused a significant reduction in tissue levels of IL-6 (Control group = 3.7 ± 0.3, Tween group = 8.1 ± 0.3, Tween + PEEP group = 5.8 ± 0.2% of square micrometers occupied by the dye/total square micrometers of the slide).
Serum/BAL TNF and IL-6
Lung Water Lung water was significantly (p < 0.05) increased in the Tween group and Tween + PEEP group as compared with the Control group (Control group = 3.5 ± 1.0, Tween group = 7.9 ± 0.7, Tween + PEEP group = 7.2 ± 0.2 wet/dry ratio).
BAL Fluid Protein
Serum/BAL Fluid Elastase and Collagenase Activity
To our knowledge, this is the first study to quantify in vivo alveolar mechanics for 4 hours and attempt to correlate alveolar instability with pulmonary, hemodynamic, histologic, morphometric, and biochemical markers of pulmonary injury. Our in vivo microscopic technique is unique in that it allowed us to positively identify alveoli with abnormal alveolar mechanics. The most significant finding in our study was that alveolar instability can mechanically injure the lung independent of inflammatory damage. This was determined by directly demonstrating that areas of lung with unstable alveoli developed injury consistent with ventilator-induced lung injury and that this injury was ameliorated by the application PEEP sufficient to stabilize alveoli. Whereas alveolar instability was associated with a modest increase in tissue and BAL IL-6 levels, the absence of a significant increase in neutrophil sequestration or protease activity suggests that the histopathologic changes observed were due largely to the mechanical shear stresses and not neutrophil-released proteases. These data are important because they demonstrate that mechanical trauma itself, which should be easily preventable with lung-protective ventilation strategies, can cause lung injury without involving a subsequent inflammatory response.
As stated previously, there is no consensus as to the effect of injurious mechanical ventilation on the release of inflammatory mediators (1–3, 9, 16–21). The reason for the discrepancy in the cytokine response to injurious mechanical ventilation is unclear. Numerous studies have demonstrated that mechanical ventilation can cause cytokine upregulation in tissue culture (9), in intact animal models (2, 3, 18, 19), and in humans (16). The majority of studies using normal isolated lungs have also found that mechanical ventilation can cause the lung to release inflammatory mediators including cytokines (1, 17, 20, 21). Normal isolated lungs subjected to high VT and low PEEP has been shown to express TNF, IL-1, IL-6, IL-10, IFN- Conversely, the study by Ricard and coworkers (4) involving injurious ventilation of normal rats demonstrated severe lung injury, without corresponding increases in TNF, IL-1, or macrophage inflammatory protein-2 in BALF. These data conflict with those generated by Tremblay and coworkers (17). Likewise, a clinical study demonstrated that high VT (15 cc/kg) and zero end-expiratory pressure did not effect an increase in plasma levels of TNF, IL-1 receptor antagonist, IL-6, or IL-10 in healthy anesthetized patients scheduled for elective surgery (22). However, ventilation with a VT of 11 cc/kg and PEEP of 6.5 cm H2O in patients with ARDS produced increased concentrations of TNF, IL-1, IL-6, and IL-1 receptor agonist in BALF and increases in plasma concentrations of TNF, IL-6, and TNF receptors compared with patients with ARDS ventilated at a VT of 7.6 cc/kg and a PEEP of 14.8 cm H2O (16). The reason for the variability in these results is unknown, but it has been suggested that the variability could be caused by different animal models (i.e., isolated nonperfused lung, isolated perfused lung, intact animals, and numerous animal species used in all models) and/or by the possibility of endotoxin contamination in some isolated lung studies. The data from the present study supports the hypothesis that a significant inflammatory response involving neutrophil sequestration and proteases release does not always occur during injurious mechanical ventilation. Our results demonstrate that the ability of PEEP to rescue the injured lung is similar to that reported in other studies (17–19). We demonstrated that PEEP, improved oxygenation, reduced BAL protein, improved histologic score, and reduced both plasma and tissue IL-6 as compared with the Tween with low PEEP group. In addition, we confirmed by direct visualization that PEEP stabilized alveoli and that alveolar stabilization was associated with decreased lung injury. The only major parameter of lung injury that PEEP did not improve was lung water. However, this could have been an artifact of the model because we instilled Tween dissolved in saline into the lung and the saline would have been measured as additional lung water. Similar to other studies (17–19), we compared the impact of cytokine production in animals with and without PEEP. Our model of lung injury (Tween instillation) did not effect a change in TNF; however, IL-6 was increased in serum, BAL, and tissue. It is well known that serum TNF levels peak early after injury, and thus the possibility exists that we missed the TNF spike by measuring serum levels only at the end of the study. However, this cyclic release of TNF into the serum should not have effected the BAL and tissue TNF concentrations. Others have shown that PEEP reduces BAL TNF from a sixfold to a threefold increase, significantly reduces IL-1 (17), prevents the loss of TNF compartmentalization (18), and significantly reduces messenger RNA levels of IL-1, IL-6, and IL-8 (19) as compared with animals ventilated with low PEEP. Our study was similar in the sense that PEEP significantly lowered both serum and tissue IL-6 levels. Although PEEP did reduce IL-6 in our study, the fact that neither the number of sequestered neutophils nor the elastase and collagenase levels were different between the high and low PEEP groups suggests that the mechanism of PEEP's protective effect was reduction of shear stress injury secondary to stabilizing alveoli, rather than reducing inflammation.
Critique of the Model However, the technique of in vivo microscopy clearly demonstrates that alveoli can become unstable. Our data show that surfactant deactivation can disrupt the structural stability of alveoli (Figure 1). The normally stable alveoli that change volume minimally during ventilation become unstable inflating and deflating with each breath, similar to a balloon. The difference in our data and that of Hubamyr may be due to the pathologic state of the alveolus in the acutely injured lung, i.e., is the alveolus fluid or air filled? Oleic acid may cause much more alveolar flooding (23) than does Tween instillation, which primarily deactivates surfactant (10–15). Thus, alveolar mechanics in patients with ARDS may depend on whether alveoli are completely fluid filled with edema (no alveolar collapse or instability) or whether the alveoli are air filled but with altered surfactant (instability and collapse). We speculate that both of the above conditions occur simultaneously in human ARDS. To our knowledge, in vivo microscopy is the only technique available to directly visualize and measure individual alveoli throughout tidal ventilation in the living animal. However, there are several problems with this technique. The only alveoli that we can film with our technique are subpleural alveoli. Therefore, our analysis is limited to a particular subset of alveoli, and we can only visualize alveoli in two dimensions. This prohibits us from observing and analyzing the dynamic changes of the entire air sac (i.e., alveoli plus the alveolar duct). Indeed, it has been hypothesized that the majority of the change in lung volume is accommodated by changes in the size of the alveolar duct with minimal change in alveolar size (24). Dynamic changes in the size of the alveolar duct and changes in alveolar size in the third dimension very likely play a significant role in both normal lung volume change and in the development of ventilator-induced lung injury. In this study, the alveolar sample size was small and focused on a specific area of lung. This experimental limitation was imposed by the questions we sought to define. In the present study we posed two questions: (1) "In a heterogeneous lung injury does alveolar instability cause tissue trauma specifically in the areas of injured lung whereas areas of lung with normal stable alveoli remain uninjured?" and (2) "Would stabilizing alveoli with PEEP reduce tissue injury specifically in areas of previously unstable alveoli?" To answer these questions we did not need to know the global effects of unstable alveoli on lung pathology but rather the specific effects on the tissue containing unstable alveoli. Thus, we could not increase our alveolar sample size beyond the alveoli that were directly under the microscope coverslip. To stay on the same microscopic field during tidal ventilation, gentle suction must be applied to hold the lung tissue under the coverslip. Thus, for the current study suction was essential. In a previous study, we compared alveolar size at end expiration and peak inspiration as well as stability of normal alveoli during mechanical ventilation with and without suction (14). We demonstrated that suction slightly but significantly increased alveolar size at both inspiration and expiration and stabilized the alveolus. These changes were very subtle with the percent change in alveolar size from expiration to inspiration being 1.1% in the suction group and 8.3% in the nonsuction group (14). This slight change in alveolar size with ventilation in normal alveoli was in sharp contrast to the change in alveolar size with ventilation after Tween, which was as high as 100% (i.e., total collapse at end expiration). Thus, these data demonstrated that suction did not fix alveolar volume, that normal alveolar stability is not an artifact of suction, and that suction does not prevent alveolar instability after surfactant deactivation.
Our study was unique in two aspects. (1) We visually confirmed that alveoli were unstable and performed histologic examination on lung tissue with confirmed alveolar instability. Our results demonstrated that alveolar instability caused tissue injury without increasing neutrophil sequestration or elevating elastase and collagenase activity and (2) PEEP converted abnormal unstable alveoli into normal functioning stable alveoli. PEEP-induced alveolar stabilization reduced lung damage, confirming that shear stress injury secondary to alveolar instability caused ventilator-induced lung injury in this model. This study is an extension of our recently published article, which showed that PEEP is necessary to stabilize newly recruited alveoli (15) and demonstrates that if alveoli are not stabilized lung injury will occur. To our knowledge, this is the first study to directly confirm that alveolar instability can independently injure lung tissue.
The authors thank Andrew Paskanik and Kathy Snyder for their expert technical assistance.
Supported by Central Surgical Association and Hamilton Medical, Inc. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Conflict of Interest Statement: J.M.S. has no declared conflict of interest; H.J.S. has no declared conflict of interest; J.M.H. has no declared conflict of interest; L.A.G. has no declared conflict of interest; H-M.L. has no declared conflict of interest; L.A.P. has no declared conflict of interest; G.F.N. received grant funding from Hamilton Medical Inc. ($24,000) and lecture fees totaling $3,000. Received in original form April 17, 2003; accepted in final form September 22, 2003
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ABSTRACT
METHODS
p Value less than 0.05 versus baseline, *p value less than 0.05 versus Control (squares) and Tween + positive end-expiratory pressure (PEEP) (circles) groups, #p value less than 0.05 versus Control and Tween (triangles) groups.
p value less than 0.05 versus Control group. Tween group represented by triangles.
, macrophage inflammatory protein-2, macrophage chemotatic protein-1, and prostacyclin (
