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Published ahead of print on December 11, 2003, doi:10.1164/rccm.200307-949OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 679-686, (2004)
© 2004 American Thoracic Society

Original Article

Preferential Diaphragmatic Weakness during Sustained Pseudomonas aeruginosa Lung Infection

Maziar Divangahi, Stefan Matecki, Roy W. R. Dudley, Stephanie A. Tuck, Weisheng Bao, Danuta Radzioch, Alain S. Comtois and Basil J. Petrof

Respiratory Division and Meakins-Christie Laboratories, McGill University Health Centre; and Centre for Host Resistance, McGill University Health Centre Research Institute, Montreal, Quebec, Canada

Correspondence and requests for reprints should be addressed to Basil J. Petrof, M.D., Respiratory Division, Room L411, Royal Victoria Hospital, 687 Pine Avenue West, Montreal, PQ, H3A 1A1 Canada. E-mail: basil.petrof{at}mcgill.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Infection with Pseudomonas aeruginosa plays a major role in the pulmonary inflammation and injury associated with cystic fibrosis. Lung inflammation may also lead to more widespread systemic effects on other organs. We tested the following hypotheses: (1) ongoing P. aeruginosa lung infection produces diaphragmatic and limb muscle weakness and (2) such muscle dysfunction is directly correlated with the level of pulmonary inflammation. Chronic bronchopulmonary infection with mucoid P. aeruginosa was induced in C57BL/6 mice. At Day 2 after infection, diaphragmatic force was decreased (37%) only in mice infected with a high dose of 1 x 106 cfu, whereas by Day 7 after infection, diaphragmatic force was similarly reduced (36%) even at a fivefold lower inoculating dose. No significant correlations were found between diaphragmatic weakness and pulmonary inflammation, as assessed by the number of neutrophils, macrophages, and lymphocytes in bronchoalveolar lavage fluid. Moreover, in marked contrast to the diaphragm, no effects of P. aeruginosa infection on contractile function were observed in prototypical slow- and fast-twitch hindlimb muscles. We conclude that sustained lung infection with P. aeruginosa induces preferential weakness of the diaphragm, which is not directly correlated with the degree of pulmonary inflammation induced under these conditions.

Key Words: respiratory muscles • sepsis • cystic fibrosis • lung inflammation • chronic Pseudomonas pneumonia

Cystic fibrosis (CF) is the most frequent autosomal recessive disorder in the white population, affecting approximately 1 in 2,500 live births. Exercise capacity is significantly reduced in patients with CF, and this is associated with a worsened prognosis (1). Interestingly, muscle weakness and a diminished capacity for performing work have been reported in patients with CF having essentially normal spirometry and nutritional status (2). This is also in keeping with the fact that abnormalities of muscle function not readily attributable to muscle atrophy have been observed (3). Therefore, additional factors beyond diminished lung function or malnutrition and muscle atrophy are likely to be involved in producing skeletal muscle weakness in patients with CF.

Patients with CF are particularly prone to chronic or recurrent pulmonary infections with the mucoid strain of Pseudomonas aeruginosa. This pathogen plays a central role in the vicious cycle of lung infection and inflammation, which ultimately culminates in irreparable lung damage, respiratory failure, and death (see Reference 4 for review). Although the role of local pulmonary inflammation in the pathogenesis of CF lung disease is well established, it is unknown whether this also contributes to skeletal muscle dysfunction. However, there is increasing recognition that lung injury and pulmonary inflammation may trigger a systemic inflammatory response (5–7). In addition, several investigators have reported that serum levels of tumor necrosis factor-{alpha}, a known inducer of muscle wasting and weakness (8, 9), are significantly elevated in patients with CF (10–12).

In this study, we hypothesized that pulmonary inflammation triggered by P. aeruginosa infection could be an important cause of diaphragmatic as well as peripheral limb muscle dysfunction, thereby contributing to the global muscle weakness found in patients with CF. To mimic the scenario found in CF, we employed a previously characterized murine model of chronic P. aeruginosa infection (13, 14). In this model, bacteria are encapsulated within agar beads to impede pulmonary clearance of the organisms, which allows for the establishment of an ongoing but clinically tolerable infection (13, 14). In this study, our specific objectives were threefold: (1) to determine the effects of a sustained pulmonary infection with P. aeruginosa on the function of the diaphragm, as well as prototypical slow-twitch (soleus) and fast-twitch (extensor digitorum longus [EDL]) hindlimb muscles; (2) to examine the relationship between alterations in respiratory or limb muscle contractile function and pulmonary mechanics, pulmonary bacterial burden, and the level of lung inflammation induced by P. aeruginosa infection; and (3) to ascertain the extent to which these responses might differ at different stages of the infection process. Some of the results of this study have been reported previously in the form of an abstract (15).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Model of Sustained P. aeruginosa Infection
Studies were performed in 8–10-week-old C57BL/6 male mice weighing 20 to 25 g (Charles River Laboratories, Saint Constant, PQ, Canada), which were used in accordance with the guidelines established by the Canadian Council on Animal Care. Under anesthesia, the trachea was intubated with a sterile cannula to deliver either Pseudomonas-laden or sterile agar bead suspension to mouse lungs. The model of chronic pulmonary infection with P. aeruginosa was performed essentially as described by Starke and coworkers (13), using a mucoid strain of the bacteria originally isolated from a patient with CF (16).

Bronchoalveolar Lavage
The trachea was cannulated with a 22-gauge catheter connected to two separate syringes via a three-way stopcock. One syringe was used to instill 5 ml of cation-free Hank's balanced salt solution (GIBCO, Burlington, ON, Canada) into the lungs, whereas the second syringe allowed the fluid to be collected by gentle aspiration. Differential cell counts were performed on cytospin preparations stained with Diff-Quick (American Scientific Products, McGaw Park, IL). A total of 300 to 400 cells were counted on each cytospin preparation, and the cells were classified as macrophages, lymphocytes, and polymorphonuclear leukocytes using standard morphologic criteria (16).

Myeloperoxidase Assay
Myeloperoxidase activity in lung and muscle was measured as described by Koike and coworkers (17), with minor modifications.

Lung Bacterial Colony Assay
Serial dilutions (1:10) of homogenized lungs were plated on Petri dishes containing trypticase soy agar. The number of P. aeruginosa cfu was counted after overnight incubation at 37°C (16).

Measurements of Respiratory Mechanics
The trachea was cannulated with a snug-fitting metal needle and connected to a computer-controlled small animal ventilator (flexiVent; SCIREQ, Montreal, PQ, Canada) for measurement of respiratory system mechanics as described previously (18). The mice were paralyzed with pancuronium chloride (0.07 mg/kg intraperitoneally) and ventilated in a quasisinusoidal fashion. Respiratory system resistance was derived from the relationship between airway opening pressure and airflow, and quasistatic deflation pressure–volume curves were collected to evaluate potential alterations in compliance.

Contractile Function of Diaphragm and Limb Muscles
Diaphragm, soleus, and EDL muscles were surgically excised for in vitro contractility measurements under isometric conditions, as described previously in detail (19). The excised diaphragm strip and limb muscles were each mounted simultaneously into separate jacketed tissue bath chambers filled with equilibrated Krebs solution. The muscles were supramaximally stimulated using square wave pulses (Model S88; Grass Instruments, West Warwick, RI). The force–frequency relationship was determined by sequentially stimulating the muscles for 1 second at 5, 10, 20, 30, 50, 100, 120, and 150 Hz, with 2 minutes between each stimulation train. Fatigability of the muscles was assessed by measuring the loss of force in response to repeated stimulations over a 3-minute period (30 Hz, 330 millisecond duration).

Statistical Analysis
All data are presented as mean values ± SE. Group mean differences were determined by analysis of variance, with post hoc application of the Tukey test where appropriate. Linear regression was performed using the least-squares method. A statistics software package was used for all analyses (SigmaStat V2.0; Jandel Scientific, San Rafael, CA). Statistical difference was defined as p value less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical Status
Mice were killed at either 2 or 7 days after infection with P. aeruginosa–laden agar beads. Two different doses of inoculating bacteria (2 x 105 and 1 x 106 cfu) were studied at 2 days after infection, whereas only the lower inoculating dose was used for the 7-day time point due to unacceptable signs of ill health at the higher dose in the 7-day group. As has been reported previously (20), body weight was slightly reduced in infected mice at Day 2 (-7 and -11% for 2 x 105 and 1 x 106 cfu, respectively) and to a lesser extent at Day 7 (-6%) compared with animals injected with sterile beads at the same time points.

Lung Bacteriology
Pulmonary bacterial counts at Days 2 and 7 after infection are shown in Figure 1 , together with values obtained from the lungs of control mice. The pulmonary bacterial burden did not differ significantly between the two inoculating doses evaluated at Day 2 after infection, although there was a trend toward increased cfu values with the higher dose (Figure 1A). In addition, there was no significant change in pulmonary bacterial load between Days 2 and 7 after infection at the lower inoculating dose of 2 x 105 cfu, indicating an inability to clear the bacteria-laden beads and persistent ongoing infection (Figure 1B). Control (CTL) mice in which no previous intervention had been made, as well as mice that had been instilled with sterile agar beads (CTL-beads), were culture negative at both time points.



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  		Figure 1. Pulmonary bacterial burden. Mice were killed to quantify numbers of bacteria in the lung at (A) Day 2 and (B) Day 7 after infection. Values are group means ± SE. *p Value less than 0.05 compared with control (CTL) mice in which no previous intervention had been made and mice that had been instilled with sterile agar beads (CTL-beads) groups.

 
Bronchoalveolar Lavage
The total number of inflammatory cells contained within bronchoalveolar lavage (BAL) fluid was markedly increased at Day 2 after infection, particularly in mice inoculated with the higher dose of 1 x 106 cfu (see Figure 2A) . This was due to increases in both polymorphonuclear leukocytes and macrophages. In contrast, by Day 7 after infection, the total number of cells found in infected mice was not significantly greater than those found in the CTL-beads group, although higher numbers of polymorphonuclear leukocytes and lymphocytes were found in infected mice (Figure 2B). There was no significant difference in BAL cell numbers between the CTL and CTL-beads groups at either time point.



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  		Figure 2. Pulmonary inflammatory response to Pseudomonas infection. Inflammatory cells in bronchoalveolar lavage (BAL) fluid at (A) Day 2 and (B) Day 7 after infection. Values are group means ± SE. p Value less than 0.05 compared with CTL and CTL-beads groups: *Polymorphonuclear leukocyte (PMN); {dagger}macrophages; {ddagger}lymphocytes; total inflammatory cells.

 
Respiratory Mechanics
To ascertain whether the instillation of agar beads (either sterile or bacteria laden) into the lungs produced changes in resistance or compliance, respiratory mechanics were measured in a subset of mice from the different experimental groups. As can be seen from Figure 3 , there were no significant differences among the various experimental groups in either the resistance or the pressure–volume relationship.



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  		Figure 3. Respiratory system mechanics. (A) Individual values for resistance of the respiratory system at the indicated time points. (B) Group mean values (± SE) for quasistatic pressure–volume curves are shown. There were no significant differences among groups.

 
Diaphragmatic Contractile Function
Figure 4 illustrates the effects of P. aeruginosa infection on the diaphragmatic force–frequency relationship at both time points. With the inoculating dose of 2 x 105 cfu, the force–frequency relationship of the diaphragm was unaltered at Day 2 after infection. However, the higher dose of 1 x 106 cfu greatly depressed the force-generating capacity of the diaphragm in comparison with the other three groups (CTL, CTL-beads, and 2 x 105 cfu). Moreover, in contrast to its lack of effect at Day 2, the lower inoculating dose of 2 x 105 cfu was associated with a marked decrease in diaphragmatic force production at Day 7 after infection (Figure 4B). On the other hand, the ability of the diaphragm to resist fatigue in vitro was not significantly affected by P. aeruginosa infection under any of the conditions studied (Figure 5) .



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  		Figure 4. Effects of Pseudomonas lung infection on the diaphragmatic force–frequency relationship. (A) At Day 2 after infection, diaphragmatic force was significantly decreased at a dose of 1 x 106 but not with the lower dose of 2 x 105 cfu. (B) At Day 7 after infection, diaphragmatic force was also decreased at the lower infecting dose of 2 x 105 cfu. Values are group means ± SE. *p Value less than 0.05 compared with CTL and CTL-beads groups.

 


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  		Figure 5. Effects of Pseudomonas lung infection on diaphragmatic endurance properties. Values are group means ± SE and are expressed as a percentage of the initial force values obtained at the onset of the fatigue protocol. There were no significant differences among groups at either (A) Day 2 or (B) Day 7 after infection.

 
Relationship between Contractile Dysfunction and Inflammation
To determine whether there was any direct relationship between the level or nature of pulmonary inflammation and the observed impairment in diaphragmatic force production after P. aeruginosa infection, correlation analysis was performed (see Table 1) . At Day 2 after infection, total BAL cell number showed the strongest correlation with diaphragmatic force impairment, although this did not achieve statistical significance (p = 0.11). At Day 7 after infection, the best correlation with diaphragmatic force impairment was obtained for total lymphocytes in BAL, but once again this failed to reach statistical significance (p = 0.21). There was also no significant correlation between diaphragmatic weakness and bacterial burden in the lungs at either Day 2 (p = 0.51) or Day 7 (p = 0.26) after infection.


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  		TABLE 1. Relationship between maximal force production by the diaphragm and bronchoalveolar lavage inflammatory cells

 
We next evaluated whether P. aeruginosa infection of the lungs was associated with inflammatory cell infiltration of the diaphragm. Diaphragm muscle sections stained with hematoxylin and eosin (not shown) did not reveal inflammatory cell infiltration at either Day 2 or Day 7 after infection. In addition, to address this issue in a more quantitative manner, we also performed myeloperoxidase assays (marker of neutrophil content) on the tissues of infected mice. In keeping with the BAL data, Figure 6A shows a large increase in myeloperoxidase activity within the lung tissue of infected mice at Day 2 after infection, with a subsequent decline toward control values by Day 7. In contrast, Figure 6B shows that myeloperoxidase activity within the diaphragm was negligible under control conditions and remained so at both Days 2 and 7 after infection.



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  		Figure 6. Myeloperoxidase (MPO) activity in lung and diaphragm after Pseudomonas infection. (A) In the lung, there was a large increase in MPO activity at Day 2 after infection, which then declined toward CTL values by Day 7. (B) In the diaphragm, there was no significant effect of Pseudomonas lung infection on MPO activity, which remained extremely low (note the difference in y-axis scale as compared with the lung). Values are group means ± SE (n = 6 per group). *p Value less than 0.05 compared with CTL.

 
Hindlimb Muscle Contractile Function
Figure 7 shows the effects of instilling P. aeruginosa–laden beads into the lungs on the force–frequency relationship of the soleus muscle at Days 2 and 7 after infection. In marked contrast to results obtained in the diaphragm, there was no significant effect of either 1 x 106 cfu (Day 2) or 2 x 105 cfu (Day 7) on specific force production by the soleus. Similarly, the endurance properties of the soleus muscle were not significantly altered by P. aeruginosa infection (see online supplement). Because the diaphragm contains a higher proportion of fast-twitch fibers than the soleus, we also determined the response of a fast-twitch limb muscle (the EDL) under the same conditions. Essentially identical results were obtained for the fast-twitch EDL, i.e., no significant effects of P. aeruginosa infection on either the force–frequency relationship or endurance properties of the muscle were found (see online supplement).



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  		Figure 7. Effect of Pseudomonas lung infection on the limb muscle (soleus) force–frequency relationship. Values are group means ± SE. Pseudomonas lung infection had no significant effects on soleus muscle force production at either (A) Day 2 or (B) Day 7 after infection.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that persistent pulmonary infection with P. aeruginosa produces significant contractile dysfunction of the diaphragm. The major findings of our study can be summarized as follows: (1) the diaphragm was preferentially susceptible to the adverse effects of P. aeruginosa infection on skeletal muscle function because hindlimb muscles displayed normal function under the same conditions, (2) the process was exacerbated by a more prolonged duration of infection, and (3) no statistically significant relationships were found between the level of diaphragmatic weakness after infection and either pulmonary bacterial burden or lung inflammatory cell counts within BAL fluid.

Pseudomonas Lung Infection Model
The Pseudomonas lung infection model used in this study offers several advantages. First, it may be more clinically relevant than the high doses of LPS typically used to induce diaphragmatic dysfunction in most studies. Second, it involves inclusion of additional virulence factors within the bacteria other than LPS (e.g., Pseudomonas exoenzyme S, a potent inducer of cytokine expression; see Reference 21), such that the full range of microbe versus host interactions can be expressed. Third, it produces a predominately neutrophilic inflammatory infiltrate within the lungs and an associated tissue damage, which are both similar to changes observed in the infected CF lung (14). Finally, by virtue of its more sustained nature, the chronic lung infection model offers the ability to study responses at different stages of the infection process. On the other hand, the model used in our study also suffers from certain limitations. In particular, it does not precisely mimic the natural history of P. aeruginosa infection in CF from airway colonization to lung injury. In addition, the mice employed in our study lack impaired pulmonary defense mechanisms and other aspects of the multiorgan dysfunction found in patients with CF.

To date, the vast majority of studies aimed at investigating the effects of sepsis on respiratory muscle function have employed LPS to produce a state of acute endotoxemia. Under these conditions, diaphragmatic dysfunction appears not to be caused by LPS itself but rather by its ability to induce the release of endogenous free radical species (22, 23) and other proinflammatory mediators (9, 24). To our knowledge, the only study examining the effects of a chronic infection on diaphragmatic function was performed by Drew and coworkers (25), who found reduced specific force generation by the fast-twitch plantaris muscle, but not the diaphragm or soleus, at 7 to 12 weeks after infecting hamsters with the protozoan parasite Leishmania donovani. In addition, despite the high frequency of pneumonia as a clinical problem, few studies have examined the effects of pulmonary infection on diaphragmatic function. Desmecht and coworkers (26) performed intratracheal instillation of Pasteurella haemolytica in calves and reported that a subset of animals displayed evidence of diaphragmatic dysfunction over a 10-hour period. Boczkowski and coworkers (27) also reported a significant reduction in diaphragmatic force production 3 days after subcutaneous inoculation of rats with Streptococcus pneumoniae, although there was no histologic evidence of pneumonia in their model.

In immunocompetent mice, direct intratracheal inoculation or aerosolization of P. aeruginosa produces only transient infection, with essentially complete bacterial clearance from the lungs within 24 to 48 hours (13, 14). To induce a more sustained infection, we employed a model in which P. aeruginosa bacteria are first embedded in agar before intrapulmonary instillation. The ability of this method to achieve a chronic Pseudomonas lung infection has been validated in several animal species (14). However, because instillation of sterile agar beads alone can cause mild and transient mononuclear cell infiltration in the lungs (14), we also ascertained the effects of this intervention on BAL cell counts and skeletal muscle function. Importantly, no significant effects of sterile agar beads on these parameters were observed. In addition, we ascertained that intrapulmonary instillation of agar beads (either alone or combined with bacteria) had no significant effects on respiratory mechanics, thus confirming a previous report (20). Therefore, we believe that the changes found in our study can be attributed to P. aeruginosa infection rather than to any nonspecific effects related to the experimental procedure.

Role of Pulmonary Inflammation
There is a large body of literature implicating local pulmonary inflammation in the deterioration of lung function observed in patients with CF (see Reference 4 for review). Recently, there has also been increased interest in the idea that pulmonary inflammation and lung injury may trigger more widespread systemic inflammatory effects (5, 7). Increased peripheral blood levels of various markers of inflammation have been documented in patients with CF as well as other forms of chronic obstructive pulmonary disease (6, 10–12, 28). In addition, circulating tumor necrosis factor-{alpha} and C-reactive protein levels in CF are further increased in the setting of symptomatic respiratory exacerbations (11, 12). Such findings have led to the suggestion that systemic manifestations of disease, including muscle wasting and weakness, may be caused by ongoing pulmonary inflammation.

In this study, we sought to determine whether there is a direct relationship between either the number or type of inflammatory cells present within the lung and P. aeruginosa–induced diaphragmatic dysfunction. Previous studies have reported a significant correlation between BAL fluid neutrophils and infection-related weight loss in wild-type mice, as well as in genetically altered CF mice, after intrapulmonary instillation of Pseudomonas-laden agar beads (20, 29). In our study, although there were trends relating total BAL cell count at Day 2 after infection and BAL lymphocyte count at Day 7 after infection with diaphragmatic weakness, none of the relationships examined was statistically significant. In addition, at the lower inoculating dose of 2 x 105 cfu, severe diaphragmatic dysfunction developed between Days 2 and 7 after infection despite a fall in BAL inflammatory cell counts over the same time period.

There are several possible explanations for these findings. For example, a better correlation may have existed between diaphragmatic weakness and the levels of certain cytokines produced by pulmonary inflammation, rather than the numbers of inflammatory cells present within the lungs of infected animals. Although we cannot exclude this possibility, in the same model, van Heeckeren and coworkers (20) reported that the correlations between infection-induced weight loss and either proinflammatory cytokine levels or absolute neutrophil counts within BAL fluid were of similar statistical strength. Another possibility is that inflammatory cells within the lung interstitium were not accurately reflected by the cells retrieved in BAL fluid and that it is the former that are most involved in the systemic inflammatory response induced by P. aeruginosa lung infection. However, BAL fluid cell counts were found previously to be significantly correlated with infection-related weight loss as mentioned earlier (20, 29). In addition, previous studies have generally reported a good relationship between BAL and whole-lung inflammatory cell characteristics in this model (30–32).

We believe that differential regulation of the inflammatory response in the pulmonary and extrapulmonary compartments is the most likely explanation for our findings. In support of this proposition, it has recently been shown that in patients with chronic obstructive pulmonary disease, there is no direct correlation between sputum and serum levels of individual markers of inflammation, despite the fact that both sputum and serum show elevated levels of these markers compared with control subjects (6). This suggests that although there is no doubt cross talk between the two compartments, the extrapulmonary systemic inflammatory response does not simply reflect spillover from the lung but is instead an independently regulated process. Moreover, it is important to note that proinflammatory mediators can be expressed by diaphragm muscle fibers themselves (9, 22, 33) and that the timing of such an expression may differ from that found in neighboring inflammatory cells (22). Therefore, differences between the pulmonary and extrapulmonary compartments in the specific mediators involved and/or the timing of their expression likely accounts for the fact that certain aspects of the systemic response, such as contractile dysfunction of the diaphragm, do not correlate well with local pulmonary inflammation.

Preferential Weakness of the Diaphragm
A particularly interesting finding in our study was the presence of muscle-specific contractile impairment, i.e., in the diaphragm but not in limb muscles (EDL and soleus) of infected animals. The EDL is adapted for relatively infrequent bursts of phasic activity, whereas the soleus is tonically activated to maintain posture. The diaphragm is essentially always active except for very short pauses, even during sleep. However, differences in fiber type composition among these muscles are unlikely to have played a role in our findings because the diaphragm is intermediate in this respect between the fast-twitch, glycolytic EDL and the slower-twitch, more oxidative soleus. In addition, our data do not support inflammatory cell infiltration into the muscle as a cause for the preferential diaphragmatic impairment because neither histologic nor biochemical (myeloperoxidase activity) examination revealed any evidence of increased diaphragmatic inflammation in the infected mice.

Several previous studies (22, 34, 35) have reported a greater susceptibility of the diaphragm to the effects of endotoxemia in comparison with limb muscles. On the other hand, Supinski and coworkers (36) found equivalent reductions in force production by the diaphragm and flexor halluces longus muscle after LPS injection. The precise reasons for these apparent discrepancies are not clear but could relate to variations in the route, timing, and dosage of LPS administration as well as species differences. In a transgenic mouse model of heart failure in which cardiac and serum (but not diaphragmatic) tumor necrosis factor-{alpha} levels are elevated, Li and coworkers (37) reported a major loss of force-generating capacity in the diaphragm, whereas the EDL and soleus muscles were unaffected. However, this same group also found no differences in the intrinsic susceptibility of isolated diaphragm and limb muscle fibers to tetanic force depression by tumor necrosis factor-{alpha} administered ex vivo (38).

We speculate that the greater activity level of the diaphragm in vivo may have contributed to its increased vulnerability to P. aeruginosa infection in our study. Muscle activity can potentially exacerbate diaphragmatic injury and weakness during sepsis through several mechanisms. These include (1) an exaggerated generation of free radical species by contracting muscle fibers (23), (2) imposition of contraction-induced mechanical stress on muscle fiber membranes made hyperfragile by exposure to free radicals (39), and (3) increased exposure of muscle fibers to force-inhibiting cytokines, either through increased endogenous production of such molecules by the muscle fibers themselves (9, 22, 33) or via augmented flow of blood-borne molecules to working muscles (40). Regarding the latter, fever and increased respiratory rates associated with sepsis, although not directly documented in our study, would be expected to further increase blood flow to the diaphragm. In addition, although our data do not indicate an increased susceptibility to in vitro diaphragmatic fatigue after infection, this may not be the case in vivo. This is because the propensity to develop fatigue is inversely related to the maximal force-generating capacity of the muscle, as reflected by an increase in the tension–time index of the diaphragm (41). Therefore, diaphragmatic weakness per se favors the onset of diaphragmatic fatigue under conditions of spontaneous breathing in vivo.

It is also possible that the close proximity between the infected lung and the diaphragm contributed to the preferential impairment of diaphragmatic contractility. The peritoneal and pleural surfaces of the diaphragm are both lined by mesothelial cells, and beneath this layer lies a network of lymphatics (42–44). On the peritoneal side, small openings (stomata) connect the peritoneal cavity with these diaphragmatic lymphatics, and tracer studies have revealed that substances injected intraperitoneally are capable of attaining the lymphatics as well as connective tissue spaces of the diaphragm (42). Similar but less frequent stomata have also been reported on the pleural surface of the diaphragm (43). Accordingly, it is conceivable that proximity and indeed direct communication between the diaphragmatic interstitial compartment and proinflammatory mediators induced within the pleural space by lung infection (45) might be involved in the loss of diaphragmatic force-generating capacity observed in our study.

Conclusions
In summary, we have shown that sustained lung infection with P. aeruginosa results in significant weakness of the diaphragm. Interestingly, even relatively mild respiratory tract infections have been found to cause further decreases in respiratory muscle strength, together with attendant hypercapnia, in patients with underlying respiratory muscle impairment (46). By impairing diaphragmatic function, chronic lung infection may similarly contribute to ventilatory insufficiency in patients with underlying lung disease from various causes, such as CF and chronic obstructive pulmonary disease. To the extent that patients with CF have a greatly reduced ability to clear P. aeruginosa from the lungs, this phenomenon could be particularly exaggerated in patients with CF. Application of the P. aeruginosa infection model in genetically altered CF mice (16, 29) could provide valuable insights into these questions.


   FOOTNOTES
 
Supported by grants from the Canadian Institutes of Health Research, the Canadian Cystic Fibrosis Foundation, and the Fonds de la Recherche en Sante du Quebec. B.J.P. is a Senior Research Scholar of the Fonds de la Recherche en Sante du Quebec. M.D. was supported by a Studentship Award from the Department of Medicine, McGill University Health Centre.

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: M.D. has no declared conflict of interest; S.M. has no declared conflict of interest; R.W.R.D. has no declared conflict of interest; S.A.T. has no declared conflict of interest; W.B. has no declared conflict of interest; D.R. has no declared conflict of interest; A.S.C. has no declared conflict of interest; B.J.P. has no declared conflict of interest.

Received in original form July 11, 2003; accepted in final form December 9, 2003


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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