INTRODUCTION

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Chronic hypercapnia in ventilatory failure may be at least partly attributable to respiratory muscle dysfunction caused by fatigue (1), weakness (2, 3), or injury of the respiratory muscles (4, 5). We have found that significant diaphragm injury and hypercapnic ventilatory failure are induced by resistive loading over a 6-d period in the hamster (4). In addition to showing muscle injury and inflammation, the diaphragm of tracheally banded (TB) hamsters is more susceptible to calpain degradation (4). Calpain, a thiol protease found in the cytosol, can degrade structural proteins such as desmin and actin in skeletal muscle. Increased activity of this enzyme and alteration of its potential substrates may be responsible for the early stages of degradation of muscle after increased contractile activity. These early degradative changes, contribute to ultrastructural changes, such as disruption of the myofibrillar apparatus, including z-band streaming, and increased permeability of the sarcolemma (6).

Examination of the time course of arterial blood gas (ABG) deterioration, increased calpain activity, and diaphragm injury during chronic resistive loading will identify the critical time points of maximal diaphragm muscle deterioration and provide some guidance about the optimal time point at which to investigate the impact of interventions to prevent diaphragm injury and ventilatory failure. The purpose of this study was to examine the time course of ABG deterioration, increased calpain activity (examined biochemically), and diaphragm injury and inflammation (examined histologically) during 4 d of increased resistive loading accomplished by tracheal banding.

	METHODS

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Because details of the methodology for cannulation and tracheal banding have been described elsewhere (7), these methods will be outlined only briefly here.

Animals and Groups

Sixty-three adult (12-16-wk-old) male Sprague-Dawley rats were obtained from Charles River Laboratories (La Prairies, PQ, Canada). Five groups of animals were studied and completed the protocol: 13 control (C) animals, 13 animals that were TB for 1 d (TB1), 13 animals that were TB for 2 d (TB2), 13 animals that were TB for 3 d (TB3), and 11 animals that were TB for 4 d (TB4). Nine percent of the rats died before completion of the protocol, probably from respiratory failure.

Experimental Protocol

The experimental protocol used in the study received ethical approval from the University of British Columbia Animal Care Committee. Rats were anesthetized by intramuscular injection of ketamine (8 mg per 100 g body weight [B.W.]) and diazepam (0.75 mg per 100 g B.W.), and also received a subcutaneous injection of glycopyrrolate (0.075 mg per 100 g B.W.) in order to minimize tracheal secretions. Through a midline incision over the trachea, the carotid artery was cannulated with polyvinyl tubing (ID = 0.23 in.; Bolab Products, Lake Havascu City, AZ), using the technique outlined previously (7). The trachea was then isolated and a water-filled polyethylene catheter (PE 50, with side holes cut on the lower 1 cm) was placed into the lower one-third of the esophagus in order to measure esophageal pressure (Pes) as an estimate of pleural pressure. The length of the catheter inserted was measured and marked for each rat. The other end of the catheter was connected to a ± 400 cm H₂O differential pressure transducer (Model 267 BC; Hewlett-Packard, Waltham, MA). This transducer was checked for sensitivity and calibrated in 5-cm H₂O increments over a 40-cm H₂O range before each experimental use. Pes during tidal breathing (Pes_VT), maximal esophageal pressure during tracheal occlusion (Pes_max), inspiratory time (TI), and total time for the repiratory cycle (Ttot) were measured, and the pressure- time index (PTI) was determined from the product of (Pes_VT/Pes_max) × (TI/Ttot). The C rats were allowed to recover from anesthesia at this point. All rats in the TB groups had a polyethylene cuff (I.D. = 1.8 mm; length = 2 mm) placed around the fifth and sixth cartilaginous rings of the trachea, and this cuff was surrounded by two silk threads that were tightened until Pes_VT was approximately 10 cm H₂O. All TB rats were allowed to recover from anesthesia.

ABG samples were taken daily from the awake rats, and ABG values obtained on the day that rats were euthanized (rather than the average of ABG values obtained daily) are shown in RESULTS. Rats were anesthetized with an anesthetic regimen similar to that described for the first procedure, at 1 d, 2 d, 3 d, or 4 d after the tracheal banding procedure for the TB1, TB2, TB3, and TB4 groups, respectively. C rats were anesthetized either 2 d or 4 d after carotid artery cannulation and the tracheal isolation procedure. Because they did not differ from one another, the C animals' data were combined for analysis. After anesthesia, Pes measurements were repeated after the esophageal catheter was inserted to the length measured on Day 0. Next, through a midline abdominal incision, the abdominal organs were reflected back, the animal was euthanized, and the trachea and diaphragm were removed. The banded region of the trachea (or a similar region in the C rats) was fixed for morphometric analysis so that the internal cross-sectional area (CSA) could be determined as another index of resistive load (7). The right hemidiaphragm was trimmed of fat and connective tissue, weighed, and quick frozen for biochemical analysis. Two 4-mm × 5-mm segments of muscle from the left lateral costal hemidiaphragm were quick frozen in isopentane cooled with liquid nitrogen for subsequent histochemical analysis. Frozen tissue was stored at 70° C until processing.

ABG Sampling

Blood samples of 0.2 ml were taken from the awake rat for ABG analysis, and the blood volume taken was replaced with saline. The volume of the cannula was replaced with concentrated heparin (1,000 U/ml). The samples were immediately analyzed with a Model 168 pH/blood gas analyzer (Corning, Corning, NY).

Muscle Histology and Point Counting of Abnormal Muscle and Connective Tissue

Cross-sections of costal biopsies were cut at a thickness of 10 µm on a cryostat-microtome (Reichart-Jung; Cambridge Instruments, Buffalo, NY) and processed for hematoxylin and eosin (H&E) staining to point count area fractions (A_A) of: (1) normal muscle; and (2) abnormal and inflamed muscle, and connective tissue (4). A_A were determined with a light microscope equipped with a camera lucida (Labophot; Nikon, Tokyo, Japan) and a computer program for point counting developed at the McDonald Research Laboratory of the University of British Columbia. The image of a 50-point grid from the computer monitor was projected via the camera lucida onto the image of the diaphragm cross-section viewed at a magnification of ×400. Points projected on the cross-section were assigned to Category 1: normal muscle; Category 2: abnormal and inflamed muscle including necrotic muscle, viable muscle with abnormal morphology, necrotic muscle with inflammatory cells, inflammatory cells (where no outline of a muscle cell was evident), and effusion; Category 3: connective tissue/fat or collagen; and Category 4: no count (if the point fell onto clear space, nerve, or vessels). The number of points in each of the first three categories was divided by the total number of points in these three categories to determine A_A.

Assay of Calpainlike Activity

Extraction and determination of calcium-dependent calpastatin-inhibited proteolytic activity (calpainlike activity) was done on diaphragm samples (8). Tissue samples (100 mg) were suspended with an Ultra-Turrax homogenizer (Model TR-10; IKA Laboratories, Wilmington, NC) for 20 s at a setting of 65 in 10 to 15 volumes of buffer containing 80 mM KCl, 20 mM Tris (pH 7.5), 5 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 2 mM dithiothreitol (DTT). The suspension was centrifuged (4° C) at 22,000 × g for 15 min (Model Z 233-M; Hermle) and the supernatant was decanted (soluble fraction) and stored in polypropylene tubes on ice (20 min) for subsequent assay of calpainlike activity. Following centrifugation, the particulate/pelleted material was homogenized in 10 to 15 volumes of the buffer described earlier, with the addition of 0.35% Triton-X 100, with 10 strokes of a Wheaton 2-ml glass homogenizer, and the homogenate was centrifuged. This procedure was judged to be adequate for removing the 80-kD protein band from the first pellet because the second pellet (following glass homogenization with Triton-X 100) contained negligible (if any) amounts of this protein as assessed with polyacrylamide gel electrophoresis. Moreover, Tan and colleagues (9) have reported that calpain activity is not altered as a result of Triton-X 100 (detergent) treatment. The second supernatant from the second centrifugation step (particulate fraction) was stored on ice and assayed for calpainlike activity.

The calcium-dependent proteolytic activity of the supernatant (soluble) and pelleted (particulate or bound) fractions was determined by microplate assay with casein as a substrate (10). Briefly, 200 ml of soluble and/or bound fractions were added to reaction mixtures containing 2 mg/ml casein, 50 mM Tris (pH 7.5), and 20 mM DTT (in duplicate). After a 5-min preincubation at 30° C, 5 mM total calcium (800 mmol/L free calcium as determined with the IONS software program [11]) was added to one of the duplicates, whereas the other contained 5 mM EGTA (as a chelating agent). After 30 min at 30° C, an aliquot (100 ml) of each sample was assayed for proteolysis in a total volume of 325 ml, using Bio-Rad G250 dye reagent concentrate (Bio-Rad Laboratories, Mississauga, ON, Canada). The protein dye reagent consists of 0.05% (wt/vol) Coomassie brilliant blue G-250, 23.5% (wt/vol) ethanol, and 42.5% (wt/vol) phosphoric acid. The enzyme activities (determined in the absence and presence of calcium) are expressed as caseinolytic activity and were calculated by taking a 0.1 change in absorbance at an optical density of 595 nm as equivalent to 1 unit of enzyme activity. The calcium-dependent caseinolytic activities of the soluble and bound fractions are expressed as calpainlike activites, because minimal activity (< 0.05%) was observed when calpastatin (a calpain inhibitor) was added to the assay mixture.

Statistical Analysis

Multivariate analyses of variance (ANOVAs) (Systat, Inc., Evanston, IL), with examination for differences between the C and TB groups of rats, were done on the following combinations of related parameters: (1) indices of respiratory muscle load (Pes_VT, Pes_VT/Pes_max, PTI, and tracheal CSA; (2) arterial blood gas measures (Pa_O2, Pa_CO2, pH, and HCO₃); (3) A_A of normal and abnormal diaphragm and connective tissue. If the multivariate analysis indicated a significant difference between C and TB rats in a group of related parameters, t tests were performed on individual parameters to seek specific differences between C and TB rats. ANOVAs were performed on calpainlike activities, TI, and Ttot to examine for differences among the animal groups. Post hoc comparisons were made with Tukey's HSD test. Differences were determined to be significant at p < 0.05. Data are expressed as mean ± SEM.

	RESULTS

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TB rats had an increased resistive loading, as shown by a 67% reduction in their internal tracheal CSA relative to C values (p < 0.001) (Table 1). We used Pes_VT and the tension-time index (TTI) as estimates of loading during the banding procedures. On Day 0, all four banded groups (TB1, TB2, TB3, and TB4) had higher Pes_VT, Pes_VT/Pes_max, and PTI values than did the C group (p < 0.05); however, on the last day of the protocol, only the TB1 group had higher Pes_VT and PTI values than those of the C group (Table 1). Pes_VT and PTI did not differ among the four banded groups on Day 0 or on the last day of the experiments. A prolonged TI was found in the TB1 and TB2 groups (0.512 ± 0.016 s, p < 0.025; and 0.510 ± 0.014 s, p < 0.03, respectively) and tended to be longer in the TB3 and TB4 groups (0.496 ± 0.014 s and 0.496 ± 0.030 s, respectively) on the last day of the protocol, as compared with the C group values (0.422 ± 0.026 s).

Increased resistive loading resulted in a lower Pa_O2 (p < 0.01) and a higher Pa_CO2 (p < 0.01) in the TB1, TB2, and TB3 rats than in the C animals (Figure 1). TB1, TB2, and TB3 rats had a lower arterial pH than did C rats (Figure 1). TB2 rats had a higher HCO₃ than did C rats (p < 0.05).

View larger version (47K): [in this window] [in a new window]   Figure 1.   ABG values for TB and C rats. Mean ± SEM. *Significantly different from C at p < 0.05, **significantly different from C at p < 0.01, and ***significantly different from C at p < 0.005.

Total calpainlike activity was increased over control levels by 13%, 24%, 25%, and 21% (p < 0.05) in the TB1, TB2, TB3 and TB4 groups, respectively. These increases in total calpainlike activity were accompanied by shifts in the subcellular localization (soluble and/or bound fractions) of enzyme activity. Increases in calpainlike activity were first observed in the soluble (or cytoplasmic) fraction (TB1) (p < 0.05) (Figure 2), and this held true for all other TB groups (p < 0.05) (Figure 2). Despite the enzyme activity increases noted for the soluble fractions in response to resistive loading, these fractions' calpainlike activities were never higher than those observed for the bound fractions (Figure 2). Moreover, there were differences noted for the subcellular distribution pattern of calpainlike activities, with the relative increases in the bound fractions being greater only in the TB2 and TB3 groups, by 27% and 16%, respectively, as compared with the C group (p < 0.05) (Figure 2). This was in contrast to the increased activities in the soluble fractions of all TB groups as compared with the C group.

View larger version (19K): [in this window] [in a new window]   Figure 2.   Calpain activity in diaphragm of TB and C rats. (Upper panel ) calpain activity in absolute terms. (Lower panel ) calpain activity as a percentage of C values. Closed bars show soluble and open bars show bound fractions. Mean ± SEM. *Significantly different from C at p < 0.05.

The A_A of normal diaphragm was lower, and the A_A of abnormal diaphragm and connective tissue was higher in the TB3 than in the C rats (p < 0.05) (Figure 3). Observation of the diaphragm cross-sections at higher power showed more mononucleated cells than polymorphonucleated cells in injured diaphragm. On the basis of morphology, it was difficult to clearly define the origin of mononucleated cells as lymphocytic, monocytic, or muscle progenitor cells.

View larger version (19K): [in this window] [in a new window]   Figure 3.   Diaphragm muscle injury in C and TB rats. Left panel shows the AA of normal muscle and right panel shows the AA of abnormal muscle and connective tissue. Mean ± SEM. *Significantly different from C at p < 0.05.

	DISCUSSION

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We found that increased resistive loading induced by tracheal banding caused a respiratory acidosis and a lower Pa_O2 during the first 3 d of tracheal banding. The activity of calpain, a nonlysosomal, Ca²⁺-specific protease, was greater in the diaphragm of rats during all 4 d after tracheal banding than in C rats, with the highest levels being shown 2 to 3 d after banding. The degree of diaphragm injury and inflammation peaked at 3 d after banding. Light microscopy showed changes similar to those previously reported in the hamster diaphragm after tracheal banding (4), including cell necrosis, cytoplasmic fragmentation, and an increased nuclearity within and between degenerating fibers. The present study is the first to show the time course of ABG deterioration, increased calpain activity, and diaphragm injury in a chronic model of low-intensity diaphragm loading.

Calpain-like activity was increased in the diaphragm of TB rats during all 4 d after tracheal banding, although the pattern of increase in the soluble and bound fractions varied over the 4 d. Our findings in the overloaded diaphragm are similar to the increased calpain activity shown by Belcastro (12) in the limb muscles of rats after an hour of level running. Similarly, Jiang and colleagues (13) showed that total calpainlike activity was increased in the rabbit diaphragm at 3 d after a 1.5-h bout of inspiratory resistive loading at a moderate level (PTI of 0.17 versus 0.23 in the high-loading group).

The primary mechanism underlying the increase in calpainlike activity induced by resistive loading could be the redistribution of a soluble (free) pool of the enzyme and/or activation of a previously bound inactive pool. It is apparent that both of these mechanisms can occur in the diaphragm. An early (TB1 group) increase in calpainlike activity in the soluble fraction of diaphragm tissue suggests a shift-induced redistribution of this activity, whereas the data for the bound fractions in the TB2 and TB3 groups suggest activation of a previously inactive bound pool of calpain. This suggestion is partly supported in the literature, where activation of calpain and its translocation between cytosolic and bound fractions (14), concurrently with increased intracellular calcium levels, has been reported in cultured cells (15), red blood cells (16), and neurons (17). These in vitro changes in localization of calpain have been associated with altered calcium levels; similarly, increased contractile activity during increased loading is accompanied by altered calcium transport functions of the sarcoplasmic reticulum (18, 19). Increased calcium levels may also contribute to the activation of calpain during continuous resistive loading induced by tracheal banding.

We did not expect parallel changes in calpain activity and diaphragm injury that we observed under the light microscope. Although the physiologic regulation of calpain activity within the cell is not fully understood, a number of factors contribute to its activity. We postulate that the redistribution of calpainlike activity plays a critical role in initiating protein degradation in the diaphragm. Moreover, the degradative action of calpain depends not only on its redistribution; its activity is further modulated by changes in: (1) intracellular calcium concentrations; (2) the state of autolysis of calpain; and (3) the localization and amounts of both calpain and calpastatin, a specific inhibitor of calpain (20, 21). The regulation of these factors is likely to depend on their spatial distribution within the cell. Since calcium levels and the activity of calpastatin, the endogenous inhibitor of calpain, were not measured in our study, it is difficult to speculate on how the increased levels of calpain resulted in an increased degradative activity in vivo. Furthermore, diaphragm injury observed under the light microscope is the result of a series of events that include not only the activity of endogenous proteases, but also the inflammatory response (see the following discussion). Thus, we expected increases in calpain activity to precede the injury observed under the light microscope, but did not expect them to occur in parallel with or to be correlated with this injury.

The 3-d peak of diaphragm injury observed light microscopically after tracheal banding is the manifestation of a cascade of events that occur during exertion-induced loading. Skeletal muscle injury results in a complex interplay of events. Initially, muscle tension may actively disrupt muscle fiber membranes (22, 23) and the cytoskeleton, including the z-line structure (5, 23). Subsequent damage can result from an increase in intracellular calcium concentration, which can activate proteases such as calpain (6, 23). Both myofilament regulatory and structural proteins are vulnerable to cleavage and loss as a result of calpain degradation. Some aspects of the inflammatory process are triggered early in exertion, such as an increase in the number of circulating white blood cells and upregulation of neutrophils. Neutrophils may be observed during the first 24 h after a bout of muscle overload (5); however, significant injury of muscle observed under the light microscope may take two to three more days (5, 24). The observed changes include fiber necrosis, infiltration of mononuclear cells, and regenerative changes (5, 23). The tendency for diaphragm injury to be less severe at 4 d after loading may reflect a decrease in injury, or an increase in regeneration, in addition to a closing up of areas in which necrotic tissue has been phagocytosed.

The 3-d peak in diaphragm injury may not only reflect a later phase in cell injury and inflammation, but may also reflect a greater pool of fatigued and injured fibers. Fatigue induced by low-intensity, repetitive contractions in the biceps (27) and dorsal interosseus muscles (28) in humans resulted in more variable recruitment (increases in firing rates and firing variability) (27, 28), followed by recruitment of additional (larger) motor units (28). As in the case of limb muscles, low-intensity tracheal banding may initially recruit a relatively small pool of fibers, with more diaphragm fibers being recruited as these initially recruited fibers become fatigued and/ or injured, with the additional fibers in turn becoming fatigued and injured. Others have shown decreased diaphragm contractile force immediately after inspiratory resistive loading (29), 3 d after inspiratory resistive loading (29), and 30 d after tracheal banding (30). Further study is needed to determine whether the number of fibers recruited and injured progressively increases during the early phase (3 d) of tracheal banding.

As with the time course of injury shown in the diaphragm, several previous studies of limb muscle injury have shown that the greatest amount of skeletal muscle injury is present 3 d after an exercise stimulus. Fridén and colleagues (24) found the greatest amount of focal disturbance of the striated band pattern in fibers at 3 d after exercise, as compared with 1 h or 6 d after intense eccentric exercise in humans. McCully and Faulkner (25) reported that lengthening contractions of mouse limb muscles resulted in infiltration of macrophages and muscle fiber degeneration peaking at 3 d, whereas muscle regeneration was evident at 4 d. Salminen (26), examining the effects on mouse limb muscles of 8 to 9 h of running, which involves a combination of concentric and eccentric contractions, found a similar pattern. Inflammation and necrotic fibers were most abundant in several limb muscles at 3 d after exertion, whereas the amount of inflammatory cells was strikingly decreased at 5 d after exertion. Thus, it would appear that diaphragm injury induced by tracheal banding showed a similar time course to that of limb muscle injury induced by eccentric or prolonged exercise.

The time course of diaphragm injury induced by tracheal banding and observed at the light-microscopic level may be similar to that observed in limb muscles; however, the pattern of loading in the two types of muscle is very different. In limb muscle loading studies (24), all loads, regardless of their duration (maximum = 9 h), were followed by periods of rest or decreased activity, whereas during tracheal banding of the rats in our study, the load was imposed during every breath for the duration of the experiment (1 to 4 d). It may seem surprising that the time course of injury was similar in the diaphragm and limb muscles, but many factors may contribute to the similar changes observed with light microscopy. First, the pattern of loading on the diaphragm may be altered during tracheal banding; after some fibers become fatigued and/or injured, other fibers in the diaphragm and other inspiratory muscles may be recruited to offload the injured diaphragm fibers. Because we did not evaluate variation of recruitment in different regions of the diaphragm or in different inspiratory muscles, this hypothesis requires further study. Second, the light microscopic changes in our study showed a peak of injury at 3 d, followed by a decrease that may have reflected regeneration and a closing up of areas in which necrotic tissue had been phagocytosed. Hamsters banded for 30 d showed hypercapnic ventilatory failure and abnormal diaphragm morphology (30). However, less fiber necrosis but more variation in fiber size and shape, as well as a shift in fiber type proportions, was observed in the diaphragm of hamsters banded for 30 d than we found in the diaphragm after short-term banding in rats. Thus, the trend toward a lesser amount of injured fibers observed at the light-microscopic level in the 4-d-banded rats in our study may reflect a shift in recruitment of other inspiratory muscles, a decrease in fiber necrosis, and a shift toward adaptive changes. Several possible changes at the molecular and cellular level require further examination to fully explain the pattern of injury observed at the light microscopic level in our study.

The loss of force that accompanies diaphragm injury may be very significant. During the 3 d after tracheal banding, diaphragm fatigue followed by progressive injury may contribute to loss of force in the diaphragm, which may in turn contribute to ventilatory failure. A bimodal decrease in force has been shown in the human quadriceps (31) and in the murine extensor digitorum longus and tibialis anterior after eccentric contractions (32); the secondary decrease has been observed at between 1 and 3 d for the former muscle and between 3 h and 1 d for the latter two muscles. Both findings (31, 32) support the hypothesis that the initial decrease in force is due to a combination of fatigue and mechanical disruption of the myofibrillar apparatus, whereas the secondary injury may be related to the inflammatory response affecting muscle function. This secondary injury associated with a loss of force has recently been shown in the diaphragm by Jiang and colleagues (29); they reported that 1.5 h of inspiratory resistive loading in rabbits resulted in diaphragm injury and inflammation with an associated loss of force immediately after this loading, and this loss of force was greater 3 d later. Further, Reid and Belcastro (30) showed that 30 d of tracheal banding in hamsters resulted in decreased stress of diaphragm strips in vitro, associated with an abnormal diaphragm morphology and hypercapnic ventilatory failure. The clinical significance of the loss of force associated with muscle injury may be substantial, because the proportion of force lost is much greater than the percentage of muscle injured (as observed under the light microscope) (29, 33), and the half-life of recovery has been documented as being as long as 5 to 6 wk (34). Although we did not measure forces of the diaphragm in vivo or in vitro, except for measurements of Pes, diaphragm force was likely to have been decreased.

The hypercapnic ventilatory failure found in the banded rats in our study may have resulted in part from an alteration in the strategy of breathing. It has been argued that fatigue (1) or weakness (3) of the respiratory muscles may result in an alteration in the breathing pattern and hypercapnic ventilatory failure. Spontaneously breathing animals show large reductions in V_T as soon as an inspiratory flow-resistive load is imposed (3). Animals breathing against high loads have been shown to reduce their PTI per breath, either as the result of muscle failure or through an adaptation in the central nervous system that reduces the outgoing signal by reducing either pressure or TI (1). We did not measure timing components of the breathing pattern except in anesthetized animals. Consequently, it is difficult to determine whether a modification of breathing pattern contributed to the poor ABG tensions measured in the awake TB rats in our study. Further study is required to determine whether decreased minute ventilation contributes to ventilatory failure in TB rats.

Breathing pattern and the associated PTI components during anesthesia were different in the banded than in the control rats. Although Pes_VT, Pes_VT/Pes_max, and PTI were increased on the day of banding in all banded groups (TB1, TB2, TB3, and TB4), only Pes_VT and PTI were significantly increased in the TB1 group at the end of the experimental protocol (Table 1). Thus, a greater PTI was produced initially against the resistive load during tracheal banding in the anesthetized rat, but this was only maintained during the first day of banding. At 2 to 4 d of banding, the values of Pes_VT and Pes_VT/Pes_max tended to be intermediate between the TB1 and C values in anesthetized rats. Analysis of TI, measured during anesthesia, showed it to be prolonged in the TB1 and TB2 groups, with a tendency to be longer in the TB3 and TB4 groups. Thus, Pes_VT tended to decrease over several days of banding, and TI tended to be longer in the TB groups. It is difficult, however, to attribute too much importance to the breathing pattern in the anesthetized rats in explaining the ventilatory failure reflected in the ABG values obtained in the awake banded rats. Breathing pattern and ventilatory muscle recruitment pattern can be altered by different anesthetic regimens (35). Our anesthetic regimen was selected to optimize the quick recovery of animals, and was not chosen to study the timing components of breathing. Therefore, we are reluctant to draw too many inferences from the PTI data. Lack of a statistically significant difference between the C and TB groups in Pes_VT, Pes_VT/Pes_max, and PTI may also be attributed to the variability of these measures and to the low statistical power of a study done with five groups of animals.

In conclusion, we found that tracheal banding resulted in a deterioration of ABG, an increase in calpain (a cytosolic, nonlysosomal protease), and diaphragm injury that peaked 3 d after tracheal banding. Future studies of the effects of calpain inhibitors or agents that modulate inflammation may provide further information about the mechanisms of respiratory muscle injury and whether this injury can be minimized or prevented.