Abstract
Chronic or prolonged low-intensity loading of the inspiratory muscles has recently been shown to produce diaphragm injury. The present study was designed to examine whether an acute episode of inspiratory resistive loading (IRL) could produce secondary diaphragm inflammation and injury. On Day 1, three groups of anesthetized and intubated New Zealand White rabbits were subjected to moderate IRL (Pao of ≈ 30 cm H2O), high IRL (Pao of ≈ 45 cm H2O), or no load for 1.5 h. On Day 3, costal and crural diaphragms, parasternals, and gastrocnemius muscles were taken to assess injury by point counting. Normal muscle, abnormal and inflamed muscle, and connective tissue on hematoxylin and eosin–stained cross-sections were expressed as percentage of the total points for that cross-section. For the costal diaphragm, both the abnormal muscle (7.3 ± 0.6% versus 1.1 ± 0.2%; p < 0.001) and connective tissue (8.0 ± 0.6% versus 5.7 ± 0.2%; p < 0.01) in the high IRL group were higher than control, whereas in the moderate IRL group they were not significantly different from control. Total calpain-like activity was increased in the moderate IRL group but not in the high IRL group. Injury was observed in the parasternal muscles but to a lesser extent. No injury was observed in the gastrocnemius muscle. We conclude that secondary diaphragm injury occurs after acute IRL but only when the IRL exceeds the fatigue threshold.
It has been well documented that both in laboratory animals (1, 2) and in humans (3-5) exercise-induced muscle injury and inflammation can occur in limb muscles following strenuous muscle activity, particularly following downhill exercise or eccentric contraction. Moreover, it has been shown that this muscle injury occurs in two phases: an acute phase during which muscle injury is seen immediately after muscle contraction, and a delayed phase (secondary muscle injury) which peaks at about 3 d afterwards (1). Histologic changes revealed by electron microscopy immediately after intense exercise include sarcoplasmic reticulum vacuolization and/or sarcometric disruptions, primarily at the level of the Z line, disintegration of the intermediate filament system, loss of the normal alignment between adjacent myofibrils (1, 2, 6), and damage to the muscle fiber plasma membrane (7). The mechanisms underlying these morphologic changes are unknown. However, it has been shown that the activation of a nonlysosomal Ca2+-activated neutral protease (calpain) is involved in exercise-induced muscle injury. Moreover, in vitro experiments indicate that the activation of a nonlysosomal Ca2+-activated neutral protease produces changes in skeletal muscle (8, 9), which are identical to those reported in vivo after exercise. Running exercise increases total nonlysosomal Ca2+-specific protease activity (10), which may contribute to exercise-induced muscle damage.
Recent evidence has revealed that the respiratory muscles, particularly the diaphragm, are not spared from muscle injury. Reid and coworkers reported that ventilatory failure induced by chronic tracheal loading (resistive load) in the hamster was associated with diaphragm injury and some of this injury was indeed linked to changes in myofibrillar complexes, specifically their susceptibility to calpain-mediated degradation (11). Other experimental studies indicate that the diaphragm is highly recruited during inspiratory resistive loading (IRL) (12) and therefore may actually reach a level required to produce respiratory muscle injury.
The characteristics of the respiratory load that leads to respiratory muscle injury, however, require more investigation. In limb muscles, even brief periods of loading or intense exercise can produce delayed injury and inflammation. Whether brief periods of loading could result in the delayed diaphragm injury and the accompanying changes in calpain-like activity is unknown. The intensities of respiratory load that are required to produce these changes and the relationship of the intensity of loading to the fatigue threshold remain to be determined.
In the present study, we hypothesized that application of a short period of intense IRL may lead to secondary respiratory muscle injury, i.e., delayed diaphragm injury and inflammation, and that the development of respiratory muscle injury would be load dependent. The present study examined the histologic changes in different respiratory and limb muscles, and the changes in total calpain-like activity in the diaphragm after a brief period of moderate or high IRL in New Zealand White rabbits.
Three groups of New Zealand White rabbits (n = 7 in each group) with comparable body weights, ranging from 3.5 to 4.5 kg, were studied. Each rabbit was randomly assigned to one of the following groups: control group, moderate IRL group with target airway opening pressure (Pao) of 30 cm H2O, and high IRL group with target Pao of 45 cm H2O. On Day 1, rabbits were anesthetized with ketamine (initial dose: 35 mg/kg body wt, intramuscularly) and xylazine (initial dose: 7 mg/kg body wt, intramuscularly). A constant level of anesthesia was maintained by giving supplementary doses of anesthetics (half of the initial dose) approximately every 30 min. Arterial blood gases were obtained from a catheter placed in an ear artery and measured by a Corning 168 pH/blood gas analyzer. A two-way valve (No. 2600; Hans Rudolph, Kansas City, MO) was connected to the endotracheal tube, and an adjustable needle valve was connected to the inspiratory port to apply the IRL.
Pao was measured from a side port inserted at a right angle into the endotracheal tube. Two 5-cm latex balloons were placed via the oropharynx, one in the stomach, filled with 1.0 ml air, to measure abdominal pressure (Pab) and the other in the mid-esophagus, filled with 0.6 ml air, to measure esophageal pressure (Pes). These balloons were connected by thin polyethylene tubing (50 cm, PE-200) to a differential pressure transducer (MP 45; Validyne Engineering, Northridge, CA). From the outputs of these two signals, transdiaphragmatic pressure (Pdi) was obtained (Pdi = Pab − Pes). Body temperature was kept between 37 and 39° C with a heating pad.
The duration of IRL was 1.5 h. The intensities of the IRL applied corresponded to a diaphragm pressure time index (PTIdi) of 0.23 ± 0.05 in the high IRL group and of 0.17 ± 0.01 in the moderate IRL group. Maximum transdiaphragmatic pressure (Pdimax) was assumed to be 65 cm H2O in the calculation of PTIdi (13). An appropriate oxygenation level in the IRL groups was ensured by applying 100% O2 at 3 L/min to the inspiratory side of the adjustable needle valve. The PaCO2 increased substantially during both moderate and high IRL. It averaged 96 ± 11 mm Hg in the moderate IRL group and 129 ± 26 mm Hg in the high IRL group at the end of the run (90 min). The PaO2 was maintained above 80 mm Hg. The animals were then extubated and allowed to recover. The rabbits in the control group received similar treatment as in the IRL group except without IRL.
On Day 3, rabbits from all three groups were euthanized. The left half of the costal diaphragm was first excised for calpain analysis. Muscle samples were trimmed of all visible fat and connective tissue and then placed immediately in liquid nitrogen with precooled tongs. All samples were stored at −71° C until analysis. The other half of the costal diaphragm, the right half of the crural diaphragm, the medial part of the third right parasternal intercostal muscle, and the whole right gastrocnemius muscle were excised and quickly frozen in isopentane precooled with liquid nitrogen and stored in a refrigerator at −71° C. These biopsies were later processed for hematoxylin and eosin (H&E) staining. For the rabbits from the moderate IRL group, only the right costal and crural diaphragms were excised and no intercostal or gastrocnemius muscles were obtained.
Cross-sections of costal and crural diaphragms, parasternal intercostals, and gastrocnemius muscles were sectioned at 10-μm thickness with a Cryostat-microtome (Reichart-Jung, Buffalo, NY) kept at −20° C and were stained with H&E. Injury of these muscles was assessed by a point-counting technique as described in principle by Weibel (14) and in practice by Reid and coworkers (11). The area fraction (AA) of normal muscle, abnormal and inflamed muscle, and connective tissue were determined from cross-sections of each biopsy sample (H&E stain). AA values were determined using a light microscope equipped with a camera lucida (Labophot; Nikon, Tokyo, Japan) and a computer program for point counting. The image of a 63-point grid from the computer monitor was projected via the camera lucida onto the image of the muscle cross-section viewed at ×400. Points projected on the cross-section were assigned to three categories: (1) normal muscle; (2) abnormal and inflamed muscle, including viable muscle with abnormal morphology, necrotic muscle, necrotic muscle with inflammatory cells, inflammatory cells (where no evident outline of a muscle cell can be seen), and effusion; (3) connective tissue (fat or collagen). The number of points in each of these three categories was expressed as a percentage of the total number of points in the three categories to determine each individual AA.
Extraction and determination of calcium-dependent calpastatin-inhibited proteolytic activity (calpain-like activity) was accomplished on costal diaphragm samples (15). Briefly, costal diaphragm samples (approximately 100 mg) were suspended with an Ultra-Turrax homogenizer (Model Tr-10; IKA Laboratories, Germany) for 20 s at a setting of 65, in 10 to 15 vol of a buffer containing 80 mM Kcl, 20 mM Tris (pH 7.5), 5 mM EGTA, and 2 mM dithiothreitol. The suspension was centrifuged (4° C) at 22,000 × g for 15 min (Model Z 233-M; Hermle, Wehingen, Germany), and the supernatant (soluble fraction) decanted and stored in polypropylene tubes on ice for subsequent assay of calpain-like activity. Following centrifugation, this particulate material was homogenized with a 2-ml Wheaton glass-homogenizer (10 strokes) in 10 to 15 vol of a similar buffer (as above) with addition of 0.35% Triton-X100 and re-centrifuged under the same previous settings. The supernatant from the second centrifugation step (particulate fraction) was stored on ice and assayed for calpain-like activity.
The total calcium-dependent proteolytic activity (total calpain-like activity) was determined by a microplate assay using casein as the substrate (16). Briefly 200 μl of extract was added to a reaction mixture containing 2 mg/ml casein, 50 mM Tris (pH 7.5), and 20 mM dithiothreitol (in duplicate). After a 5-min pre-incubation at 30° C, 5 mM total calcium (800 μmol/L free calcium as determined by IONS software program) was added to one of the duplicates while the other contained 5 mM EGTA. After 30 min at 30° C, an aliquot (100 μl) of each sample was assayed for calcium proteolysis in a total volume of 325 μl using a Bio-Rad protein dye reagent concentrate (Bio-Rad Laboratories). The protein dye reagent is composed of 0.05% (wt/vol) Coomassie brilliant blue G-250, 23.5% (wt/vol) ethanol, and 42.5% phosphoric acid. The enzyme activities (carried out in the absence and presence of calcium) are expressed as caseinolytic activity and calculated based on a 0.1 absorbance change at an optical density of 595 nm being equivalent to 1 U of enzyme activity. The calcium-dependent, caseinolytic activities of the soluble and particulate fractions are expressed as calpain-like activities (U/g wet muscle mass), because minimal activity (< 5%) was observed when calpastatin (a calpain inhibitor) was added to the assay.
Analysis of variance plus Tukey test was used for comparisons of area fractions and total calpain activity among groups and for comparisons of area fractions among four different muscles for the control and high IRL groups. Values represent means ± SE unless otherwise stated.
Examination of the micrographs taken from the rabbits exposed to high IRL showed marked costal diaphragm injury, characterized by necrotic diaphragm fibers, flocculent degeneration, and profound influx of inflammatory cells both in the necrotic fibers and in the interstitial tissues. The inflammatory cells were both neutrophils and mononuclear cells. The interstitial space in the muscle was also widened. Representative light micrographs showing these changes are presented in Figure 1. We observed that the extent of diaphragm fiber injury varied among the rabbits. For instance, the injured fibers were more widespread throughout the diaphragm muscle cross-section in some cases, whereas in others the injured fibers were localized. Similar muscle injury was also present in the crural diaphragms but to a lesser extent compared with that in the costal diaphragm. A small number of inflammatory cells in the interstitial tissue and a few scattered necrotic fibers were observed in the parasternal intercostals as well but to a much lesser extent compared with that in the costal diaphragm. No injury was observed in the gastrocnemius muscle.
Fig. 1. Light micrographs of H&E-stained cross-sections of costal diaphragm from control rabbit (A, ×400) and high IRL rabbits (B, ×400; C, ×100; and D, ×200). The scale bar in B indicates 25 μm, and the scale bar in C indicates 100 μm. Note necrotic fibers, flocculent degeneration, and profound influx of inflammatory cells.
[More] [Minimize]Comparison of normal muscle, abnormal muscle, and connective tissue of the costal diaphragm among three groups is shown in Figure 2. The AA of normal muscle, abnormal muscle, and connective tissue in the control group averaged 93.2 ± 0.2%, 1.1 ± 0.2%, and 5.7 ± 0.2%, respectively. In the high IRL group, the AA of abnormal muscle (7.3 ± 1.3%) was greater (p < 0.001) and the AA of normal muscle (84.7 ± 1.7%) was less (p < 0.001) than control. The AA of the connective tissue in the high IRL group (8.0 ± 0.6%) was also greater than that in the control group (p < 0.01). In the moderate IRL group, however, the AA of abnormal muscle (1.8 ± 0.1%) was not different from that in the control group. The AA of normal muscle (91.3 ± 0.2%) and the AA of the connective tissue (7.0 ± 0.3%) did not differ from control.
Fig. 2. Area fractions of normal muscle, abnormal muscle, and connective tissue of the costal diaphragm among control, moderate IRL, and high IRL groups. ** (p < 0.01) and *** (p < 0.001) indicate significant increases or decreases in area fraction compared with those in the control group. Bars represent means ± SE.
[More] [Minimize]The AA of normal muscle, abnormal muscle, and connective tissue for the crural diaphragm are shown in Figure 3. In the high IRL group, the AA of abnormal muscle (3.5 ± 1.3%) tended to be greater than control (1.0 ± 0.2%), although the difference did not reach a significant level probably because of variations in the AA of abnormal muscle in the high IRL group. The AA of normal muscle (89.6 ± 1.6%) and connective tissue (6.9 ± 0.3%) also did not differ from control (93.1 ± 0.3% and 5.8 ± 0.2%, respectively). In the moderate IRL group, the AA of normal muscle (91.6 ± 0.3%), abnormal muscle (1.6 ± 0.1%), and connective tissue (6.7 ± 0.2%) did not differ from control.
Fig. 3. Area fractions of normal muscle, abnormal muscle, and connective tissue of the crural diaphragm among control, moderate IRL, and high IRL groups. There were no significant differences in area fractions of normal muscle, abnormal muscle, or connective tissue among the three groups. Bars represent means ± SE.
[More] [Minimize]For the parasternal intercostals in the IRL group, the AA of normal muscle (91.1 ± 0.6%) was significantly smaller than control (93.5 ± 0.2%; p < 0.05) and the AA of abnormal muscle (2.8 ± 0.3%) was significantly greater than control (0.9 ± 0.04%; p < 0.001). The AA of connective tissue (6.1 ± 0.3) did not differ from control (5.6 ± 0.2%). For the gastrocnemius muscle, there were no differences in AA of normal muscle (94.6 ± 0.2% versus 94.1 ± 0.1%), abnormal muscle (0.4 ± 0.1% versus 0.6 ± 0.1%), and connective tissue (5 ± 0.2% versus 5.3 ± 0.1%) between the control group and the high IRL group.
The AA of normal muscle, abnormal muscle, and connective tissue among the costal diaphragm, crural diaphragm, and the parasternal intercostals in the high IRL group are compared in Figure 4. In this figure, the AA of normal muscle, abnormal muscle, and connective tissue of the crural diaphragm and the parasternals are compared with those of the costal diaphragm. There were no significant differences in AA of normal muscle, abnormal muscle, and connective tissue between crural diaphragm and costal diaphragm, although for the crural diaphragm the AA of normal muscle tended to be greater than that of the costal diaphragm, and the abnormal muscle and connective tissue of the crural diaphragm tended to be smaller compared with the costal diaphragm. Muscle injury was observed in the parasternals (p < 0.001), but it was significantly smaller compared with that of the costal diaphragm (p < 0.05).
Fig. 4. Area fractions of normal muscle, abnormal muscle, and connective tissue among the costal diaphragm, crural diaphragm, and parasternal intercostals for the high IRL group. * (p < 0.05) indicates significantly greater or smaller area fraction in the crural diaphragm and the parasternal intercostals compared with the costal diaphragm. Bars represent means ± SE.
[More] [Minimize]Three days after the exposure to IRL, the total calcium-dependent calpastatin-inhibited (calpain-like) activity in the costal diaphragm from the control rabbits averaged 15.7 ± 1.5 U/g wet muscle weight. In the rabbits exposed to the moderate IRL, the total calpain-like activity (24.7 ± 2.1 U/g wet muscle weight) was significantly greater than that from the control rabbits (p < 0.05). However, the total calpain-like activity (13.4 ± 1.2 U/g wet muscle weight) from the rabbits exposed to the high IRL did not differ from that in the control group.
The present study provides morphologic evidence that delayed diaphragm injury and inflammation can be produced 3 d after a short period of high-intensity IRL, especially in the costal region; and that this diaphragm injury is load dependent. Muscle injury was present in the other primary muscles of inspiration as well, such as in parasternal intercostals, but to a lesser extent. There were no histologic changes showing injury in the gastrocnemius muscle, a nonrespiratory muscle.
In the present study, the diaphragm, the parasternal intercostals, and the gastrocnemius muscles were excised 3 d after a short period of IRL to assess muscle injury. We chose this time regimen based on the following observations. After a period of vigorous limb muscle contraction, injury occurs in two phases, an acute phase and a delayed phase. The acute phase starts immediately after the injurious muscle contraction, whereas the delayed phase (secondary muscle injury) peaks at about 3 d after muscle contraction, particularly eccentric muscle contraction (1, 17, 18). Indeed, McCully and Faulkner (19) reported that 1 d after lengthening contractions, muscles had many fibers that appeared abnormal and 2 to 4 d after lengthening contractions, infiltration with macrophages and muscle fiber degeneration were seen. However, these events reached a peak at 3 d. The other reason that muscle biopsies were sampled on Day 3 is that degenerating limb muscle fibers can be better distinguished from intact fibers at this time as it has been observed that muscle fiber regeneration begins at Day 4 (19). Further evidence in support of the concept of secondary muscle injury is the delayed rise in serum LDH and CPK in plasma of rats exposed to downhill exercise or eccentric contraction. Armstrong and colleagues (1) reported that there was an initial peak in these plasma enzyme levels immediately after the exercise; however, there was another large peak on Day 3.
Most of the previous studies examining secondary muscle injury and inflammation following exercise have been performed in limb muscles (19, 20). However, respiratory muscles, particularly the diaphragm, are not spared from muscle injury. Reid and coworkers (11) found that ventilatory failure was associated with diaphragm injury following a period of chronic tracheal loading (resistive load) in the hamster (11). There are also reports that signs of muscle fiber injury can be found in the respiratory muscles of patients with chronic obstructive pulmonary disease (21). One study has shown that diaphragm contraction band necrosis was a frequent finding both in infant and adult patients who died suddenly, with the lesion being most pronounced in a patient who died of status asthmaticus (22).
The mechanisms underlying the exercise- or load-induced muscle injury are not clearly understood. However, potential mechanisms may include high intramuscular tension, metabolic events due to raised intramuscular temperature, elevated oxygen free radical production, lower pH, or activation of calcium-activated proteases such as calpain (6, 18). Elevated intramuscular calcium may play an important role in producing exercise-induced muscle injury (23, 24). The lengthening of muscle fibers that occurs during eccentric muscle contraction may lead to physical disruption of the sarcolemma and to sarcomere disruption. We speculate that the mechanisms underlying the respiratory muscle injury in response to IRL may be similar to those leading to limb muscle injury after exhaustive exercise. This muscle injury may be mediated by endogenous proteases, including lysosomal and nonlysosomal systems such as the Ca2+-activated neutral protease, calpain. Reid and coworkers found that the diaphragm injury and inflammation elicited by chronic tracheal loading was associated with an increase in total calpain-like activity (11). We found that the total calpain-like activity in the costal diaphragm was significantly higher in the rabbits exposed to moderate IRL, suggesting that an increased activation of calpain-like activity had occurred. However, the total calpain-like activity in the costal diaphragm was not increased in the high IRL group, in which marked diaphragm injury was present. In the present study, we did not measure the total calpain-like activity at different periods of recovery after the IRL and the total calpain-like activity might have been increased in the early stage of the recovery process in the high IRL group. There is a report of increased protein turnover after exercise and that the rate of protein synthesis is greater with intense exercise (25). Therefore, we speculate that there was less stimulation to increase the rate of protein synthesis in the moderate IRL group and accordingly the higher total calpain-like activity may represent gradual changes in protease activation. On the other hand, the relatively normal level of total calpain-like activity in the high IRL group may be the result of greatly increased protein synthesis during the early phase of recovery or advanced autolysis of the protease that occurred early after high-intensity IRL. Further studies are needed to clarify this issue.
We observed marked costal diaphragm injury on histologic examination, characterized by necrotic diaphragm fibers, flocculent degeneration, and profound influx of inflammatory cells both in the necrotic fiber and in the interstitial tissues in the rabbits exposed to high IRL. Our point-counting data indicate that in the rabbits exposed to high IRL the abnormal tissue was significantly increased in the diaphragm and to a lesser extent in the parasternals. The abnormal tissue (7.3% of total area) observed in the diaphragm mainly consisted of necrotic diaphragm fibers and inflammatory cells. Because there was a profound influx of neutrophils and mononuclear cells (or macrophages) to the necrotic diaphragm fibers and interstitial tissue, we believe that inflammation in the muscle (including inflammatory cells and mediators) probably contributed to the delayed muscle injury. Thus, the inflammatory response may have mediated some of the diaphragm and parasternal injury we observed. Although the present study did not address the role of inflammation in mediating respiratory muscle injury, other models of exercise overload have shown that the administration of anti-inflammatory medications reduces muscle injury (26, 27).
We reported that the area fraction of abnormal muscle in the control costal diaphragm was 1%, which mainly consisted of changes in fiber size and shape (0.3%) as well as enlarged and round-shaped nuclei (0.7%) in the interstitial tissue. These nuclei may well be the resident mononuclear or macrophages in normal skeletal muscles but no information is available on the diaphragm at this time. A similar fraction of abnormal muscle has been observed in the control hamster diaphragm in another study (11).
In addition, one may argue that hypercapnia and the attendant respiratory acidosis that occur during IRL alone may induce diaphragm muscle injury independently of diaphragm force developed. The arterial blood gases at the end of high IRL were as follows: PaCO2 , 129 ± 26 mm Hg (mean ± SD); pH, 7.05 ± 0.14; and PaO2 , 84 ± 25 mm Hg. However, our data indicate that the respiratory acidosis that occurred during IRL did not play a significant role in producing muscle injury. First, this argument is supported by the data obtained from the limb muscle (gastrocnemius), which, similar to the diaphragm, was also subjected to the effect of respiratory acidosis in the high IRL group. However, although marked injury and inflammation was found in the diaphragm in the high IRL group, no muscle injury was found in the gastrocnemius muscle. Second, we found that, although moderate IRL also resulted in a significant rise in PaCO2 (96 ± 11 mm Hg; p < 0.001) and a corresponding significant decrease in pH (7.15 ± 0.11; p < 0.001) at the end of the IRL run, there was no evidence of histologic injury and inflammation. Therefore, it appears that the respiratory acidosis produced does not play a role in IRL-induced muscle injury.
We observed most of the injury in the costal diaphragm. Although the parasternals and crural diaphragm demonstrated injury, it was more evident in the costal diaphragm. The costal diaphragm may be more susceptible to injury for a number of reasons. First, in this rabbit model, activation of the costal diaphragm is high (12, 28, 29). Second, compared with the crural diaphragm, the costal diaphragm shortens less (29, 30) under no added load and its shortening is markedly inhibited by loads placed at the airway (29, 30). Assuming that muscle shortening is influenced by the afterload, these observations may be explained by the mechanical linkage of the crural diaphragm and the chest wall, such that loads placed on the abdominal compartment rather than the airway impede its contraction (31). Third, the costal diaphragm consists of many long fibers not all extending from origin to insertion, and muscle fiber contraction may be inhomogeneous (29). Although eccentric contractions of the diaphragm have not been observed during unloaded breathing, lengthening in regional segments of the costal diaphragm has been reported during inspiratory loading (29). It is known that eccentric contractions are more injurious. Thus, the observation of more injury in the costal diaphragm may provide some information on how the muscle contracts during loading as well as how muscle injury develops. There was a significant increase in injury to the parasternals as compared with control in the high IRL group but to a lesser extent compared with the costal diaphragm. We speculate that one of the reasons that there was less injury in the parasternals compared with the costal diaphragm may be related to the fact that during IRL the parasternal intercostals (medial part) shorten and no lengthening of the parasternals has been observed during IRL (32).
Finally, regarding the fiber specificity in muscle injury, previous studies have shown that a greater degree of histologic injury occurs in type II, particularly type IIb, fibers in human limb muscles (3, 4). Similar observations were also made in rabbit tibialis anterior muscle (33, 34), suggesting the importance of fiber oxidative capacity in eccentric contraction– induced muscle injury. However, data on the fiber specificity in diaphragm muscle injury are lacking, except that one study performed in the awake dog after 4 d of intermittent inspiratory resistive breathing found that type I diaphragm fibers are more susceptible to sarcolemmal disruptions (35). In the present study, we observed marked diaphragm injury in the high IRL group. Because the injury process itself often results in not only fiber necrosis but also fiber regeneration, it is difficult to distinguish which fiber type had been affected in the injured muscle with traditional histochemical fiber-typing techniques and more studies are required to clarify this issue.
The respiratory muscle injury observed in this study may have implications to respiratory muscle fatigue or respiratory failure. Our data indicate that a short period of high-intensity IRL can produce respiratory injury but moderate-intensity IRL did not produce a significant amount of muscle injury. However, our data do not exclude the possibility that application of a prolonged period of moderate-intensity IRL could produce significant respiratory muscle injury. Indeed, Reid and coworkers used continuous loading with pressure time index (PTIdi) estimated at 0.06 and have shown injury in the diaphragm (11). We chose a short period of high-intensity loading with PTIdi well above the fatigue threshold (high IRL group with PTIdi of 0.23) and one very close to it (moderate IRL group with PTIdi 0.17). Our data suggest that short periods of loading require high-intensity activation well above the fatigue threshold to produce secondary injury. Calpain activity was elevated in the moderate IRL group, indicating some degradative changes can occur in the absence of overt inflammation. Overt inflammation is usually required before functional change in the muscle can be recognized, since secondary injury and inflammation is known to be associated with severe force loss in limb muscles (17, 19). Therefore, according to this information, one would expect that the delayed or secondary respiratory muscle injury, particularly diaphragm injury, produced by high-intensity IRL could reduce the force development of the respiratory muscles and may have a significant impact on respiratory muscle contractility. Clearly, more studies are required to examine the relationship between respiratory muscle injury, respiratory muscle force development, and possibly ventilatory failure.
Supported by a grant from the Medical Research Council of Canada.
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