Abstract
Three groups of NZW rabbits were studied to examine the role of free radical scavengers in preventing diaphragm injury produced by inspiratory resistive load (IRL): control, IRL, and scavenger groups. An IRL (Pao: 45–55 cm H2O) was applied to the IRL and the scavenger groups on Day 1. Free radical scavengers (polyethylene glycol superoxide dismutase, N-acetylcysteine, and mannitol) were given (intravenously) to the scavenger group both before and after the IRL. All rabbits were killed on Day 3 to collect diaphragms. Point counting H&E-stained diaphragm x-sections indicated that abnormal diaphragm muscle in the IRL group was significantly greater than control (p < 0.01). However, it was significantly lower in the scavenger group than the IRL group (p < 0.05) and it did not differ from control. In vitro diaphragm physiological studies found that the twitch tension (p < 0.05) and maximal tension (p < 0.01) in the IRL group were significantly lower than control. The maximal tensions (p < 0.05) in the scavenger group were lower than control. After the fatigue protocol, diaphragmatic contractility in the scavenger group was similar to control and was better maintained compared with the IRL group. We conclude that free radical scavengers can prevent the development of diaphragm injury as evidenced by histology but the protection of diaphragm function is limited.
Keywords: diaphragm injury; inspiratory resistive loading; diaphragm contractility; free radicals; scavengers
Skeletal muscle injury can occur following strenuous muscle activity, particularly following downhill exercise or eccentric contraction in both experimental animals (1, 2) and human studies (3-5). Our previous studies have also shown that diaphragm injury can be produced 3 d following a short period of high-intensity inspiratory resistive loading (IRL) (6, 7) and that this IRL-induced diaphragm injury significantly reduces in vivo diaphragm force production (7).
The development of diaphragm injury following a short period of IRL is likely to result from a complex interplay of several biophysical processes (6-8). Investigators have found that the production of oxygen-derived free radicals in the diaphragm is increased by loaded breathing (9-10) and have postulated that these free radicals somehow produce muscle fatigue. Borzone and coworkers (10) demonstrated an increase in electron spin resonance (ESR) signals in the diaphragm of anesthetized rats in which the diaphragmatic workload was increased by applying a large inspiratory resistive load. Recent experimental data also indicate that oxygen-derived free radicals may play a role in mediating the development of diaphragmatic fatigue following periods of sustained or repetitive high-intensity contraction. Moreover the development of respiratory failure in conditions in which the respiratory workload is increased may be mediated, in part, by free radical-induced respiratory muscle dysfunction (9-11).
Administration of free radical scavengers has been shown to reduce the rate of development of diaphragm fatigue during loaded breathing (12) and to protect the diaphragm from the effects of endotoxin (13). Similarly, other studies have shown that antioxidant treatment in vitro (14) and in situ (15) can attenuate fatigue of the diaphragm. Diaphragm injury is a recently described pathophysiological process with no established relationship to muscle fatigue. Although muscle fatigue recovers with rest, muscle injury may progress over several days and is associated with an attendant marked decrease in contractility. There is no information on the relationship between free radical production and diaphragmatic injury or on the extent that free radical scavengers could prevent the development of diaphragm injury produced by IRL.
Our previous measurements of force before and after diaphragm injury were measured in vivo and in the current study we specifically used in vitro contractility as our standard because in vivo diaphragmatic force production may not necessarily reflect in vitro muscle function due to several extraneous factors such as muscle shortening, which reduces transfer of tension to pressure. Based on these considerations, we speculated that in vivo measurements of diaphragm force reduction after injury could underestimate the reduction in in vitro measures of diaphragm contractility associated with muscle injury.
We hypothesized that elevated free radical production remains present in the diaphragm 3 d following an acute period of intense IRL at a time when diaphragm injury is evident. We also hypothesized that the IRL-induced diaphragm injury would significantly reduce diaphragm contractility measured in vitro. Finally we hypothesized that the use of free radical scavengers would reduce the production of free radicals in the diaphragm during and after IRL and therefore attenuate diaphragm injury. The present study was designed to test these hypotheses in the New Zealand White (NZW) rabbit.
The study was approved by our Committee on Animal Care and conformed to the animal care guidelines of the Canadian Council on Animal Care. Three groups of NZW rabbits (six in each group) were studied: control, IRL, and scavenger groups. The anesthesia, intubation, blood gas monitoring, IRL set up, airway opening pressure (Pao), and transdiaphragmatic pressure (Pdi) measurements were done as described in our previous publications (7).
An IRL (Pao 45∼55 cm H2O) was applied to the IRL and the scavenger groups for 1.5 h. The control group breathed against no added load. The following free radical scavengers were given (intravenously) to the scavenger group: polyethylene glycol superoxide dismutase (PEG-SOD) 2000 IU/kg 30 min before IRL and 36 h after IRL. This dose was chosen based on a previous study (16) and the timing was modified according to its half-life (30–40 h) in order to keep a relatively constant effective level; N-acetylcysteine (NAC) 150 mg/kg/d immediately before IRL, at 24 h, and 48 h after IRL. The dosage and the timing given were also chosen based on previous studies (15-18); mannitol 0.5 g/kg immediately before and after the IRL (see 19, 20).
All rabbits were euthanized. Diaphragms were excised for in vitro diaphragm physiological study, thiobarbituric acid reactive substances (TBARS) analyses, and H&E stains for assessing diaphragm injury as described previously (6, 7).
TBARS measurements. Concentrations of TBARS, an index of free radical-mediated lipid peroxidation, were determined on diaphragm samples. Immediately after removal, the excised diaphragm samples were quickly frozen in liquid nitrogen, stored at −70° C, and were later assayed for TBARS concentrations.
Diaphragm samples were thawed on ice. One-half gram (0.5 gm) of each tissue was weighed, minced, and homogenized on ice in 5 ml 50 mM Tris–0.1 mM EDTA buffer with pH 7.6 at 50% of the Polytron maximum for 2 × 15 s. Samples were centrifuged 5 min (microcentrifuge, 12,000 × g, 4° C) and 0.8 ml supernatant aliquoted. Then 0.4 ml 0.5% thiobarbituric acid (in 0.025 M NaOH) was added to the supernatant. Samples were then boiled for 15 min, cooled, and the absorbance at 532 nm was measured. A sample of homogenate was also centrifuged and the supernatant was assayed for protein, using the Bradford dye-binding method. The TBARS (absorbance at 532 nm) was expressed as a percentage of its protein concentration.
Thin cross sections were sectioned from costal diaphragm samples of 10 μm thickness with a Cryostat-microtome (Reichart-Jung) kept at −20° C and were stained with hematoxylin and eosin (H&E). Diaphragm injury was assessed by point counting the H&E-stained cross sections as described in our previous studies (6, 7). Briefly, the diaphragm cross sections were viewed using a light microscope equipped with a camera lucida (Labophot, Nikon) and a computer program was used 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 diaphragm 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, and necrotic muscle with inflammatory cells; and (3) interstitial space. The area fractions (AA) of each category were expressed as a percentage of the total number of points counted for the whole section.
After removal, diaphragm muscle samples were immediately immersed in aerated Krebs solution. Two diaphragm muscle strips, approximately 2–3 mm in width and 2 cm in length, were dissected from the middle region of the costal diaphragm for each rabbit. The strip was tied at both its ends (the central tendon and the muscle end with silk sutures, Ethicon 5-0 silk).
The isolated muscle strips were transferred to the experimental tissue bath containing Krebs solution, bubbled continuously with 95% O2 and 5% CO2 throughout the experiment. The Krebs solution consisted of the following ingredients (in mM): 137 NaCl, 5 KCl, 24 NaHCO3, 1 NaH2PO4, 2 CaCl2, 1 MgSO4, 11 glucose, and 0.025 d-tubocurare chloride (0.2%). The pH of the Krebs solution was adjusted and maintained at or very close to 7.4. The strips were mounted horizontally in the tissue bath. The tissue bath (20 ml) was water jacketed and thermostatically controlled at a constant temperature of 37° C. The tissue bath was flushed with Krebs solution at a constant flow rate of 5 ml/min.
After equilibration with the Krebs solution for 15 min, the passive length–tension curves for each strip were obtained. Maximum stimulation voltage (three times the threshold voltage) and the optimal length (Lo) for each strip were then established and all subsequent stimulations were performed at the determined Lo and the supramaximal voltage. Bipolar platinum electrodes were used for stimulation and the tension generated was recorded with a computerized data acquisition system (“Direct,” RayTech, Vancouver, B.C., Canada). For each strip, the following measurements were made:
1. Twitch characteristics: five twitch stimulations were performed at intervals of 1 min. From the twitch profile, time to peak tension (TPT), peak twitch tension (PT), and half-relaxation time (1/2 RT) were measured.
2. Force–frequency curves: tetanic stimulations were performed at frequencies of 10, 20, 30, 50, 80, and 100 Hz. Peak tetanic tension at 100 Hz was taken as the maximal tension generated by the strip. The pulses were square waves of 0.2 ms duration with a train duration of 0.4 s for tetanic stimulation.
3. After baseline force–frequency curves were obtained, all muscle strips were subjected to a fatigue protocol, that is, all muscle strips were stimulated at 50 Hz, 30 stimulations per minute and 40% Ti until the force decreased to 50% of its baseline value (modified from references 21 and 22).
4. Immediately after the fatigue protocol, the twitches and forces at 20 Hz and 100 Hz were performed. Finally, the twitches and the force–frequency curve measurements were repeated after 30 min recovery.
At the end of each experiment, the optimal length (cm) for each diaphragm strip and muscle mass (g) were measured and cross-sectional areas (CSA) were calculated. The density of the muscle was assumed to be 1.056 mg/cm3 (23). Data recorded with a computerized acquisition system were analyzed with software (“Anadat,” RHT Info-Dat Inc.) and peak twitch and tetanic tensions were measured. The tension was expressed as Newtons/cm2 (N/cm2). One-way ANOVA plus Tukey test were used for statistical analyses. Values are presented as means ± SE.
Light microscopic examination of the H&E-stained diaphragm cross sections taken from the IRL group showed marked diaphragm injury, characterized by necrotic diaphragm fibers, flocculent degeneration, and influx of inflammatory cells both in the necrotic fibers and in the interstitial tissues (Figure 1B and 1C), whereas much less abnormal muscles can be seen in the cross sections taken from the scavenger rabbit (Figure 1D). The inflammatory cells were both neutrophils and mononuclear cells. The interstitial space in the muscle was also widened. Figure 2 shows the area fractions of normal muscle, abnormal muscle, and interstitial space of the diaphragm among three groups. The area fraction of normal muscle in the IRL group (85 ± 2%) was significantly lower than those in the control group (93 ± 0.2%, p < 0.001) and in the scavenger group (92 ± 1%, p < 0.01). The area fraction of abnormal muscle in the IRL group (7.3 ± 1%) was significantly greater than the control group (1 ± 0.1%, p < 0.01) and the scavenger group (2.7 ± 1%, p < 0.05), but there was no difference between the control and the scavenger groups. The area fraction of interstitial space in the IRL group (8 ± 0.6%) was significantly greater than those in the control group (6 ± 0.2%, p < 0.01) and in the scavenger group (5.3 ± 0.2%, p < 0.01).
Fig. 1. Light micrographs of H&E-stained cross sections of costal diaphragm from control rabbits (A, original magnification: ×400), high IRL rabbits (B, original magnification: ×100; C, original magnification: ×400), and scavenger rabbits (D, original magnification: ×200). The scale bars in A and C indicate 25 μm, the scale bar in B indicates 100 μm, and the scale bar in D indicates 50 μm. Note necrotic fibers, flocculent degeneration, and profound influx of inflammatory cells in the diaphragm cross sections from the IRL group (B and C ), whereas much less abnormal changes were observed from the scavenger group (D).
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Fig. 2. Area fractions of costal diaphragm normal muscle, abnormal and inflamed muscle, and connective tissue (interstitial space, % total cross-sectional area) measured by the point counting technique for the control, the IRL, and the scavenger groups. **(p < 0.01) indicates significant increases or decreases in area fraction in the IRL group compared with the control group. Values represent means ± SE.
[More] [Minimize]The TBARS concentration in the diaphragm for the three groups is presented in Figure 3. This figure shows that the TBARS concentration (absorbance at 532 nm) in the IRL group (0.83 ± 0.06% protein concentration) was significantly higher than that in the control group (0.6 ± 0.01%, p < 0.05), whereas the TBARS concentration in the scavenger group did not differ from that in the control.
Fig. 3. Concentrations of thiobarbituric acid-reactive substance (TBARS) measured as absorbance at 532 nm and expressed as % of protein concentration, in the rabbit diaphragms taken from the control, the IRL, and the scavenger groups. *Significant increases in diaphragm TBARS concentration compared with the control group. Values represent means ± SE.
[More] [Minimize]Twitch characteristics. Table 1 presents the twitch characteristic data obtained before and after the fatigue run for the three groups examined. Table 1 shows that at baseline the peak twitch tension (Pt) in the IRL group was significantly lower than the control group (p < 0.05), whereas there was no decrease in Pt in the scavenger group compared with the control group. After the fatigue protocol, the Pt in the control group (p < 0.001), in the IRL group (p < 0.01), and in the scavenger group (p < 0.01) all decreased significantly compared with their baseline values. Pt in the scavenger group after the fatigue protocol did not differ compared with the other two groups. There were no differences in baseline time to peak tension (TTP) before or after fatigue protocol among the three groups examined. The 1/2 RTs in the IRL and the scavenger groups at baseline were not different compared with control. After the fatigue protocol, the 1/2 RT was prolonged in the control group (p < 0.01), the IRL group (p < 0.01), and the scavenger group (p < 0.01) compared with their baseline values.
| Control Group | IRL Group | Scavenger Group | ||||
|---|---|---|---|---|---|---|
| Pt/CSA, N/cm2 | ||||||
| Baseline | 5.3 ± 0.2 | 3.5 ± 0.5* | 4.1 ± 0.4 | |||
| After fatigue | 1.0 ± 0.1† | 0.9 ± 0.2‡ | 1.5 ± 0.2‡ | |||
| TTP, ms | ||||||
| Baseline | 33 ± 1 | 34 ± 2 | 35 ± 2 | |||
| After fatigue | 34 ± 2 | 36 ± 2 | 38 ± 3 | |||
| 1/2 RT, ms | ||||||
| Baseline | 48 ± 4 | 61 ± 12 | 59 ± 8 | |||
| After fatigue | 127 ± 11‡ | 80 ± 13§ | 90 ± 14§ | |||
| Po/CSA, N/cm2 | ||||||
| Baseline | 27.0 ± 1.9 | 16.3 ± 1.5‖ | 19.9 ± 1.0* | |||
| After fatigue | 16.9 ± 1.5‡ | 10.4 ± 1.1* ,‡ | 14.7 ± 1.3‡ |
Tetanic tensions. The baseline maximal tetanic tension (Po) (Table 1) decreased significantly in both the IRL group (p < 0.001) and the scavenger group (p < 0.05) compared with control group. After the fatigue protocol, the Po decreased significantly in all three groups compared with their baseline values (p < 0.01). The Po in the IRL group after the fatigue run was also significantly lower than control (p < 0.05).
Figure 4 presents the force–frequency relationships obtained at baseline (left panel) and after the fatigue run (right panel) for the diaphragm muscle strips taken from the control, the IRL, and the scavenger groups. At baseline, the forces in the scavenger group were lower compared with control values at 50 Hz (p < 0.05), 80 Hz (p < 0.05), and 100 Hz (p < 0.01), whereas the tetanic tensions in the IRL group at these frequencies were much lower than control values (50 Hz: p < 0.01; 80 Hz: p < 0.001; 100 Hz: p < 0.001). However, there was no significant difference in diaphragm force between the IRL and scavenger groups and between the scavenger and the control groups.
Fig. 4. Force–frequency relationships obtained at baseline (left panel ) and after the fatigue run (right panel) for the diaphragm strips taken from the control, the IRL, and the scavenger groups. *(p < 0.05), **(p < 0.01), and ***(p < 0.001) indicate significantly less force in the IRL and the scavenger groups compared with the control group. Values represent means ± SE.
[More] [Minimize]After the fatigue protocol, the tetanic tensions at all frequencies were significantly decreased relative to prefatigue values for the control group (p < 0.01), the IRL group (p < 0.01), and the scavenger group (p < 0.01). Moreover, the tetanic tensions in the IRL group were lower than those in the control group, particularly at 10 Hz (p < 0.05), 20 Hz (p < 0.01), and 100 Hz (p < 0.05), whereas there were no differences in the tetanic tensions at most of the frequencies between the scavenger group and the control group.
The diaphragm muscle endurance was assessed by the fatiguing time, that is, the time required for the force generated during continuous 50 Hz stimulation to drop to 50% of its baseline value. There were no significant differences in diaphragm muscle strip endurance time among the three groups (control: 144.6 ± 5.8 s; IRL: 137.6 ± 9.7 s; scavenger: 136.4 ± 16.2 s) (p > 0.05).
The present study demonstrates that (a) the production of oxygen-derived free radicals is mildly increased at 3 d after a period of loaded breathing; (b) free radical scavengers can prevent this increase in free radical generation in the diaphragm and the development of diaphragm injury as evidenced by histology; and (c) the diaphragm injury and inflammation produced following a short period of high intensity of IRL significantly impairs in vitro diaphragm contractility and the use of free radical scavengers partly prevented the reduction in diaphragmatic contractility induced by IRL.
The main focus of the present study was to assess the effects of free radical scavengers on the diaphragm injury produced by an initial IRL. There are many commonly measured indices of free radical production, such as indicators of glutathione oxidation, lipid peroxidation, and protein oxidation in exercised or contracting muscle or exhalation of small-molecular-weight hydrocarbons following exercise (12, 24, 25). The TBARS assay was chosen to study the lipid peroxidation in the diaphragm in this study as it is one of the simple and most commonly measured indices of lipid peroxidation in the diaphragm involving loaded breathing (9, 16). Nevertheless, some concerns regarding the TBARS assay should be mentioned. Although the TBARS assay has been widely used for studying lipid peroxidation in both laboratory animals and in humans with disorders, concerns regarding its analytical specificity have been expressed. It has been shown that in a complex biological system the thiobarbituric acid (TBA) reacts, not only with the aldehyde end products of lipid peroxidation, such as malondialdehyde (MDA), but also with a wide variety of other chemical species that can also produce a pink to red color that absorbs maximally at 532 nm (26). It is also possible that MDA formation could occur during the assay procedure itself rather than in vivo experimental manipulation, making results derived from experiments in vivo difficult to interpret. Clearly, the use of the TBARS assay as evidence for lipid peroxidation in complex biological material has limitations due to its lack of specificity and the factors affecting the results. However, many of these potentially disturbing substances do not occur in appropriately high concentrations in tissue extracts under normal conditions. Slater (27) had compared the TBA reaction with other methods of measuring lipid peroxidation, such as diene conjugation, chemiluminescence, oxygen uptake, PUFA loss, and lipid hydroperoxide content. The comparisons were found satisfactory enough to warrant continued usage of the TBARS assay.
The present study demonstrates that the TBARS concentration in the diaphragm of the IRL rabbits was mildly elevated even on the third day after the initial loaded breathing as indicated in Figure 3. Figure 3 also shows that the use of free radical scavengers prevented the increase in TBARS concentration in the diaphragm. Previous studies have shown that TBARS levels in the diaphragm were elevated immediately following resistive loaded breathing (9, 16), and other investigators have reported increases in diaphragm 8-isoprostane levels accompanying both acute and chronic respiratory loading (25, 28). Our present data do not provide information regarding the time course of TBARS concentration in the diaphragm following the initial IRL. The above and other previous studies suggest that increased production of free radicals in the diaphragm occurs from an early stage following IRL (9, 16, 28, 29). It remains possible that the naturally occurring free radical scavengers might reduce that level. However, the present data indicate that there was still a mild elevation of TBARS at Day 3 following the initial loading, suggesting the possibility of ongoing excessive lipid peroxidation due to the increased free radical production. Another possibility is that the inflammatory process associated with the diaphragm injury at Day 3 after the IRL caused the overproduction of the free radicals from cells such as neutrophils and monocytes. A recent study has shown that white blood cells in sepsis are a source of free radicals, which can produce diaphragm dysfunction (30).
The diaphragm injury produced at 3 d following an initial IRL was termed “delayed or secondary diaphragm injury” in previous studies (6, 7). Our data indicates that the use of free radical scavengers attenuated this delayed or secondary diaphragm injury. The free radical scavengers may have acted by preventing the initial “primary” injury and thus preventing the early and perhaps more extensive insult of free radicals, which then would limit the development of the secondary diaphragm injury. However, the present data do not allow us to draw any conclusion regarding the effect of free radical scavengers on the relationship of initial injury to secondary injury.
We speculate that oxygen-derived free radicals could produce respiratory muscle dysfunction by several possible mechanisms. First, oxygen free radicals could damage diaphragm fibers directly by causing lipid peroxidation of muscle membranes (e.g., sarcolemmal membranes and sarcoplasmic reticulum) or by damaging muscle contractile proteins. Second, free radicals could also damage the endothelium of the respiratory muscle microcirculation, altering the blood supply to some myofibers. Third, free radicals may also alter muscle structure by triggering or potentiating muscle proteolysis, a process activated during loaded breathing as demonstrated by the increase in calpain activity in the rabbits subjected to high IRL in our previous experiments (6).
We observed that 3 d after the initial IRL the diaphragm endurance time in the scavenger group was similar to that in the IRL group. Diaphragm endurance had therefore not improved following the use of free radical scavengers. Hence, force–frequency curves after the endurance run showed that the IRL group was reduced at 10, 20, and 100 Hz compared with control whereas the scavenger group was not different from control. Thus, the scavenger group did demonstrate greater resistance to fatigue than the IRL group at a few frequencies. Previous data (16) have shown a protective effect of free radical scavengers on endurance but that study used 20 Hz stimulation and studied initially fresh muscles.
We used three free radical scavengers in this study (PEG-SOD, N-acetylcysteine, and mannitol) in an attempt to block all three major species of free radicals (superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide). We observed that the use of these free radical scavengers did prevent the development of diaphragm injury in terms of histology but it did not completely prevent the reduced diaphragm contractility in the scavenger group. Although we tried to block all three major free radical species, it is possible that the blockade of free radicals was incomplete. Furthermore, as mentioned above free radicals are probably not the only mechanism responsible for the development of diaphragm injury and the decrease in diaphragm contractility produced by IRL and other factors may also have contributed to the development of diaphragm injury and decrease in contractility. For instance, it has been reported that contraction-induced increases in intracellular hydrogen and phosphate ions can result in reductions in maximal contractile protein force-generating capacity and a shift in the myofilament calcium sensitivity (31). Moreover, vigorous muscle contraction can result in activation of muscle proteases such as calpain, leading over time to protein degradation, muscle wasting, and force loss (32). Strong muscle contraction can also result in cytokine activation, white blood cell (WBC) infiltration into muscle, and subsequent alterations in muscle function as the result of other WBC-mediated inflammatory changes (33). Another possibility is that diaphragm inflammation as measured by histology and force loss may not be linked. Because the mechanisms underlying the development of diaphragm injury following an IRL are complex and increased production of free radicals is only one of them, it is not surprising that the use of free radical scavengers prevented the development of diaphragm injury in terms of histology but the prevention of diaphragm dysfunction produced by IRL was incomplete.
The present study also demonstrates that diaphragm injury and inflammation occurring following a short period of high- intensity loading is associated with impaired in vitro diaphragm contractility. Histological examination of the diaphragm from the IRL group revealed muscle injury, which was clearly different from control diaphragm and in vitro diaphragm contractility was significantly reduced compared with the control group. This observation is similar to our previous observation made in an in vivo experimental model in which the rabbits underwent the same experimental protocol without administration of free radical scavengers (7). However, the in vivo diaphragmatic force production mainly reflects diaphragmatic shortening against a low afterload rather than being a measure of force or tension production at a fixed length (34). Therefore, we speculated that the in vivo measurements of diaphragm force reduction (Pdi) after injury would underestimate the reduction in in vitro measures of diaphragm contractility. We compared the in vitro measurements of diaphragm contractility (tetanic tension at different stimulation frequencies) with in vivo diaphragm force generation (Pdi–frequency curves) from our previous study (7) and found that the reduction in diaphragm force in the IRL rabbits only underestimated the reduction in in vitro diaphragm contractility at low frequencies after the fatigue protocol (Figure 5). We conclude that in vivo contractility is a useful reflection of in vitro contractility in this muscle with this caveat. Presumably reduced pressure production in vivo reflects reduced shortening against the same afterload. Thus reduced tension-generating capacity and shortening appear linked.
Fig. 5. Reduction in diaphragmatic force (in vitro: tetanic tension; in vivo: Pdi) at different frequencies in the IRL groups (expressed as percentages of baseline control means), obtained at baseline (left panel ) and after the second IRL (in vivo) (16) and fatigue protocol (in vitro) (right panel ). The in vivo diaphragmatic force measurements tended to underestimate the reduction in vitro diaphragmatic force production. Values represent means ± SE.
[More] [Minimize]In conclusion, increased production of oxygen-derived free radicals can be observed during the secondary injury phase in the diaphragm and the use of free radical scavengers can prevent histological evidence of diaphragm injury produced by IRL and partly preserves in vitro diaphragm contractility. The present study also confirms our previous observations that the imposition of a high intensity of IRL for a short period of time produces secondary diaphragm muscle injury but adds that this injury can impair in vitro diaphragm contractility in a similar way to in vivo contractility.
This study was supported by an MRC research grant (Canada).
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