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Research Reports |
JM Eason, PT, PhD, is Assistant Professor, Department of Physical Therapy, School of Allied Health Professions, Louisiana State University Health Sciences Center, 1900 Gravier St, New Orleans, LA 70112 (USA) (jeason{at}lsumc.edu). At the time this study was done, she was a doctoral student in the Department of Exercise Science, University of Florida.
SL Dodd, PhD, FACSM, is Associate Professor, Department of Exercise and Sport Sciences, University of Florida, Gainesville, Fla
SK Powers, EdD, PhD, FACSM, is Professor, Department of Exercise and Sport Sciences, University of Florida
AD Martin, PT, PhD, FACSM, is Associate Professor, Department of Physical Therapy, University of Florida
Address all correspondence to Dr Eason
Submitted December 28, 1998;
Accepted September 29, 1999
 | Abstract |
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Key Words: Contractile properties • Glucocorticoid • Muscle atrophy • Rat • Skeletal muscle
| Introduction |
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Glucocorticoids are used to treat a variety of pulmonary disorders; therefore, it is possible that the diaphragm may develop muscle atrophy. Because the diaphragm is considered the primary inspiratory muscle, any decrease in force production as a result of muscle atrophy may have deleterious effects in people with lung disease. The decreased force production in the diaphragm may have clinical implications. Impairment in force production of the diaphragm may place these patients at risk for prolonged mechanical ventilation and length of stay in an intensive care unit. Physical therapy intervention may need to include treatments designed to increase the force generated by the diaphragm.
It is generally well accepted that prolonged treatment (8 –14 days) with glucocorticoids at either moderate or high doses results in a reduction of both diaphragm muscle mass2–5 and force production3,4,6 in the rat diaphragm. Moore et al2 and Dodd et al3 found 28% and 15% reductions in diaphragm mass in rats treated with glucocorticoids for 10 days. Dodd et al3 and Sasson et al4 found 26% and 12% declines in diaphragm force production in rats treated with glucocorticoids. Furthermore, administration of glucocorticoids results in atrophy of type IIb and IIx fibers6–8 in the rat diaphragm but not type I or IIa fibers. Van Balkom et al6 reported that the cross-sectional areas of type IIx and IIb fibers from glucocorticoid-treated animals were 62% and 67% of the cross-sectional areas of the same fibers from control animals. Type IIx fibers are classified as fast fibers and possess biochemical and contractile characteristics intermediate between type IIa and IIb fibers.9,10
High doses of glucocorticoids for short periods of time (5 days or less) are commonly used in the treatment of both lung transplant rejection and status asthmaticus.11–13 Very little is known regarding the potential effects that short-term, high doses of glucocorticoids may have on respiratory muscles. Nava et al14 reported that very high doses of glucocorticoids over a 5-day period resulted in a decrease in diaphragm mass but did not affect maximal specific isometric tetanic tension (Po). Those researchers, however, used glucocorticoid dosages that were 5 times greater than the dosage commonly used in clinical practice to treat patients with status asthmaticus.15 Problems may occur, however, even when glucocorticoids are administered at dosages used in clinical practice. These problems are related to possible differences in how the drug is metabolized in individuals once it has been transported into the cell. Furthermore, it is often difficult to separate the iatrogenic effect of these drugs from the effects of the disease.
We became interested in the potential effects of the short-term administration of clinically recommended high doses of glucocorticoids on the diaphragm muscle. Thus, the purpose of our study was to assess the effects of the administration of short-term, high doses of glucocorticoids for 5 days on the morphologic and contractile properties of the rat diaphragm. We hypothesized that administration of glucocorticoids at a dosage of 5 mg/kg/d for 5 days would result in a decrease in both body and diaphragm weights. Furthermore, we hypothesized that this drug regimen would result in a decrease in diaphragm Po.
| Method |
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TopAbstractIntroductionMethodResultsDiscussionConclusionReferences |
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Prednisolone was chosen because it is prototypical of the nonfluorinated glucocorticoids used to treat human disease. The dosage of glucocorticoids used in this study is one that is commonly given to patients in clinical practice for the treatment of status asthmaticus.12 The rat diaphragm is an appropriate model to study characteristics of the diaphragm because the fiber type composition is similar to that of humans.16,17 Prednisolone was suspended in 0.9% saline and injected subcutaneously daily for 5 days. All injections were done at approximately the same time of the day. Animals were weighed prior to initiation of injections. Thereafter, they were weighed daily. The final weight was obtained 24 hours following the final injection. Guidelines for animal use established by the American Physiological Society18 were followed.
Measurement of Contractile Characteristics
Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg) 24 hours following the final injection, and the entire diaphragm was removed and placed in a dissecting dish containing a Krebs-Henseleit solution equilibrated with a 95% O2/5% CO2 gas mixture.2,4,6,8,14 A small strip of the costal diaphragm was cut with a portion of the central tendon on one end and rib attachment on the other end. The strip was used to determine in vitro contractile measurements of peak isometric twitch specific tension (Pt), time to peak tension (TPT), one-half relaxation time (
RT), and Po. The twitch contractile measurements of Pt, TPT, and RT are generally indicative of calcium handling by the muscle. The measurement of TPT is the amount of time it takes the muscle to develop Pt and is generally indicative of calcium release by the sarcoplasmic reticulum. The measurement of RT is the time it takes for Pt force to return to one half of the force generated during the peak isometric twitch following removal of the stimulus. This measurement is generally thought to be indicative of calcium uptake by the sarcoplasmic reticulum. The measurement of Po is the amount of tetanic tension developed by the muscle when a stimulus of sufficient frequency and duration is delivered to the muscle.
All contractile measurements were collected with the muscle at optimal length (Lo).2,4,6,8,14 Optimal length was defined as the muscle fiber length at which maximal twitch tension was generated by a 2-millisecond pulse. The Lo was obtained by systematically lengthening the muscle with a micrometer while evoking single twitch contractions. The muscle was considered to be at Lo when maximal twitch tension was generated. Following contractile measurements, the mass of the muscle strip and its length (Lo) were measured in order to determine the muscle cross-sectional area (CSA). Expressing tension as the amount of force generated per muscle CSA allows for comparison of force generation between muscle strips of different sizes. Muscle CSA was determined using the following formula19:
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The remaining costal diaphragm was trimmed of connective tissue and fat, blotted, and weighed on an analytical balance. Total weight of the diaphragm was obtained by adding the weight of the muscle strip to the weight of the remaining costal diaphragm. Because the diaphragm contains a mixture of muscle fiber types, we were also interested in comparing the effects of short-term, high doses of glucocorticoids on muscles composed primarily of one fiber type. Thus, the soleus and extensor digitorum longus (EDL) muscles were excised, cleaned of connective tissue and fat, and weighed on an analytical balance. These muscles are composed primarily of type I and IIb muscle fibers, respectively. The animal was then sacrificed with an overdose of pentobarbital.
Experimental Protocol
The experimental protocol for collection of contractile data was similar to that described by previous investigators.2,4,6,8,14 These methods are the standard methods of measuring these muscle characteristics and can be judged to yield valid and reliable measurements based on their extensive use in the literature. Briefly, the dissected muscle strip was suspended vertically between 2 Plexiglas* clamps in a jacketed tissue bath containing Krebs-Henseleit solution and connected to a force transducer (model 300B
). In order to produce complete neuromuscular blockade, 12-µM d-tubocurarine was added to the tissue bath. The jacketed tissue bath was aerated with gas (95% O2/5% CO2), pH was maintained at 7.4, and the osmolality of the bath was approximately 290 mOsm. The temperature in the organ bath was maintained at 25°C. The muscle strip was stimulated along its entire length with platinum wire electrodes using a modified Grass Instruments S48 stimulator.
After a 15-minute equilibration period, the muscle strip was adjusted to Lo, and maximal twitch tension was obtained by applying a supramaximal voltage (140 V) to stimulate the strip.
The force transducer output was amplified and differentiated by operational amplifiers and underwent analog-to-digital conversion for analysis using a computer-based data acquisition system (GW Instruments Series II
). All contractile measurements were measured in triplicate and averaged, and the mean was used for statistical analysis. The twitch contractile measurements of Pt, TPT, and
RT were obtained by applying twitches of 2 milliseconds duration to the strip at supramaximal voltage. The tetanic contractile measurement of Po was produced by using a supramaximal stimulus train of 80 Hz and 330 milliseconds duration. Maximum tension generated during both twitch and tetanic contractions was normalized to muscle CSA.
Data Analysis
Comparisons between the control and prednisolone groups were made by unpaired t tests. Variables measured were diaphragm and body weights, Po, Pt, TPT, and
RT. A repeated-measures analysis of variance was used to test changes in weight across groups over time. Data were analyzed by the Statview 4.1 statistical package|| on a Macintosh computer.# Significance level was set at P <.05.
| Results |
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TopAbstractIntroductionMethodResultsDiscussionConclusionReferences |
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±SEM) of the animals are summarized in Table 1. Mean body weights were similar in both the control and prednisolone groups prior to administration of glucocorticoids (280.3±2.4 g versus 280.2±4.4 g). The Figure illustrates the decrease in body weight over the 5-day treatment period. By the third day of drug treatment, the body weights of the rats in the prednisolone group were 7% less than those of the rats in the control group. The body weights of the rats in the prednisolone group decreased by 2% to 3% daily throughout the remainder of the drug treatment protocol. Thus, by the end of the 5-day drug treatment, the body weights of the rats in the prednisolone group were 15% less than those of the rats in the control group.
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Contractile Properties
Contractile properties of both groups are summarized in Table 2. Due to technical difficulties at the time of data collection, we can only report contractile data on 13 control group animals and 11 prednisolone group animals. Maximal specific isometric tetanic tension was lower in the prednisolone group as compared with the control group (18.6±0.7 N·cm–2 versus 21.4±0.7 N·cm–2). There were no differences in twitch properties (Pt, TPT,
RT) between the 2 groups.
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| Discussion |
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TopAbstractIntroductionMethodResultsDiscussionConclusionReferences |
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The validity of the twitch properties data is questionable because statistical power calculations indicate that the sample size was too small to determine whether a meaningful difference existed between these 2 groups. We believe that a power of approximately 0.80 or greater indicates that a true difference existed. Power analyses for the twitch data produced values ranging from approximately 0.04 to approximately 0.10, thus indicating an inability to detect a difference. These data were collected to supplement the Po data. Thus, we believe these data are less important to the overall question of this study. A power analysis, however, revealed a power of 0.80 to detect a difference in Po between the 2 groups at the .05 level. Therefore, we believe that the Po data indicate that a true difference existed between the 2 groups and that the magnitude of this difference might have been greater with a larger sample size.
Our data show that short-term, high doses of prednisolone result in a 15% loss of body weight. Several studies have used pair-fed controls to test the notion that decreases in body and muscle weights are due to a reduction in caloric intake. The results of studies by Sasson et al4 and Van Balkom et al,6 however, suggest that glucocorticoids have a detrimental effect on body and muscle weights that cannot be accounted for by a caloric deficit. In both of these studies, glucocorticoid-treated animals lost more weight than did pair-fed controls, leading the authors to suggest that administration of glucocorticoids has a catabolic effect on muscle independent of atrophy due to decreased caloric intake.4,6 Furthermore, the results of studies by Moore et al2 and Gardiner et al20 showed that pair-fed control rats actually gained weight during the course of the studies, whereas the glucocorticoid-treated animals lost weight. Because we did not measure caloric intake in our study, we acknowledge that a caloric deficit may have played a role in the weight loss we observed in the glucocorticoid-treated animals. Based on the evidence of previous investigators,2,4,6,20 however, we believe that the decrease in body weight observed in our study was primarily the result of the effects of glucocorticoids.
The study of Nava et al14 is most similar to our study in terms of treatment duration. Nava and colleagues, however, used a drug dosage (ie, 80 mg/kg/d) that was much greater than our dosage (ie, 5 mg/kg/d). They reported a 20% decrement in body weight after 5 days of glucocorticoid treatment. Comparison with other studies is difficult due to differences in type of glucocorticoid administered (fluorinated versus nonfluorinated) and duration and dosage of treatment.
Both the diaphragm and the EDL muscle weights of the prednisolone group were less than those of the control group. There was no difference, however, in soleus muscle weight between the 2 groups. The degree of muscle atrophy was not assessed in individual muscle fibers, but these results indicate that it is likely that atrophy of both type IIx and type IIb fibers occurred. The EDL muscle contains a predominance of type IIb fibers, whereas a large proportion of the fibers of the diaphragm are type IIx fibers. Studies6,8,21 have shown that glucocorticoids cause a preferential atrophy of both type IIx and type IIb fibers. In regard to the soleus muscle, our results agree with data from previous studies2,4,21 that showed that the soleus muscle is not affected by glucocorticoids. These results are consistent with the notion that type I muscle fibers are generally resistant to the atrophic effects of glucocorticoids.
An important finding of this study was that the Po of the diaphragm in the prednisolone group was 13% less than that of the control group. These results are in agreement with those of other studies3,4,6,21 that have shown a decrease in Po of the diaphragm following administration of glucocorticoids. Other studies,2,8,14 however, have shown no decrease in diaphragm Po. These conflicting results, in our opinion, are due largely to differences in drug dosage and duration and type of glucocorticoid administered. The study that is most similar to ours is that of Nava et al.14 Despite using doses of glucocorticoids much greater than ours for the same treatment duration, they found no decrease in diaphragm Po. The mechanisms to explain this difference are unclear at this time.
A reduction in Po indicates that intrinsic changes occurred in the muscle fibers. These changes could be due to several factors. The first possibility is that a reduction in the myofibrillar protein density in the diaphragm may have occurred due to glucocorticoid administration. Kayali et al22 showed that corticosterone at a dosage of 10 mg/kg for 10 days resulted in a selective loss of myofibrillar proteins from the plantaris muscle. Such an alteration could result in a decrease in maximal isometric tetanic tension by reducing the number of crossbridges available to generate force. Further studies are necessary to confirm this possibility.
An alteration in calcium handling is another factor that may affect maximal isometric tetanic tension. A change in calcium handling would result in a modification in crossbridge cycling kinetics and could possibly affect tension development. The few studies examining the effects of glucocorticoids on calcium handling in skeletal muscle, however, demonstrated conflicting results.23,24 The results of our study appear to indicate that this glucocorticoid regimen does not affect the calcium-handling capabilities of treated muscle and support the notion that glucocorticoids directly affect the muscle fibers, as evidenced by the decrease in Po.
In our study, there were no differences in Pt, TPT, or
RT between the 2 groups, and this finding could have been due to our small sample. Mixed results regarding twitch characteristics have been reported. Several investigators reported no change in Pt in glucocorticoid-treated animals,7,8,21 a decrease in Pt in glucocorticoid-treated animals,2,4 or an increase in Pt.14 Similar differences exist when examining TPT and RT measurements. The study by Nava and colleagues14 is most similar to our study and, with a large sample size, their results indicate a prolongation of both TPT and RT. Again, comparison of studies is difficult considering the type and dosage of glucocorticoid used as well as the time over which the drug was administered.
We believe that the results observed in our study may be clinically relevant. A decrease in diaphragm muscle force may contribute to difficulty in weaning patients with cardiopulmonary dysfunction from mechanical ventilation. Acute diaphragm myopathy has been reported in people with asthma hospitalized with severe exacerbations of their disease and requiring high doses of intravenous glucocorticoids (
1,000 mg/d) for short periods of time.11–13 These patients had difficulty weaning themselves from the ventilator and, therefore, required an increased length of mechanical ventilation and intensive care unit stay.11 Caution should be used when applying these results because patients may have concomitant underlying medical problems such as hypoxemia, malnutrition, and hyperinflation that may also lead to diaphragmatic dysfunction. It is likely, however, that the negative effects of steroids are further enhanced in these patients. Furthermore, the activity level of these patients is decreased, which leads to the development of disuse atrophy. An increase in cytosolic glucocorticoid receptors has been reported in patients with disuse muscle atrophy.25 Shee26 hypothesized that steroid-induced changes in diaphragm function occur early due to inactivity of the diaphragm as a result of mechanical ventilation, especially when patients are medically paralyzed. Physical therapists and other health care providers should be cognizant of the role glucocorticoids may play in the development of diaphragm dysfunction in patients who are mechanically ventilated, especially those who are medically paralyzed.
There are several limitations to our study. We did not examine the endurance characteristics of the rat diaphragm. Although diaphragm force is important, endurance characteristics are also critical to successful weaning from mechanical ventilation. Future studies should address the implications of glucocorticoids on the endurance of the diaphragm. Furthermore, we did not examine recovery from the morphological and contrac-tile dysfunction we observed in this study. Dekhuijzen et al27 examined the recovery from glucocorticoid-induced dysfunction in rats treated with low doses of glucocorticoids for 4 months and found that many of the contractile characteristics had returned to control levels 2 months following discontinuation of glucocorticoids. The animals, however, continued to display mild type IIb fiber atrophy, and the authors concluded that recovery from steroid-induced muscle atrophy is a prolonged process. We examined the effects of high doses of glucocorticoids for shorter periods of time, and it is unknown how quickly reversal of these muscle changes would occur following cessation of glucocorticoids.
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TopAbstractIntroductionMethodResultsDiscussionConclusionReferences |
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| Footnotes |
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This project was approved by the University of Florida Institutional Animal Care and Use Committee.
A portion of these results were presented at the Experimental Biology Meeting, New Orleans, La, April 1997.
This study was supported by a grant from the Foundation for Physical Therapy.
* Rohm & Haas Co, Independence Mall W, Philadelphia, PA 19105. 
Cambridge Technology Inc, 109 Smith Pl, Cambridge, MA 02138.
Grass Instruments, Div of Astro-Med Inc, 600 E Greenwich Ave, West Warwick, RI 02893.
GW Instruments, 35 Medford St, Somerville, MA 02143.
|| Abacus Concepts Inc, 1918 Bonita Ave, Berkeley, CA 94704.
# Apple Computer Inc, 1 Infinite Loop, Cupertino, CA 95014.
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  J. M Eason, S. L Dodd, and S. K Powers Use of Anabolic Steroids to Attenuate the Effects Glucocorticoids on the Rat Diaphragm Physical Therapy, January 1, 2003; 83(1): 29 - 36. [Abstract] [Full Text] [PDF]
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