Weight-bearing effects on skeletal muscle during and after simulated bed rest

541

Weight-Bearing Effects on Skeletal Muscle During and After Simulated Bed Rest

Marybeth Brown, PhD, Eileen M. Hasser, PhD

ABSTRACT. Brown M, Hasser EM. Weight-bearing effects on skeletal muscle during and after simulated bed rest. Arch Phys Med Rehabil 1995;76:541-6. • The detrimental consequences of bed rest include a rapid loss of muscle mass and strength. Yet, the utility of treatment to offset the effects of bed rest has not been well established. It was the purpose of this study to examine the effects of therapeutic intervention on simulated bed rest in rats. Simulated bed rest was accomplished by unweighting the hindlimbs of rats for 2 weeks via a suspension apparatus attached to the tail and midriff. Weight- bearing (eg, standing, walking) effects on hindlimb unweighting (HLU) were investigated under three conditions: (1) 1 hour of weight bearing per day during HLU; (2) a week of natural cage recovery after HLU; and (3) a combination of 1 hour of weight bearing per day during HLU and a week of recovery after HLU. Muscle contractile function and fiber atrophy were examined in the soleus (SOL), a postural muscle, and the extensor digitorum longus (EDL), a nonpostural muscle. The unweighted SOL showed a 37% loss in wet weight and a 61% decline in peak tetanic tension (P0). The SOL in rats allowed 1 hour of weight bearing per day lost 22% of its mass and 38% of its P0, a 38% attenuation. One week of cage recovery after HLU resulted in a SOL wet weight that was 26% less and Po that was 42% less than controls. Animals that received the combination of 1 hour of weight bearing per day and 1 week of cage activity nearly recovered muscle mass, but P0 still was 19% less than controls. The EDL was much less affected by HLU than the SOL. Results strongly support early intervention, particularly weight bearing, during a period of bed rest to modify the decline in strength and muscle mass in postural muscles such as the SOL. Further, results suggest that simply sending a patient home from the hospital to recover from bed rest delays return of strength unnecessarily. © 1995 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation

Bed rest in humans results in a rapid and significant loss of skeletal muscle strength, particularly in postural muscles such as the quadriceps and soleus. 1'2 An animal model of bed rest, hindlimb unweighting (HLU), also results in a rapid decline in muscle mass and contractile tension in those muscles used primarily for weight bearing. The soleus muscle, in particular, has been studied extensively. 3-11

A number of interventions or countermeasures employed during HLU have modified the rapid decline in rat skeletal muscle. T M The loss of soleus (SOL) and gastrocnemius muscle mass and fiber area is attenuated by centrifugation, la weight lifting, 14 and treadmill walking for 10 minutes every 6 hours during the unweighting period. 16'17 With the possible exception of weight lifting, these interventions are not very feasible within a rehabilitation setting. Consequently, it was the intent of this study to examine the utility of the therapeutic intervention that is most likely to be employed for a bed-rested patient: normal weight bearing (eg, standing and walking). Contractile function was the primary variable of concern because most previous studies have assessed the

From the School of Medicine (Dr. Brown), Washington University, St. Louis, MO; and Veterinary Biomedical Sciences (Dr. Hasser), School of Veterinary Medicine, University of Missouri, Columbia, MO.

Supported in part by grants BRSG RR05389, NIH HL43700, and NIA AG00585. Submitted for publication June 23, 1994. Accepted in revised form January 24,

1995. No commercial party having a direct financial interest in the results of the research

supporting this article has or will confer a benefit upon the authors or upon any organization with which the authors are associated.

Reprint requests to Marybeth Brown, PhD, PT, Program in Physical Therapy, Box 8052, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110.

© 1995 by the American Congress of Rehabilitation Medicine and the American Academy of Physical Medicine and Rehabilitation

0003-9993/95/7606-3121 $3.00/0

utility of intervention on the basis of muscle weight and fiber area only. v'13'14"18 Of primary interest in this investigation was to compare the efficacy of intervention during simulated bed rest (HLU), after HLU, and after a combination of intervention during and after HLU. It was hypothesized that a combination of weight-bearing intervention during and after HLU would be the most effective for attenuating loss of muscle mass and contractile tension.

The reported consequences of unweighting on the nonpostural extensor digitorum longus (EDL) have been variable. This muscle has been reported to hypertrophy ~9 and atrophy 2° and to be unaffected by unweighting. 6 Whether unweighting affects mechanical properties of the EDL is not clear. Thus, a secondary aim of this investigation was to examine HLU and intervention effects on the EDL.

M E T H O D S

A n i m a l s

Adult (6 to 7 months) male Fisher 344 rats weighing between 330 and 420g were obtained from Sasco Inc a and assigned randomly to a control, hindlimb unweighted, or one of three weight-bearing (treatment) groups. The control group (CON, n = 9) was studied for the duration of the study and weighed weekly. The hindlimb unweighted or suspended group (HS, n = 11) was placed in a modified tail suspension apparatus 6 for 2 weeks. Treatment animals were placed into one of three groups. The first group, hindlimb suspended-down (HS-D, n = 8) was HLU for 2 weeks, but rats were allowed 1 hour of weight bearing per day. To accomplish this, all hindlimb unweighted animals were taken from the suspension apparatus and placed in a 3x4ft box

Arch Phys Med Rehabil Vol 76, June 1995

542 WEIGHT-BEARING EFFECTS ON BED-RESTED MUSCLE, Brown

where they stood in place or walked. Walking was encour- aged by repeatedly placing rats into a corner as far away from their companions as the box would allow. The second group, hindlimb suspended-recover (HS-R, n = 9), was unweighted for 2 weeks followed by 1 week of normal cage recovery. Recovery was believed to be somewhat analagous to being discharged directly home from a hospital setting. The third treatment group, hindlimb suspended-down-recover (HS-DR, n = 9) was hindlimb unweighted for 2 weeks, but animals were allowed ! hour of weight bearing per day during the period of unweighting and 1 week of normal cage activity after the 2 weeks of unweighting. Pilot data for control rats that wore the suspension apparatus for 2 weeks but were not unweighted showed that differences between animals that wore the suspension apparatus (n = 8) and those that did not were insignificant.

Rats were housed one per cage (18xl8xl6in) in an approved facility where temperature and light/dark cycles were controlled (light 6AM to 6PM), and food and water were available ad libitum. All protocols were performed in accor- dance with the Guide for the Care and Use of Laboratory Animals as approved by the Council of the American Physio- logic Society and by the Animal Use Review Boards of both universities conducting these studies.

After a 3-day period of familiarization to the unweighting procedure for 1 hour per day, rats were suspended for 14 days using a modification of the procedure described by Jaspers and Tischler. 6 To accomplish the unweighting, two small casts were applied, one to the tail and another around the thorax to eliminate excessive lumbar lordosis. Wires from the tail and body casts were fed through a leader that was attached to a swivel hook at the top of the cage, thus allowing animals 360 ° of rotation. Rats moved freely about the entire cage using their forelimbs.

Contractile Properties Rats were deeply anesthetized with an intraperitoneal in-

jection of sodium pentobarbital (50mg/kg) with 5.0rag injec- tions administered approximately every 30 minutes to main- tain anesthesia. SOL and EDL muscles were surgically exposed. The distal tendons of each muscle were dissected free, tied with 2.0 silk, and attached to a Grass b force transducer. The tibial and peroneal nerves were stimulated directly.

Before contractile properties were obtained, animals and muscles were allowed to thermoequilibrate for 30 minutes. Body temperature was maintained by keeping the anesthetized rat on a heating pad. A rectal thermometer was used to monitor body temperature. Muscles were exposed mini- mally to room air, and the exposed portion was bathed continuously with 37-degree mineral oil.

A length-tension curve was performed, muscles then were adjusted to their optimal lengths (Lo), and peak isometric twitch tension was obtained by a supramaximal 0.5-ms square wave pulse (Grass Instruments $48c). Peak twitch tension (Pt), time to peak twitch tension (TTP), and half relaxation time (twitch) (½RT) were determined. Peak tetanic tension (Po) was elicited by a 0.5-ms supramaximal pulse at 100Hz (450ms) for the SOL and 150Hz (250ms) for the EDL. SOL and EDL fatigue characteristics were assessed by

a 15-minute bout of repeated tetanic contractions as follows: 250ms trains (stimulation rate 100Hz) delivered at 0.75Hz for the SOL and 250ms trains at 0.5Hz (stimulation rate 150Hz) for the EDL. During stimulation, the leg was rigidly immobilized.

After muscle contractile characteristics were obtained, rats were given an overdose of sodium pentobarbital, and SOL and EDL muscles were dissected out, weighed, and prepared for histochemistry.

Histochemistry SOL and EDL muscles were fixed at in situ length, embed-

ded in OCT, frozen in liquid nitrogen, and stored at -80°C until analysis. Serial transverse sections of 10# were cut on a cryostat and stained for myofibrillar adenosine triphospha- tase (ATPase) 21 for determination of fiber types. Acid (pH 4.3, 4.65) and alkaline (pH 9.4) preincubations for distin- guishing fiber types I, IIa, IIb, and IIc were performed for 5 minutes before incubation in a buffer containing 18mmol/ L CaCI2 and 2.7mmol/L ATP at 37°C for 20 minutes. NADH-TR (nicotinamide adenine dinucleotide hydro- genase-tetrazolium reductase) 22 as an index of oxidative activity, alkaline phosphatase for determination of capillary density, and hematoxylin and eosin (H&E) for routine mor- phology were performed on alternate sections.

Fiber type distribution was determined from ATPase- stained sections with fibers designated as follows: major fiber types I, Ha, and IIb, and transitional fibers, IIc. A minimum of 1,000 fibers was counted for determination of fiber-type distribution in each muscle. ATPase-stained sections also were used for determination of fiber area. Areas of 100 fibers of each fiber type, if present, were calculated using a computerized planimetry system. The number of capillaries surrounding a minimum of 125 fibers was determined from alkaline-phosphatase-stained sections.

Data Management Results were analyzed using a one-way analysis of vari-

ance (ANOVA). If significance was achieved (p < .05), pairwise comparisons were performed using Tukey's HSD method.

RESULTS

Muscle Weight, Fiber Area Muscle weights are presented in table 1. After 14 days of

HLU, SOL mass was 37% less than CON values (p < .05). Rats weight bearing 1 hour per day (HS-D) and those allowed 1 week of normal cage recovery (HS-R) after HLU had an attenuated decline in SOL wet weight. The hour a day of weight bearing (HS-D) and the week of normal cage recovery (HS-R) resulted in a 22% and 26% loss in muscle weight, respectively.

The decline in SOL muscle wet weight was owing almost entirely to a reduction in fiber area (table 1). A comparable amount of atrophy was observed in SOL fiber types I and IIa in all unweighted groups. A complete recovery in fiber cross-sectional area occurred in those animals (HS-DR) permitted weight bearing during and after the 14 days of HLU.

Absolute EDL muscle mass decreased 15% with hindlimb


WEIGHT-BEARING EFFECTS ON BED-RESTED MUSCLE, Brown

Table 1: SOL and EDL Wet Weights and SOL Fiber Area (Mean ± SEM)

543

Groups (n) CON (9) HS (11) HS-D (9) HS-R (8) HS-DR (9)

SOL wet wt. (rag) 125.0 ± 8.2 79.2 ± 4.9* 97.4 _+ 3.6* 92.2 + 5.8* 115.1 _+ 8.1 SOL fiber area (~m 2)

Type I 2,597 ± 128 1,462 +_ 113" 1,894 _+ 65* 1,975 ± 103" 2,517 _+ 181 Type IIa 2,227 ± 123 1,199 +_ 82 1,470 ± 93 1,733 ± 109 2,257 _+ 156

EDL wet wt. (mg) 135.6 ± 3.4 114.9 ± 4.4* 114.3 ± 2.2" 113.5 + 2.4* 132.7 _+ 6.7

* p = .0001 when compared with CON.

unweighting (table 1), which was accounted for entirely by a reduction in type II fiber area (data not shown). The weight- beating intervention had no affect on the mass of this muscle.

Contractile Tension Summaries of the changes in contractile properties are

presented in table 2. A 58% decrease in Pt (from 4l to 17g) and a 65% decline in Po (from 161 to 57g) occurred in HS rats as compared with controls (p < .001). In animals that were permitted 1 hour of weight bearing per day (HS-D), Pt and Po were reduced by 37% and 38%, respectively. One week of recovery (HS-R group) brought SOL twitch and tetanic tension to within 41% of control values. For rats that had 1 hour of cage activity per day and 1 week of recovery (HS-DR), Pt was recovered but Po remained 19% below control values. Thus, fiber area and muscle mass recovered in HS-DR animals but peak tetanic tension did not. When values for twitch and tetanic tension were expressed per gram of muscle wet weight, significant differences persisted for HS rats only (table 2).

For the EDL, Pt did not change with unweighting or treatment, whereas Po was significantly increased in the HS-DR group (table 3).

Fiber-Type Distribution SOL fiber-type distribution did not change with un-

weighting or the interventions employed (table 4). A greater percentage of transitional type IIc fibers was noted in the SOL of HS rats, but differences were not statistically significant. A trend toward a higher percentage of type IIa and fewer IIb fibers was noted in the EDL of unweighted animals, but differences did not reach statistical significance.

Contraction Times TTP for the SOL was quite variable, ranging from 32 to

50ms. Half relaxation times (1RT) also were variable (range 47 to 67ms) but significant differences were not apparent. The only difference among the groups was TTP for the HS- DR animals (32ms), which was significantly less than TTP for HS-R rats (50ms). EDL contraction times ranged from 20 to 27ms (TTP) and from 18 to 22ms (1RT), but differences among groups were not significant.

Fatigue Protocol The decline in Po after 15 minutes of tetanic stimulation

ranged from 5% to 10% for the SOL and from 59% to 64% for the EDL, with none of the differences between groups reaching statistical significance (table 5).

Capillary/Fiber Ratios Differences in the number of capillaries per fiber were

not apparent when comparisons were made among CON, HS, or treatment groups. Cappillary/fiber ratios ranged from 3.9 to 4.3 (SOL only).

Histology A notable increase in intramuscular fat and connective

tissue was observed in the SOL of HS animals, less so in SOL muscles from rats that had been permitted weight bearing. Also apparent in HS rats was a rounding of the muscle cell nuclei and an increase in the size of muscle fiber nuclei. Between fasciculi were numerous other cells, some with a fibroblastlike appearance, but some of these nuclei may have been satellite cells or macrophages. Approximately 5% of the muscle fbers in the suspended SOL were degenerating, as evidenced by pyknotic nuclei and absence of cytoplasmic staining, or regenerating, with central nuclei. In animals permitted weight bearing during unweighting and 1 week of recovery (HS-DR), few changes were noted. HS-D and HS- R rats did show some intramuscular fat and connective tissue and cellular infiltrate but not to the degree that HS rats did. The only change finding in the EDL was muscle fiber atrophy.

DISCUSSION

Results from this study strongly support the importance of weight bearing on the SOL muscle during HLU. Results indicate that the earlier that weight-bearing intervention is prescribed, the more rapid the recovery. Findings also suggest that patients sent home to recovery "natural ly" from hospitalization are likely to experience an unnecessary delay in return of strength. Results indicate that early intervention during the period of bed rest should provide a patient with a a higher level of strength and function to build on during recovery.

Table 2: Pt and Po/MUscle Weight for SOL (Mean ± SEM)

Groups (n) CON (9) HS (11) HS-D (9) HS-R (8) HS-DR (9) Pt/mwt 353 + 57 216 ± 21" 264 ± 23 265 + 28 378 ± 52 Po/mWt 1,349 ± 158 715 + 59* 861 + 142 1,015 ± 91 1,141 ± 85

Abbreviation: mwt, muscle wet weight. * p < .05 when compared with CON and HS-DR.



Table 3: Peak Twitch (Pt) and Peak Tetanic Tension (Po) for EDL (Mean _+ SEM)


Pt (g) 44.0 _+ 2.6 54.0 _+ 4.8 50.1 _+ 3.1 55.6 -+ 3.1 51.7 _+ 3.3 Po (g) 244.6 _+ 12.9 251.3 _+ 7.6 245.3 _+ 6.7 256.3 _+ 19.2 320.9 _+ 21.0"

* Po for HS-DR rats was significantly higher than values for all other groups (p = .005).

Rats in this study were young, and compromise in physical function after HLU was confined to climbing. The authors have since hindlimb unweighted old rats (28 and 36 months), and the loss of function in these animals that already had muscle strength compromised by age-related change was enormous. After HLU, without intervention, most of the older animals were unable to ambulate but a few feet, and their climbing ability was lost (unpublished). Old rats given just an hour a day of weight-bearing activity did not show an inability to ambulate or climb. Thus, for older adults, all of whom require a higher proportion of available strength just to accomplish activities of daily living, early intervention during bed rest is probably critical.

Rats allowed 1 hour of weight bearing a day (HS-D) had a comparable amount of peak contractile tension, muscle mass, and fiber area as rats permitted a full week of recovery (HS-R). Rats in the HS-D group had a total of 14 hours of weight bearing compared with 168 hours of potential weight bearing for the HS-R animals, yet both had similar SOL muscle profiles. Thus, therapeutic intervention during a period of weightlessness is far more beneficial than waiting until after a period of unweighting or bed rest. No intervention at all during 2 weeks of bed rest may delay recovery by as much as 1 week. This is assuming that conditions for recovery are optimal; the patient is young and disease free. For the older adult, the potential for a spontaneous and timely recovery might be quite poor. Given the likelihood of disease, and preexisting age-related decline in muscle, full recovery, without intervention, might not occur.

Weight beating was chosen as the intervention because of its reasonable application to bed-rested humans, particularly older adults. Standing and walking do not appear to attenuate the decline in SOL muscle mass as effectively as centrifugation 12 or weight lifting ~4 but it does have the advantage of being achieved readily. Combining more than one form of intervention in future studies may accomplish the desired end of preventing the decline in SOL (and other weight- bearing muscles) function. Results from this study also suggest that to attenuate skeletal muscle decline, patients should be stood at bedside as many times a day as possible, rather than being placed in a bedside chair for 1 or 2 hours.

The decline in SOL muscle mass and fiber area with HLU was comparable with that reported by others. 2°'23"24 The attenuated decline in muscle wet weight and fiber area ( - 5 0 % ) in the rats that were returned to normal weight bearing for

1 hour per day (HS-D group) was similar to what was observed by Hauschka and others who incorporated treadmill walking as a countermeasure during 7 days of HLU.13'17 Rats in this study did not walk continuously and spent most of their time standing. Thus, it would appear that the duration of weight-bearing activity rather than intensity of weight- bearing activity is the more important determinant in the maintenance of muscle mass as suggested by D'Aunno and colleagues. 12

Results indicate that intervention during hindlimb unweighting, as expected, promotes a more rapid recovery. Assuming a linear rate of decline in Po, HS animals lost tetanic tension at an estimated rate of 7g per d; those in the HS-D group lost tetanic tension at an estimated rate of 4.8g per day, an attenuated loss of ~30%. Rats allowed 1 week of spontaneous recovery regained an average of 6g of tension per day. If a linear rate of gain in tetanic tension occurred during spontaneous recovery, HS-R animals would have re- quired 17 days to reach control values. HS-DR rats, those allowed 1 hour of weight bearing per day and 1 week of spontaneous recovery, would have regained pre-HLU values for tetanic tension by the 1 lth day. Thus, weight-bearing intervention can facilitate the return to pre-HLU values. Re- gardless of whether the loss and recovery in contractile tension is linear, it is apparent that the decline in SOL Po with HLU occurs more quickly than recovery and that early intervention is important.

As observed by others, 8'16'z5 the percent decline in Pt and Po in HS rats was greater than the percent decline in muscle wet weight and fiber area. This finding further supports the suggestion that the processes responsible for the maintenance of contractile proteins are affected to a greater degree by unweighting than the processes governing muscle mass. 1°'26 Even in HS-D, HS-R, and HS-DR rats, the loss in Po exceeded the loss in muscle mass and fiber area. The dissociation between the decline in muscle mass and muscle contractile tension was attenuated but not eliminated by weight bearing, particularly in HS-SR animals.

The finding in this study of intramuscular fat and connective tissue in SOL muscles of unweighted animals suggests that muscle weight may have been influenced by the presence of this material. The presence of new and necrotic fibers in the SOL appears to indicate that HLU has a disturbing effect on this muscle.

Numerous investigators have reported a change in SOL

Table 4: Fiber-Type Distribution for SOL (Mean __ SEM)


% Type I 87.6 _+ 1.1 84.5 _+ 2.3 84.3 _+ 3.4 83.8 -4- 1.3 88.9 -4- 1.7 % Type IIa 10.9 _+ 1.1 10.8 _+ 2.2 12.2 _+ 3.2 I0.1 _+ 1.5 9.3 -+ 1.9 % Type IIc 1.5 _+ 0.2 4.7 _+ 1.3 3.5 _+ 0.5 6.1 _+ 1.4 1.8 + 0.2

NOTE. No significant differences.


WEIGHT-BEARING EFFECTS ON BED-RESTED MUSCLE, Brown 545

Table 5: % Decl ine in Po With 15 -Minute Fatigue Test (Mean _+ SEM)

Groups CON HS HS-D HS-R HS-DR (n) (9) (11) (9) (8) (9)

SOL 8 + 3 5_+2 8 + 3 7_+3 10-+2 EDL 64 -+ 2 59 + 3 59 -+ 3 56 _+ 2 63 -+ 2

NOTE. Differences between groups were not significant.

fiber-type distribution with H L U . 1l'20"23'24 Little change in SOL fiber-type distribution occurred in this study, which supports the possibility that changes in SOL fiber-type distribution and contraction times with unweighting may be owing to the age of the animals used or to differences between species, w'27 However, the trend toward higher percentages of transitional or IIc fibers among HS rats does suggest that change in fiber types may have been occurring. Takahashi and colleagues, for example, did not find an increase in fast myosin heavy chain isoforms in the 14-day suspended SOL but did find significant differences after 21 days of suspension. 28 The lack of decline in percent SOL type I fibers in this study was reflected in measures of contraction time and fatigue, which also did not change.

Curiously, intramuscular fat and connective tissue did not appear to have much affect on TTP or ½RT in unweighted rats. The longest contraction time was found in the HS-R group, which did not exhibit a noticeable increase in fat and connective tissue.

One week of recovery was not adequate in this study to fully restore muscle wet weight, fiber area, or peak tetanic tension after 14 days of unweighting. Even with 1 hour of weight bearing a day and 1 week of recovery, contractile function had not fully returned to control values. Bed-rested humans also do not recover contractile function as quickly as they lost it, particularly in weight-bearing muscles such as the S OL) In humans, the SOL has more than twice the physiological cross-sectional area of the gastrocnemius and is a key muscle in gait. Thus, an exaggerated loss of SOL muscle mass and strength in humans has greater functional consequence than it does in rats. Findings from this study highlight the importance of intervention during a period of unweighting or bed rest and further emphasizes the need for more effective countermeasures.

SUMMARY

Weight bearing as a therapeutic intervention during and after simulated bed rest significantly attenuated the decline in SOL muscle mass and contractile function. Values for SOL wet weight, fiber area, and Po for animals that allowed 1 hour of weight bearing per day (14 hours total) were comparable with values for rats given a full week of cage recov- cry (168 potential hours of weight bearing). An hour of weight bearing per day and 1 week of recovery restored muscle mass and fiber area but did not result in full recovery of SOL contractile function. The EDL was little affected by HLU or the weight bearing measures employed.

Acknowledgment:Sincere appreciation to Sarah Friskey for superb tech- nical assistance.

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Suppliers a. Sasco Inc, Rural Route 1, O'Fallon, MO. b. Grass force transducer; Grass Instrument Co., PO Box 576, 101 Old

Colony Avenue, Quincy, MA 02169. c. Square wave pulse; Grass Instrument Co., PO Box 576, 101 Old Colony

Avenue, Quincy, MA 02169.

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Weight-bearing effects on skeletal muscle during and after simulated bed rest