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Short-time recovery skeletal muscle from dexamethasone-induced atrophy and weakness in old female rats

Open AccessPublished:October 25, 2022DOI:https://doi.org/10.1016/j.clinbiomech.2022.105808

      Highlights

      • Biomechanical parameters (tone, stiffness, elasticity, relaxation time, creep) were examined.
      • Steroid-induced myopathy and following recovery has impact on biomechanical parameters.
      • Mild therapeutic exercise did not improve recovery from steroid myopathy.
      • The myometric monitoring of muscle allows to assess the recovery from steroid myopathy.

      Abstract

      Background

      Several pathological conditions (atrophy, dystrophy, spasticity, inflammation) can change muscle biomechanical parameters. Our previous works have shown that dexamethasone treatment changes skeletal muscle tone, stiffness, elasticity. Exercise training may oppose the side effects observed during dexamethasone treatment. The purpose of this study was to examine the changes in biomechanical parameters (tone, stiffness, elasticity) of skeletal muscle occurring during dexamethasone treatment and subsequent short-time recovery from glucocorticoid-induced muscle atrophy and weakness, as well as the effect of mild therapeutic exercise.

      Methods

      17 old female rats, aged 22 months were used in this study. The hand-held and non-invasive device (MyotonPRO, Myoton Ltd., Tallinn, Estonia) was used to study changes in biomechanical properties of muscle. Additionally, body and muscle mass, hind limb grip strength were assessed.

      Findings

      Results showed that dexamethasone treatment alters muscle tone, stiffness and elasticity. During 20-day recovery period all measured parameters gradually improved towards the average baseline, however, remaining significantly lower than these values. The body and muscle mass, hind limb grip strength of the rats decreased considerably in the groups that received glucocorticoids. After 20 days of recovery, hind limb grip strength of the animals was slightly lower than the baseline value and mild therapeutic exercise had a slight but not significant effect on hind limb grip strength. Biomechanical parameters improved during the recovery period, but only dynamic stiffness and decrement retuned to baseline value.

      Interpretation

      The study results show that monitoring muscle biomechanical parameters allows to assess the recovery of atrophied muscle from steroid myopathy.

      Keywords

      1. Introduction

      Skeletal muscles account for approximately half of the total body weight of adults and are the organs involved in motion and posture maintenance. Muscle atrophy occurs in various catabolic conditions and causes loss of muscle mass, resulting in muscle weakness and fatigue (
      • Cohen S.
      • Nathan J.
      • Goldberg A.
      Muscle wasting in disease: molecular mechanisms and promising therapies.
      ;
      • Powers S.K.
      • Lynch G.S.
      • Murphy K.T.
      • Reid M.B.
      • Zijdewind I.
      Disease-induced skeletal muscle atrophy and fatigue.
      ).
      Several studies have shown that dexamethasone (DEX) treatment promotes the degradation of proteins related to muscle mass (
      • Desler M.M.
      • Jones S.J.
      • Smith C.W.
      • Woods T.L.
      Effects of dexamethasone and anabolic agents on proliferation and protein synthesis and degradation in C2C12 myogenic cells.
      ;
      • Yang H.
      • Wei W.
      • Menconi M.
      • Hasselgren P.O.
      Dexamethasone-induced protein degradation in cultured myotubes is p300/HAT dependent.
      ;
      • Zhao W.
      • Qin W.
      • Pan J.
      • Wu Y.
      • Bauman W.A.
      • Cardozo C.
      Dependence of dexamethasone-induced Akt/FOXO1 signaling, upregulation of MAFbx, and protein catabolism upon the glucocorticoid receptor.
      ). DEX is a synthetic glucocorticoid (GC) and a well-known anti-inflammatory drug that is indicated in several medical conditions. Its long-term use, dosage administration route, and type lead to progressive negative effects comprising in the whole-body physiology that affect several organ systems, including musculoskeletal systems (
      • Schäcke H.
      • Döcke W.D.
      • Asadullah K.
      Mechanisms involved in the side effects of glucocorticoids.
      ;
      • Schakman O.
      • Kalista S.
      • Barbé C.
      • Loumaye A.
      • Thissen J.P.
      Glucocorticoid-induced skeletal muscle atrophy.
      ). These side effects include GC-induced muscle atrophy that is one of most common drug-induced myopathies in humans and animals (
      • Seene T.
      • Kaasik P.
      • Pehme A.
      • Alev K.
      • Riso E.-M.
      The effect of glucocorticoids on the myosin heavy chain isoforms’ turnover in skeletal muscle.
      ;
      • Attaix D.
      • Mosoni L.
      • Dardevet D.
      • Combaret L.
      • Mirand P.P.
      • Grizard J.
      Altered responses in skeletal muscle protein turnover during aging in anabolic and catabolic periods.
      ;
      • Gupta A.
      • Gupta Y.
      Glucocorticoid-induced myopathy: pathophysiology, diagnosis, and treatment Indian.
      ) characterized by preferential atrophy of fast-twitch glycolytic muscle fibres (
      • Schakman O.
      • Gilson H.
      • Thissen J.P.
      Mechanisms of glucocorticoid induced myopathy.
      ), thickening of sarcolemma and capillary basement membranes (
      • Djaldetti M.
      • Gafter U.
      • Fishman P.
      Ultrastructural observations in myopathy complicating Cushing’s disease.
      ), intramuscular fat deposition (
      • Prineas J.
      • Hall R.
      • Barwick D.
      • Watson A.J.
      Myopathy associated with pigmentation following adrenalectomy for Cushing’s syndrome.
      ), mitochondrial dysfunction and oxidative damage (
      • Djaldetti M.
      • Gafter U.
      • Fishman P.
      Ultrastructural observations in myopathy complicating Cushing’s disease.
      ).
      DEX-induced muscle atrophy can be inhibited in several ways. Most studies on the topic have concluded that muscle atrophy can be prevented by increasing protein synthesis through the Akt/mTOR pathway and by suppressing the protein levels of Atrogin-1 and MuRF1 (specific markers of muscle atrophy) (
      • Castillero E.
      • Alamdari N.
      • Lecker S.H.
      • Hasselgren P.-O.
      Suppression of atrogin-1 and MuRF1 prevents dexamethasone-induced atrophy of cultured myotubes.
      ;
      • Clarke B.A.
      • Drujan D.
      • Willis M.S.
      • Murphy L.O.
      • Corpina R.A.
      • Burova E.
      • Rakhilin S.V.
      • Stitt T.N.
      • Patterson C.
      • Latres E.
      • Glass D.J.
      The E3 ligase MuRF1 degrades myosin heavy chain protein in dexamethasone-treated skeletal muscle.
      ;
      • Zhao W.
      • Qin W.
      • Pan J.
      • Wu Y.
      • Bauman W.A.
      • Cardozo C.
      Dependence of dexamethasone-induced Akt/FOXO1 signaling, upregulation of MAFbx, and protein catabolism upon the glucocorticoid receptor.
      ). GC-induced myopathy is reversible, after cessation of treatment the recovery of muscle mass and force occurs but may be protracted. The recovery from GC-induced weakness may take from months to one year (
      • Bodine S.C.
      • Furlow J.D.
      Glucocorticoids and skeletal muscle.
      ;
      • Owczarek J.
      • Jasińska M.
      • Orszulak-Michalak D.
      Drug-induced myopathies. An overview of the possible mechanisms.
      ;
      • Shee C.D.
      Risk factors for hydrocortisone myopathy in acute severe asthma.
      ).
      Since known pharmacotherapies cannot accelerate recovery from steroid-induced myopathy, the only proven way to resolve the respective symptoms is to stop taking the drug. Exercise therapy, taking into account baseline functional status, is effective in attenuating muscle atrophy and recommended to treat glucocorticoid-induced myopathy (
      • LaPier T.K.
      Glucocorticoid-induced muscle atrophy. The role of exercise in treatment and prevention.
      ).
      Exercise is considered to be an effective intervention for treatment and prevention of several diseases. Exercise training may oppose the side effects observed during DEX treatment (
      • Barel M.
      • Perez O.A.
      • Giozzet V.A.
      • Rafacho A.
      • Bosqueiro J.R.
      • do Amaral S.L.
      Exercise training prevents hyperinsulinemia, muscular glycogen loss and muscle atrophy induced by dexamethasone treatment Eur.
      ;
      • Czerwinski S.M.
      • Kurowski T.G.
      • O’Neill T.M.
      • Hickson R.C.
      Initiating regular exercise protects against muscle atrophy from glucocorticoids.
      ;
      • Falduto M.T.
      • Czerwinski S.M.
      • Hickson R.C.
      Glucocorticoid-induced muscle atrophy prevention by exercise in fast-twitch fibers.
      ;
      • Nicastro H.
      • Gualano B.
      • Moraes W.M.
      • de Salles Painelli V.
      • da Luz C.R.
      • dos Santos Costa A.
      • de Salvi Guimaraes F.
      • Medeiros A.
      • Brum P.C.
      • Lancha Jr., A.H.
      Effects of creatine supplementation on muscle wasting and glucose homeostasis in rats treated with dexamethasone.
      ;
      • Seene T.
      • Kaasik P.
      Physical activity in prevention of glucocorticoid myopathy and sarcopenia in aging.
      ). There are currently no special rehabilitation programmes, whereas studies in rats are promising. Aerobic exercise programme can be effective in retarding GC-induced muscle atrophy or preventing the muscle mass reduction induced by GC (
      • Barel M.
      • Perez O.A.
      • Giozzet V.A.
      • Rafacho A.
      • Bosqueiro J.R.
      • do Amaral S.L.
      Exercise training prevents hyperinsulinemia, muscular glycogen loss and muscle atrophy induced by dexamethasone treatment Eur.
      ). Other studies have demonstrated that resistance training attenuates DEX-induced muscle atrophy via the mammalian target of rapamycin (mTOR) pathway and a small increase in the Muscle RING finger protein 1 (MuRF1) protein level (
      • Krug A.L.
      • Macedo A.G.
      • Zago A.S.
      • Rush J.W.
      • Santos C.F.
      • Amaral S.L.
      High-intensity resistance training attenuates dexamethasone-induced muscle atrophy.
      ).
      In rats, ladder exercise training is associated with muscle hypertrophy, changes in muscle fibre phenotype, and neuromuscular adaptations (
      • Carroll T.J.
      • Riek S.
      • Carson R.G.
      Neural adaptations to resistance training: Implications for movement control.
      ;
      • Hornberger Jr., T.A.
      • Farrar R.P.
      Physiological hypertrophy of the FHL muscle following 8 weeks of progressive resistance exercise in the rat.
      ).
      Biomechanical parameters of muscles can be used as markers for muscle health. Several pathologic conditions, such muscle atrophy, dystrophy (
      • Cornu C.
      • Goubel F.
      • Fardeau M.
      Muscle and joint elastic properties during elbow flexion in Duchenne muscular dystrophy.
      ), spasticity (
      • Fridén J.
      • Lieber R.L.
      Spastic muscle cells are shorter and stiffer than normal cells.
      ), inflammation (
      • Song Y.
      • Lee S.
      • Yoo D.H.
      • Jang K.S.
      • Bae J.
      Strain sonoelastography of inflammatory myopathies: comparison with clinical examination, magnetic resonance imaging and pathologic findings.
      ) can change biomechanical parameters.
      The myometer is a simple, reliable, non-invasive tool for objective measuring of biomechanical parameters of muscles (
      • Bizzini M.
      • Mannion A.F.
      Reliability of a new, hand-held device for assessing skeletal muscle stiffness.
      ). It has been shown in our previous works that muscle tone and biomechanical parameters, such as stiffness and elasticity are either weakened or reinforced by DEX treatment (
      • Alev K.
      • Vain A.
      • Aru M.
      • Pehme A.
      • Purge P.
      • Kaasik P.
      • Seene T.
      Glucocorticoid-induced changes in rat skeletal muscle biomechanical and viscoelastic properties: aspects of aging.
      ;
      • Aru M.
      • Alev K.
      • Gapeyeva H.
      • Vain A.
      • Puhke R.
      • Pehme A.
      • Kaasik P.
      • Selart A.
      • Seene T.
      Glucocorticoid-induced alterations in titin, nebulin, myosin heavy chain isoform content and viscoelastic properties of rat skeletal muscle.
      ). Non-invasive quantifying of biomechanical parameters of muscles after DEX treatment can provide information about recovery of the muscle.
      In the present study, hand-held non-invasive digital device (MyotonPRO) was used to measure biomechanical parameters of gastrocnemius muscle in rats before and after DEX treatment, and during recovery period with and without mild therapeutic exercise. More specifically, we examined the effect of mild therapeutic exercise (ladder climbing) on recovery from CG-induced myopathy.
      The objective of this study was to examine the changes occurring in short-time recovery of skeletal muscle from DEX-induced atrophy, weakness and effect of mild therapeutic exercise. We hypothesised that mild therapeutic exercise (low intensity resistance training) accelerates recovery from muscle atrophy.

      2. Methods

      2.1 Animals

      The animals used in this study were bred at the local animal house, using Wistar rat parents (Harlan Laboratories, the Netherlands). 17 old female rats, aged 22 months were used in this study. The rats were housed in group cages under controlled environmental conditions (room temperature 21 °C, 12 h light-dark cycle) and received food and water ad libitum. Animal care and all experimental protocols involving animals were in accordance with the European Directive 2010/63/EU and were approved by the Animal Experimentation Committee at the Estonian Ministry of Rural Affairs.

      2.2 Dexamethasone treatment and experimental groups

      The study consisted of two experimental periods, DEX administration and recovery. The DEX treatment was begun following a short acclimatization period with periodic body weight checks. After a short acclimatization period with periodic body weight checks, the DEX treatment was started. At this point the body weight of rats was 347–359 g.
      DEX doses 50 μg/ 100 g body mass (bm) were established according to the previously used protocol published by
      • Savary I.
      • Debras E.
      • Dardevet D.
      • Sornet C.
      • Capitan P.
      • Prugnaud J.
      • Mirand P.P.
      • Grizard J.
      Effect of glucocorticoid excess on skeletal muscle and heart protein synthesis in adult and old rats.
      who found that rats can withstand doses of DEX not exceeding 2.0 mg/kg bm/day for young or 0.5 mg/kg bm/day for old rats.
      DEX (Dexafort 3 mg/mL; International B.V. Boxmeer, the Netherlands) was diluted to 200 μg/mL with 0.15 M NaCl and administered intraperitoneally daily for 10 days, 50 μg/ 100 g bm. After 10 days of treatment, one group of rats (Dex, n = 5) were euthanized by excess of anaesthesia. The remaining animals (n = 12) were distributed into two recovery groups, the 10 days (REC 10, n = 4) and 20 days (REC 20, n = 8) recovery group, respectively.
      For investigating the effects of passive and active recovery from atrophy, the REC 20 group rats were further divided into passive (REC 20, n = 4) and active recovery (REC 20TEx, n = 4) subgroup. The active recovery group rats were subjected to mild therapeutic exercise described below, whereas animals of the passive recovery group stayed in their cage. After the recovery period rats were euthanized by excess of anaesthesia. On Fig. 1a are shown the details of the animal experiments and the parameters evaluated in this four groups of animals (Dex, REC 10, REC 20, REC 20TEx).
      Fig. 1
      Fig. 1Experimental design. Dexamethasone treatment and recovery schedule (a), mild therapeutic exercise protocol (b). Dex – 10 days DEX treatment; REC 10–10 days without DEX treatment/recovery; REC 20–20 days without DEX treatment/recovery; REC 20TEx – 20-day recovery/mild therapeutic exercise.

      2.3 Mild therapeutic exercise (climbing on vertical treadmill)

      For determining the effects of mild therapeutic exercise on recovery from myopathy, the animals climbed on a vertical treadmill without extra load. The vertical treadmill consists of a vertical ladder (length 1500 mm; width 300 mm) with stainless rungs (5 mm diameter) with 8 mm distance between each (
      • Pehme A.
      • Seene T.
      A model for strength exercise of skeletal muscles in the rat.
      ). Mild therapeutic exercise protocol started on 4th recovery day and consisted of 8 sessions (3–5 climbs repetitions per session) per recovery period. Animals were exercised on vertical treadmill at a speed 12 m/min (1st-4th exercise session) 1.0 m during 5 s and 18 m/min (5th–8th exercise session) 1.5 m during 5 s (Fig. 1b). This exercise speed on treadmill was tolerated by all rats of the group.

      2.4 Body and muscle mass

      The body mass of rats was measured during the short acclimatization, before (baseline) and after 10 days of DEX treatment, 10 days and 20 days of recovery (passive and active). Animals in the REC 20 and REC 20TEx groups had their body weight measured on each training day. After the experimental procedures, all animals were euthanized by excess of anaesthesia. Entire fast-twitch gastrocnemius muscles were surgically removed, trimmed clean of visible fat and connective tissue, weighed and immediately frozen.

      2.5 Hind limb grip strength

      Grip strength of hind limb was tested using a grip strength meter for rodents (Grip Strength Meter 0167-004 L, Columbus Instruments, US). Hind limb grip strength was measured before (baseline), after 10 days of DEX treatment, 10 days and 20 days of recovery (passive and active), thus before and at the end of the experimental protocol. Additionally, rat hind limb grip strength was measured in REC 20 and REC 20TEx groups before each training day. All rats performed three sequential trials. The average of three trials (strength in N) was included in the statistical analysis.

      2.6 Biomechanical parameters assessment

      In the present study a hand-held and non-invasive device (MyotonPRO, Myoton Ltd., Tallinn, Estonia) was used to study changes in biomechanical parameters of muscle (
      • Bizzini M.
      • Mannion A.F.
      Reliability of a new, hand-held device for assessing skeletal muscle stiffness.
      ;
      • Vain A.
      Estimation of the functional state of skeletal muscle.
      ;
      • Vain A.
      Device and method for real-time measurement of parameters of mechanical stress state and biomechanical properties of soft biological tissue.
      ). The central part of the gastrocnemius muscle (the major calf muscle on the posterior surface of the lower hind limb) belly of the rats was tested.
      The method is based on recording oscillation acceleration signals and subsequent computing of five parameters: 1) Tone (frequency of natural oscillation) (Hz), 2) stiffness (the ability of tissue resistance to contraction or external force that deforms the muscle's initial shape) (N/m), 3) elasticity (logarithmic decrement of damping oscillation's amplitude), 4) mechanical stress relaxation time (ms) and 5) gradual elongation of muscle over time under constant tensile stress (creep characterized by Deborah number).
      The higher the values of frequency of natural oscillation and stiffness are, the greater are the tension and stiffness of the examined muscle in the determined point. The lower the logarithmic decrement value (expressed in arbitrary units) is, the smaller is the dissipation of mechanical energy during oscillation and the higher is the elasticity of the muscle (
      • Gavronski G.
      • Veraksitš A.
      • Vasar E.
      • Maaroos J.
      Evaluation of viscoelastic parameters of the skeletal muscles in junior triathletes Physiol.
      ). The lower the relaxation time value is, the higher is the tension or stiffness. In this study was used MultiScan pattern of 20 measurements, and the mean was calculated.

      2.7 Statistical analysis

      Descriptive statistics were performed, reporting means and standard error (mean ± SE) values. Data were analysed with t-test and two-way analysis of variance (ANOVA), statistically comparing the results obtained before and after the DEX treatment and the recovery period. Statistical significance was defined as P < 0.05.

      3. Results

      3.1 Body and gastrocnemius muscle mass

      The groups began the experiment with similar body mass indicating similar physical activity. Over the treatment period, DEX induced a significant body and muscle mass loss. After 10 days of DEX treatment the rats had lost 23.4% of their initial body mass (P < 0.05) (Table 1).
      Table 1Body and gastrocnemius muscle mass (g) of DEX-treated rats after the treatment and recovery periods (mean ± SE).
      ABLDexREC 10REC 20REC 20TEx
      n = 5n = 4n = 4n = 4n = 4
      Body mass (g)351.00 ± 2.95265.80 ± 9.69 *284.28 ± 17.86 *307.15 ± 10.40 * #306.75 ± 7.33 * #
      Change %−23.4%−21%−11.5%−12-12,5%
      Muscle mass (g)1.52 ± 0.020.96 ± 0.03 *1.06 ± 0.09 *1.23 ± 0.01 * #1.10 ± 0.03 * # ¤
      Change %−37%−31%−20% -20%−28%
      ABL – average baseline, before DEX treatment; Dex – 10 days DEX treatment; REC 10–10 days without DEX treatment/recovery; REC 20–20 days without DEX treatment/recovery; REC 20TEx – 20-day recovery/mild therapeutic exercise. * P < 0.05 in comparison with ABL; # P < 0.05 in comparison with Dex; ¤ P < 0.05 REC 20 in comparison with REC 20 TEx.
      After 10 days of recovery, the body mass of the animals had not changed significantly compared to the corresponding value measured after DEX administration (21% and 23,4% below the baseline value, respectively). During the recovery period, the total body mass of rats reached the near pre-DEX treatment level (Table 1), however, remaining significantly lower in comparison with the baseline value (P < 0.05).
      Mild therapeutic exercise on vertical treadmill during the recovery period did not affect body mass recovery. At the end of 20-day recovery period total body mass in the studied groups was not completely restored.
      The results concerning the gastrocnemius muscle mass are consistent with the body weight curve data, with significant loss of muscle mass in the group that received GC in relation to the baseline level (P < 0.05) (Table 1).
      After 10 days of DEX administration the mass of gastrocnemius muscle constituted 63% of the initial level, during the subsequent 10-day recovery period the muscle mass had recovered to 70% of the baseline value, but significant muscle atrophy persisted (P < 0.05) (Table 1).
      After 20 days of recovery the muscle mass was not completely restored. Muscle atrophy had decreased but muscle mass remained significantly lower in comparison with the baseline value (Table 1). Similarly, significantly lower muscle mass was noted after 20-days of recovery compared to ABL, but it increased significantly compared to Dex in both groups. The muscle mass was significantly higher in REC 20 group compared to REC 20TEx group (Table 1).

      3.2 Hind limb grip strength

      As expected, DEX treatment caused significant decrease in hind limb grip strength. Grip strength was significantly lower in the Dex group compared to ABL (76% of baseline) (P < 0.05) and decreased further on the following 10 days, accounting for 68% of the initial level (Fig. 2).
      Fig. 2
      Fig. 2Changes in rat hind limb grip strength (percentage from the average baseline value) at different time points during experimental period. Data are presented as mean ± SE. ABL – average baseline, before treatment; Dex – 10 days DEX treatment; REC 10–10 days without DEX treatment/recovery; REC 20–20 days without DEX treatment/recovery; REC 20TEx – 20-day recovery/mild therapeutic exercise. * P < 0.05 in comparison with ABL; # P < 0.05 in comparison with Dex; ‡ P < 0.05 in comparison with REC 10.
      Fig. 3 shows the dynamics of body mass and hind limb grip strength during the 20-day active (mild therapeutic exercise) and passive recovery period.
      Fig. 3
      Fig. 3Dynamics of rat body mass (g) and hind limb grip strength (N) during DEX treatment and 20-day active and passive recovery period. Data are means ± SE. Bm REC 20 – body mass, 20 days without DEX treatment/recovery; Bm REC 20TEx – body mass, 20-day recovery/mild therapeutic exercise; ST REC 20 – hind limb grip strength, 20 days without DEX treatment/recovery; ST REC 20TEx – hind limb grip strength, 20-day recovery/mild therapeutic exercise. * P < 0.05 in comparison with the baseline value.
      Although body mass and hind limb grip strength were in the mild therapeutic exercise group animals slightly higher, no significant differences in body mass and hind limb grip strength were observed between the two recovery groups.

      3.3 Myometry

      This study evaluated the recovery of gastrocnemius muscle tone, and biomechanical and viscoelastic properties caused by myopathy.
      Fig. 4 illustrates the pattern of change in five measured parameters: tone (oscillation frequency) (Fig. 4a), dynamic stiffness (Fig. 4b), elasticity (logarithmic decrement) (Fig. 4c), mechanical stress relaxation time (Fig. 4d) and creep (Fig. 4e).
      Fig. 4
      Fig. 4Changes in gastrocnemius muscle tone (frequency) (a), stiffness (b), elasticity (decrement) (c), mechanical relaxation time (d) and creep (e) of rats at different time points during the experimental period. Data are presented as mean ± SE. ABL – average baseline, before DEX treatment; Dex – 10 days DEX treatment; REC 10–10 days without DEX treatment/recovery; REC 20–20 days without DEX treatment/recovery; REC 20TEx – 20-day recovery/mild therapeutic exercise. * P < 0,05 in comparison with ABL; # P < 0.05 in comparison with Dex; ‡ P < 0.05 in comparison with REC 10; ¤ p < 0.05 REC 20 in comparison with REC 20 TEx.
      The results showed a significant difference in muscle tone, elasticity, stiffness, relaxation time, and creep after DEX treatment. Muscle tone (oscillation frequency) (Fig. 4a) and stiffness (Fig. 4b) of the gastrocnemius muscle were significantly higher and muscle elasticity (logarithmic decrement) (Fig. 4c) was significantly lower than the ABL level. Mechanical stress relaxation time (Fig. 4d) and creep (Fig. 4e) values were significantly lower than the ABL values after DEX administration.
      At the end of recovery period all five measured parameters had gradually shifted towards the baseline values, however, the tone, stiffness, mechanical stress relaxation time and creep remained significantly lower than the initial values. Changes in muscle tone and stiffness occurred already 10 days after DEX administration, remaining significantly reduced at the end of 10-day recovery in comparison with DEX level (Fig. 4a and b).
      Significant differences in tone (oscillation frequency), stiffness and mechanical stress relaxation time occurred between the passive (REC 20) and active (REC 20 TEx) recovery. Gastrocnemius muscle tone and stiffness increased, mechanical stress relaxation time decreased with active recovery (mild therapeutic exercise), no significant differences were found in muscle elasticity and creep (Fig. 4a and b).
      Biomechanical parameters improved during the recovery period, but only dynamic stiffness and decrement returned to baseline value.

      4. Discussion

      In this study have been examined muscle atrophy caused by DEX administration, and the subsequent recovery of body mass, muscle mass, muscle tone and biomechanical parameters after withdrawal from DEX treatment.
      GCs has been shown to have a direct catabolic effect on muscle, specifically causing muscle atrophy and weakness (
      • Cohen S.
      • Nathan J.
      • Goldberg A.
      Muscle wasting in disease: molecular mechanisms and promising therapies.
      ;
      • Powers S.K.
      • Lynch G.S.
      • Murphy K.T.
      • Reid M.B.
      • Zijdewind I.
      Disease-induced skeletal muscle atrophy and fatigue.
      ). Among drug-induced myopathies, steroid myopathy is the most common and serious side effect of synthetic glucocorticoids (
      • Barel M.
      • Perez O.A.
      • Giozzet V.A.
      • Rafacho A.
      • Bosqueiro J.R.
      • do Amaral S.L.
      Exercise training prevents hyperinsulinemia, muscular glycogen loss and muscle atrophy induced by dexamethasone treatment Eur.
      ;
      • Egerman M.A.
      • Glass D.J.
      Signaling pathways controlling skeletal muscle mass Crit.
      ;
      • Ma K.
      • Mallidis C.
      • Bhasin S.
      • Mahabadi V.
      • Artaza J.
      • Gonzalez-Cadavid N.
      • Arias J.
      • Salehian B.
      Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression.
      ). The risk of steroid myopathy is enhanced with increased doses and prolonged duration of use.
      Administration of high-dose DEX causes not only decreased muscle mass but also muscle dysfunction characterized by reduced force and weakness (
      • Macedo A.G.
      • Krug A.L.
      • Souza L.M.
      • Martuscelli A.M.
      • Constantino P.B.
      • Zago A.S.
      • Rush J.W.
      • Santos C.F.
      Time-course changes of catabolic proteins following muscle atrophy induced by dexamethasone.
      ).
      The present study demonstrated that 10 days after DEX treatment developed significant atrophy of gastrocnemius muscle and 20-day recovery was not sufficient for reversing the muscle atrophy process. Our data showed that DEX treatment induced a significant body and muscle mass loss (23.4% and 37% respectively), and at the end of recovery period the body mass was not completely restored. In case of partial muscle atrophy evoked by 3–4 day DEX treatment, the loss of muscle mass is mostly reversible. After 20 days of recovery, muscle atrophy decreased but muscle mass remained significantly lower in comparison with the baseline value. The experimental protocol indicates that mild therapeutic exercise does not slow down weight loss of gastrocnemius muscle.
      • Rieu I.
      • Sornet C.
      • Grizard J.
      • Dardevet D.
      Glucocorticoid excess induces a prolonged leucine resistance on muscle protein synthesis in old rats.
      have examined muscle atrophy caused by DEX administration and the subsequent recovery of muscle mass after withdrawal from DEX treatment. During the recovery period an increase in body and muscle mass on the last day of the protocol was observed; these results are in agreement with previous publications (
      • Barel M.
      • Perez O.A.
      • Giozzet V.A.
      • Rafacho A.
      • Bosqueiro J.R.
      • do Amaral S.L.
      Exercise training prevents hyperinsulinemia, muscular glycogen loss and muscle atrophy induced by dexamethasone treatment Eur.
      ;
      • Macedo A.G.
      • Krug A.L.
      • Herrera N.A.
      • Zago A.S.
      • Rush J.W.
      • Amaral S.L.
      Low-intensity resistance training attenuates dexamethasone-induced atrophy in the flexor hallucis longus muscle.
      ).
      As we have shown in a previous study, hind limb DEX treatment caused significant decrease in hind limb grip strength (
      • Aru M.
      • Alev K.
      • Pehme A.
      • Purge P.
      • Õnnik L.
      • Ellam A.
      • Kaasik P.
      • Seene T.
      Changes in body composition of old rats at different time points after dexamethasone administration.
      ).
      During the following 10-day period, the gastrocnemius muscle mass and hind limb grip strength decreased to 69% and 68% of the baseline value, respectively. The decrease in muscle mass and hind limb strength is probably related to the cumulative effect of DEX. An increase in muscle mass and hind limb strength may be observed not before 10 days after stopping glucocorticoid treatment.
      After 20 days of recovery, the animals' hind limb grip strength was slightly lower than the baseline value and mild therapeutic exercise did not induce increase in grip strength. Interestingly, in the exercise group gastrocnemius muscle mass was significantly lower than in the passive recovery group while the hind limb grip strength was greatest in the active recovery group. The increased force production during the short-term recovery phase is probably nerve-mediated.
      • Seo Y.
      • Lew J.
      The effect of red ginseng on sarcopenic rat.
      have also shown in their study that after cessation of DEX treatment the recovery of body and muscle mass and grip strength occurs but this takes weeks.
      Different physical activity programmes (aerobic and resistance exercise) as non-pharmacological interventions to prevent muscle atrophy have been tested in animal and human studies (
      • Horber F.F.
      • Scheidegger J.R.
      • Grünig B.E.
      • Frey F.J.
      Evidence that prednisone-induced myopathy is reversed by physical training.
      ;
      • LaPier T.K.
      Glucocorticoid-induced muscle atrophy. The role of exercise in treatment and prevention.
      ;
      • Seene T.
      • Kaasik P.
      Role of exercise therapy in prevention of decline in aging muscle function: glucocorticoid myopathy and unloading.
      ), whereas resistance training and weight lifting have been shown to be the best way for muscle regeneration. Moderate exercise has also been shown to decrease GC-induced muscle atrophy (
      • LaPier T.K.
      Glucocorticoid-induced muscle atrophy. The role of exercise in treatment and prevention.
      ).
      In this study, a rat climbing model on vertical treadmill was used as therapeutic resistance exercise stimulating several muscle groups, including extensor digitorum longus, soleus, flexor hallucis longus and gastrocnemius muscles. Studies have reported that ladder climbing regimen increases muscle mass and strength, depending on the duration, frequency and intensity of the training (
      • Duncan N.D.
      • Williams D.A.
      • Lynch G.S.
      Adaptations in rat skeletal muscle following long-term resistance exercise training.
      ;
      • Hall K.E.
      • McDonald M.W.
      • Grise K.N.
      • Campos O.A.
      • Noble E.G.
      • Melling C.W.
      The role of resistance and aerobic exercise training on insulin sensitivity measures in STZ-induced type 1 diabetic rodents.
      ;
      • Ho K.W.
      • Roy R.R.
      • Tweedle C.D.
      • Heusner W.W.
      • Van Huss W.D.
      • Carrow R.E.
      Skeletal muscle fiber splitting with weight-lifting exercise in rats.
      ;
      • Klitgaard H.A.
      A model for quantitative strength training of hind limb muscles of the rat.
      ).
      In the present study, mild therapeutic exercise after DEX treatment did not attenuate body and muscle mass loss and the body mass remained lower throughout the experimental protocol.
      • Macedo A.G.
      • Krug A.L.
      • Herrera N.A.
      • Zago A.S.
      • Rush J.W.
      • Amaral S.L.
      Low-intensity resistance training attenuates dexamethasone-induced atrophy in the flexor hallucis longus muscle.
      have shown that low-intensity resistance exercise, performed together with DEX treatment, does not affect body mass.
      • Lee S.
      • Farrar R.P.
      Resistance training induces muscle-specific changes in muscle mass and function in rat.
      found in their study that 8-week vertical climbing does not change the mass of plantaris muscle. To our knowledge, there are no studies on exercise programmes that can reverse muscle atrophy and induce muscle recovery after stopping DEX treatment, whereas several studies have demonstrated that increased physical activity after immobilisation can reverse the muscle atrophy caused by inactivity (
      • Appell H.J.
      Morphology of immobilized skeletal muscle, the effects of a pre and post immobilization training program.
      ;
      • Kannus P.
      • Jozsa L.
      • Renström R.
      • Järvtoen M.
      • Kvist M.
      • Lehto M.
      • et al.
      The effects of training, immobilization and remobilization on musculoskeletal tissue. Remobilization and prevention of immobilization atrophy.
      ;
      • Song Y.
      • Lee S.
      • Yoo D.H.
      • Jang K.S.
      • Bae J.
      Strain sonoelastography of inflammatory myopathies: comparison with clinical examination, magnetic resonance imaging and pathologic findings.
      ).
      In the present study, hand-held and non-invasive digital device MyotonPRO was used to measure changes in biomechanical parameters of gastrocnemius muscle in rats before and after DEX treatment and the recovery period. MyotonPRO provides a reliable and sensitive way for objective and non-invasive digital palpation of superficial skeletal muscles. Muscle tone and biomechanical parameters such as stiffness and elasticity are either weakened or reinforced by DEX treatment, thus being potential targets for DEX-induced changes in biomechanical parameters (
      • Alev K.
      • Vain A.
      • Aru M.
      • Pehme A.
      • Purge P.
      • Kaasik P.
      • Seene T.
      Glucocorticoid-induced changes in rat skeletal muscle biomechanical and viscoelastic properties: aspects of aging.
      ).
      Muscle elasticity is a key determinant of muscle performance and force. The assessment of muscle elasticity in vivo can help to improve the understanding of muscle functions. Stiffness is induced by changes in the extracellular matrix and endo-, peri- and epimysium elasticity. Extracellular matrix is also influenced by GCs (
      • Oikarinen A.I.
      • Uitto J.
      • Oikarinen J.
      Glucocorticoid action on connective tissue from molecular mechanisms to clinical practice.
      ). Excess of GCs has to some extent similar effect on the intracellular and extracellular compartments of skeletal muscle, like decreased synthesis of proteins (
      • Seene T.
      Turnover of skeletal muscle contractile proteins in glucocorticoid myopathy.
      ;
      • Ahtikoski A.M.
      • Riso E.-M.
      • Koskinen S.O.A.
      • Risteli J.
      • Takala T.E.S.
      Regulation of type IV collagen gene expression and degradation in fast and slow muscles during dexamethasone treatment and exercise Pflüg.
      ). Increased amount of connective tissue within extracellular matrix of muscle increases muscle stiffness and reduces muscle strength.
      Reduced muscle elasticity, increased tone and stiffness with concomitant decrease of cytoskeletal proteins titin, nebulin and contractile protein myosin content are accompanied by muscle atrophy (
      • Alev K.
      • Vain A.
      • Aru M.
      • Pehme A.
      • Purge P.
      • Kaasik P.
      • Seene T.
      Glucocorticoid-induced changes in rat skeletal muscle biomechanical and viscoelastic properties: aspects of aging.
      ;
      • Aru M.
      • Alev K.
      • Gapeyeva H.
      • Vain A.
      • Puhke R.
      • Pehme A.
      • Kaasik P.
      • Selart A.
      • Seene T.
      Glucocorticoid-induced alterations in titin, nebulin, myosin heavy chain isoform content and viscoelastic properties of rat skeletal muscle.
      ), caused by DEX treatment.
      It is unknown how long after cessation of DEX treatment the recovery of muscle's biomechanical and viscoelastic properties, and functional parameters can take and whether mild therapeutic exercise affects the recovery of these parameters.
      Eccentric exercise affects muscle tone, elasticity and stiffness (
      • Hoang P.D.
      • Herbert R.D.
      • Gandevia S.C.
      Effects of eccentric exercise on passive mechanical properties of human gastrocnemius in vivo.
      ;
      • Whitehead N.P.
      • Weerakkody N.S.
      • Gregory J.E.
      • Morgan D.L.
      • Proske U.
      Changes in passive tension of muscle in humans and animals after eccentric exercise.
      ). In case of decreased muscle elasticity muscles are easily fatigued and their movement speed is limited (
      • Gapeyeva H.
      • Vain A.
      Methodical Guide: Principles of Applying Myoton in Physical Medicine and Rehabilitation.
      ).
      Values of tone, biomechanical and viscoelastic numerical parameters differed between the REC 20 group (20 days of reconditioning) and the Dex group. At the end of the recovery period all five measured parameters gradually shifted towards the average baseline but remained significantly lower than the initial values, which is a likely indicator for recovery taking effect in gastrocnemius muscle within the second week of recovery from myopathy. Changes in muscle mass were accompanied by changes in endo-, peri-, and epimysium-located collagen helix. These changes show effectiveness of transmission of mechanic elastic energy liberated from muscle contraction, from sarcomere to the skeleton (
      • Vain A.
      Estimation of skeletal muscle elasticity on subtonic tension level.
      ;
      • Vain A.
      The phenomenon of mechanical stress transmission in skeletal muscles.
      ).
      The ability to monitor patients being treated with GC may enable clinicians to detect evolving changes of steroid myopathy prior to clinically significant weakness. These findings suggest that myometer may quantify skeletal muscle alterations associated with corticosteroid use. The recording of biomechanical parameters allows to evaluate the efficiency of the recovery process in real time and manage the recovery through load selection.

      5. Study limitations

      The findings of the present study have several limitations. First, the generalizability of the obtained results is limited due to the small sample size and too short recovery period. Second, we did not study sex –related differences in the assessed parameters, only female rats were included in our study. Third, mild therapeutic exercise model should be improved, taking into account baseline functional status, for managing and regulation recovery from steroid-induced myopathy.

      6. Conclusions

      It is concluded that all evaluated parameters altered in rats by DEX administration were not completely restored after 20 days since DEX discontinuation. The present study demonstrated that 20-day free movement in cage and mild therapeutic exercise were not sufficient for reversing steroid myopathy in rats. According to the results of the present study this period is too short for full recovery of the evaluated parameters, and mild therapeutic exercise carried out after interruption of DEX was insufficient for reversing the GC-induced atrophy.
      The biomechanical parameters assessment clearly and easily indicated the direction and magnitude of change in muscle tissue after DEX administration and can be useful for detecting and tracking recovery from GC-induced myopathy.

      Funding

      No sources of funding were used to assist in the preparation of this article.

      Authors' contributions

      The authors confirm contribution to the paper as follows:
      study conception and design: P.K. and A. V.;
      data collection, analysis: K.A., M.A., A.P., A.V.;
      analysis and interpretation of results: K.A., M.A., A.P., P.K.;
      writing of the article, reviewing and revising the text and figures: T.S., K.A., A.V.
      All authors reviewed the results and approved the final version of the manuscript.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgements

      We would like to thank Mare Vene for language editing.

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